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The protein-only RNase P PRORP1 interactswith the nucleaseMNU2inArabidopsismitochondriaG. Bonnard, M. Arrivé, A. Bouchoucha, A. Gobert, C. Schelcher, F.Waltz,P.Giegé�Institut de biologiemoléculaire des plantes, CNRS,Université de Strasbourg,Strasbourg,FranceThe essential endonuclease activity that removes 5’ leader sequences fromtransfer RNA precursors is called RNase P.While ribonucleoprotein RNase Penzymescontainingaribozymearefoundinalldomainsoflife,anothertypeofRNasePcalled“PRORP”,for“PROtein-onlyRNaseP”,onlycomposedofproteinoccursinawidevarietyofeukaryotes,inorganellesandthenucleus.AlthoughPRORP proteins function as single subunit enzymes in vitro, we find thatPRORP1occurs inprotein complexes and is present inpolysome fractions inArabidopsis mitochondria. The analysis of immuno- precipitated proteincomplexes identifies proteins involved in mitochondrial gene expressionprocesses.Inparticular,directinteractionisestablishedbetweenPRORP1andMNU2anothermitochondrialnucleaseinvolvedinRNA5’processing.AspecificdomainofMNU2andaconservedsignatureofPRORP1arefoundtobedirectlyaccountableforthisproteininteraction.Altogether,resultsrevealtheexistenceofanRNA5’maturationcomplexinArabidopsismitochondriaandsuggestthatPRORPproteinsmightcooperatewithothergeneexpressionregulatorsforRNAmaturationinvivo.

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P1.2CytoRP,acytosolicRNasePtotargetTLS-RNAphytovirusesA.Gobert1,Y.Quan1,I.Jupin2,P.Giegé11Institut de biologie moléculaire des plantes, CNRS, Université deStrasbourg,Strasbourg,France�1InstitutJacquesMonod,CNRS,UniversitéParisDiderot,Paris,France�Inplants,PRORPenzymesareresponsibleforRNasePactivitythatinvolvestheremovalofthe5’extremityoftRNAprecursors.WeshowedpreviouslythatinadditiontothematurationoftRNAprecursors,PRORPenzymescleavetRNA-like structures (TLS) present on mitochondrial mRNA.�Many plant viruses(phytoviruses)areRNAvirusescontainingTLSstructures.TheseRNAelementsareinvolvedinvirusgenomereplication.ThebestcharacterizedTLSisfoundintheTurnipYellowMosaicVirus(TYMV)genome.TLSbasedvirusesarefoundinnumerous clades of viruses. Their impact on crop production is major withconsiderablecropyielddecreasesworldwide.OurprojectisbasedonthemodificationofPRORPenzymesinordertobeabletotargetTLSRNAvirusestoobtainplantresistancetovirusinfections.Forthis,weproducedaPRORPvariantcalled“CytoRP”,localizedinthecytosol,wherevirusreplicationtakesplace.�CytoRPisabletocleavetheTYMVTLSinvitro.WehaveproducedtransgenicplantsexpressingCytoRPandacisgenicplantwherethe nuclear localization signal of PRORP2 was deleted using CrispR/Cas9technology.WeshowthatthenuclearlocalizationofthesemutantsislostandCytoRPaccumulatesinthecytosol.TheabilityoftheseCytoRPplantstohinderTYMVinfectioninvivoiscurrentlyinvestigated.

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Structuralprobingofstaphylococcal-specificglyST-boxdomainsandmodulationoftranscriptionbyproteinsynthesisinhibitorsNikoleta Giarimoglou1#, Vassiliki Stamatopoulou1#, Shuang Li2,MariaMaria Apostolidi3, Eleni Kaliatsi1, Jinwei Zhang2 and ConstantinosStathopoulos11Department of Biochemistry, School of Medicine, University of Patras,Greece�2Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD,USA�3Department ofMolecular and Cellular Physiology, School ofMedicine,YaleUniveristy,WestHaven,CT,USAStaphylococcus aureus glyS T-box contains the species-specific interveningsequencestemSaaspartoftheantiterminator/terminatorstem,whichpossiblyconfers selectivity for five different tRNAGly isoacceptor ligands. In addition,stem Sa was found to accommodate binding of protein synthesis inhibitorswhich induce antiterminationof glyS transcription. Structural probing and invitroanalysisofaseriesofmutantscontainingswapsofstemIorapical loopbetweenO. iheyensis and S. aureus and stem Sa deletions revealed diverseeffectsinthepresenceoftigecyclineandlinezolid.DeletionofStemSareducedthe in vitro transcription independently of the presence or absence ofantibiotics, suggesting that stem Sa is important for T-box-mediatedtranscription.Moreover,tRNAGlymutationsincrucialnucleotidesofthe“elbow”showedthatstemIbecomesinsensitiveofdiscriminatingtheisoacceptorsandthisphenotypeisreversedwhenstemSaisdeleted.Takentogether,ourdatasupportourpreviousobservationsontheroleofstemSaintheequilibriumoftranscription termination/antitermination conformations, when all tRNAisoacceptorscompeteforT-boxriboswitchbinding.#equalcontribution

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P1.4Effects of Lupus antigen (La) expression in transcription andtranslationcomponentsinlungcancerEleniKaliatsi1,Athanasios-NasirShaukat1,IliasSkeparnias1,AikateriniI.Argyriou2,GeorgiosA.Spyroulias2andConstantinosStathopoulos1�1DepartmentofBiochemistry,SchoolofMedicine,UniversityofPatras,Greece2DepartmentofPharmacy,UniversityofPatras,GreeceLaprotein is themajorantigen in lupusandSjogren’s syndrome, involved infoldingoftRNAsandsafeguardingmiRNApathwayfromtRNAfragmentsduringselection of small non-coding RNAs for silencing events. Although aberrantexpressionofLahasbeenlinkedwithmalignancy,itsexactroleinvariouscancertypesremainsuncharacterized. In thepresentstudy,wescreened theNSCLCcell lineA549andvariousNSCLCbiopsysamplestoverifyupregulationofLa.ExpressionofLainA549cellsundervariousstressconditions,showedconstantlocalizationinthenucleusandnucleolus,incontrasttowhathasbeenreportedfor HeLa cells. Interestingly, expression of La both at transcriptional andtranslationallevelisincreasedunderglucosedeprivationbutdecreasedunderoxidativestress.ExpressionofLaleadstoupregulationofgenesinvolvedincellcycle,tRNAmaturationandtransportandmiRNAbiogenesisandupregulationof specific miRNAs and tRFs species. Finally, FACS analysis showed that Laexpressiondoesnotleadtocellcyclearrest.Instead,woundhealingandCFSEassaysshowedthatLaexpressionacceleratescellmotility,suggestingpossibleinvolvementinmetastasis.

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NuclearregulationofmitochondrialtRNAproduction�OliverRackham�HarryPerkinsInstituteofMedicalResearchandCentreforMedicalResearch,6 Verdun Street, Nedlands, Western Australia 6009, Australia�School ofMolecular Sciences, TheUniversity ofWestern Australia, Nedlands,WesternAustralia6009,AustraliaThe size and organization of the animal mitochondrial genome has beenreduced and compacted significantly since its endosymbiosis from an α-proteobacterial ancestor. This compaction has necessitated the evolution ofuniquemechanismstofacilitaterapidchangesingeneexpressioninresponsetothechangingenergydemandsofthecell.Themitochondrialtranscriptomeencodes proteins that are subunits of the respiratory chain, responsible formost of the energy production required by the cell. Consequently thecoordinatedregulationofthemitochondrialtranscriptomebythenucleusisofparticular importance for the maintenance of cell health and energymetabolism.WehavebeeninvestigatingtheunusualfeaturesofmitochondrialtRNAsandtheRNA-bindingproteinsthatcontroltheirproduction,maturation,translationandstabilizationtounderstandtheregulationofmitochondrialgeneexpression and its contribution to health and disease. I will highlight thedevastatingconsequencesofdysregulatedmitochondrialgeneexpressionandproteinsynthesisinanewmodelofdiseasecausedbygeneticdisruptionofatRNA-bindingprotein.Mousemodelsofdiseasehaveenabledustounderstandthe in vivo role of fundamental processes that regulatemitochondrial tRNAmetabolism and the pathogenesis of diseases caused by impaired proteinsynthesis.ThisworkillustratesthatbalancedmitochondrialtRNAproductionisrequiredfortheregulationofnucleargeneexpression,aswellastranslationinmultiplecellularcompartments.

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Characterizationofprotein-onlyRNasePincomplexwithtRNA.C.Schelcher1,A.Gobert1,C.Sauter2,P.Giegé1

1Institutdebiologiemoléculairedesplantes,Strasbourg,France2Institutdebiologiemoléculaireetcellulaire,Strasbourg,FranceRNasePactivityistheessentialcleavagethatremoves5'-leadersequencesfromtransfer RNA precursors. “PRORP” (PROteinaceous RNase P) is a category ofprotein-onlyRNaseP.BeforethecharacterizationofPRORP,RNasePenzymeswere thought to occur universally as ribonucleoproteins (RNP). ThecharacterizationofPRORPrevealedanenzymewithtwomaindomains,anN-terminaldomaincontainingfivePPRRNAbindingmotifsandaC-terminalNYNdomainholdingcatalyticactivity(1,2).We used a combination of biochemical and biophysical approaches tocharacterize the PRORP / tRNA precursor complex. Analyticalultracentifugation,microscalethermophoresisandactivityassaysshowthatthesole PPR motifs PPR2 and PPR3 are involved in PRORP/ tRNA precursorinteraction.TheseresultswerecombinedwithsmallangleXrayscaterringdataof PRORP alone and with tRNA to build a model of the PRORP2 pre-tRNAcomplex(3). Inthismodel,PPR2and3 interact, inaccordancewiththe“PPRcode”,repectivelywithC56andG18spacialylocalizedatthecornerofthetRNA.This shows that PRORP target recognitionprocess is in accordancewithPPRproteins canonical mode of action (4). Altogether, our analysis reveals aninterestingcaseofconvergentevolution.ItsuggeststhatPRORPhasevolvedanRNArecognitionprocesssimilartothatofRNPRNaseP.References:1. Gobert et al. (2010). A single Arabidopsis organellar protein has RNase P

activity. Nat. Struct. Molec. Biol., 17(6), 740–744.�2. Gobert et al. (2013).Structuralinsightsintoprotein-onlyRNasePcomplexedwithtRNA.Nat.comm,

4,1353.3.Pinker&Schelcheretal.(2017).BiophysicalanalysisofArabidopsis

protein-onlyRNasePaloneandincomplexwithtRNAprovidesarefinedmodel

of tRNA binding. J. Biol. Chem.: jbc.M117.782078.�4. Barkan et al. (2012). AcombinatorialaminoacidcodeforRNArecognitionbypentatricopeptiderepeat

proteins.PLoSGenetics,8(8),e1002910.

117

P1.7

Substrate recognition by the novel single-subunit protein-onlyRNasePHARPI.Schencking†,N.B.Wäber,M.Gößringer,R.K.HartmannInstitute of Pharmaceutical Chemistry, Department of Pharmacy, Philipps-UniversityMarburg†Correspondingauthor:isabell.schencking@pharmazie.uni-marburg.deRNasePistheessentialtRNA5'-endmaturationendonuclease.Recently,anewformofRNA-freeRNasePwasdiscoveredinthehyperthermophilicbacteriumAquifex aeolicus, termed AaRP (Aquifex aeolicus RNase P) [1]. Homologs ofAquifexRNaseP(HARP)wereidentifiedinmanyArchaeaandsomeBacteria,ofwhichallArchaeaandmostBacteriaalsoencodeanRNA-basedRNaseP.WehavebeguntostudysubstraterecognitionbyAq_880incomparisonwithHARPsfrom Methanothermobacter thermautotrophicus (archaeon) andThermodesulfatator indicus (bacterium) using a series of substrate variantsalreadyused inthestudyofsubstraterecognitionbyA.thalianaPRORP3[2]:

precursor-tRNAGlyderivativeswith(i)varying5'-leaderlength,(ii)with3'-CCAdeletionand3'-extensions,(iii)withotherstrategicdeletions,suchasthoseofanticodon andD armand (iv) length variation of acceptor/T stems. ThiswillprovideadetailedandrepresentativepictureofsubstraterecognitionbyHARPsincomparisontoPRORPsandRNA-basedRNasePenzymes.[1]A.I.Nickeletal.(2017)Proc.Natl.Acad.Sci.USA114:11121[2]N.Brillanteetal.(2016)NucleicAcidsRes.44:2323

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P1.8

AblationofRNaseZ5viaCRISPR-Cas9resultsinviablecellswithalteredphenotypeAthanasios-Nasir Shaukat, Eleni Kaliatsi, Ilias Skeparnias andConstantinosStathopoulosDepartmentofBiochemistry,SchoolofMedicine,UniversityofPatras,GreeceRibonucleaseZ(RNaseZ)isresponsibleforthe3’maturationofprecursortRNAsandtRF-1production. Inhuman,bothactivitiesareservedpredominantlybyRNaseZL(ELAC2),whiletheroleofRNaseZ5(ELAC1)whichis localizedinthecytoplasm, has been overlooked.Overexpression of both genes in A549 andHEK-293TcellsrevealeddifferentialresponsesontheexpressionofAGOgenes,p21, p27, p53 and CCN genes and DANCR, GAS5, HULC, HOTAIR, MALAT1lncRNAs, indicatingapossibleroleofbothenzymesinlncRNAbiogenesisandturnover.Inaddition,ELAC1wasupregulatedtogetherwithANGundervariousstress conditions, indicating a possible concerted action in stress inducibletiRNAmetabolism.TogaininsightsonthebiologicalroleofRNaseZ5weusedCRISPR-Cas9tosuccessfullyknockoutthesolecopyofELAC1encodedinHEK-293Tcells.AblationofELAC1didnotaffectcellviabilityandELAC2expressionbutupregulatedANG. Finally, it led to increasedG1phase, similar apoptoticbehavior,reducedproliferationandmotility,vulnerabilitytoserumdeprivation,higherresistancetoglucosedeprivation,increasedmitochondrialmasspercellandoverallalteredmorphology.

119

P1.9Role of CCA adding enzyme in the mitochondrion ofTrypanosomabruceiShikhaS,SchneiderA�DepartmentofChemistryandBiochemistry,UniversityofBernTbruceiwhichisasinglecelleukaryotepresentsauniquesituationwhereallthetRNAgeneshavebeenthrownoutof themitochondrialgenomeandareimportedfromthenuclearencodedcytosolicpoolofmaturetRNAs.Hence,thetRNAprocessingenzymesarenotneededinitsmitochondria.Yet,recentresultfromourlabshowsthatCCAaddingenzymethataddsCCAtailtothetRNAsinalleukaryotesispresentinthemitochondriaoftrypanosomes.WefoundthatCCA addition activity is also present in this mitochondrion along with thecytosol. And this activity is abolished by the knockdown of the single geneencodingforCCAaddingenzymeintrypanosomes.Additionally,theknockdownshows shorter tRNA accumulation in mitochondria. Though it is not clearwhethertheCCA-lesstRNAsareimportedintothemitochondriabecauseoftheknockdownor lackofenzymeleadstoaccumulationofCCA-lesstRNAduringinternaltRNAturnoveroftheorganelle.Throughdissectingitsduallocalisationmechanism we aim to create a mitochondrial knockout of this enzyme andascertain its dispensability for the mitochondria and/or involvement in anyotherfunctionapartfromitscanonicalroleinTbrucei.

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P1.10

Novel protein-onlyRNasePs in prokarya -comparisonofAaRPandarchaealHARPs�Wäber, Nadine B1*; Nickel, Astrid I1.;Gößringer, Markus1;Lechner,Marcus1;Schwarz,Thandi3;Weidenbach,Katrin4,Schmitz-Streit,Ruth4;Marchfelder,Anita3;Rossmanith,Walter2;HartmannRolandK.11Institute of Pharmaceutical Chemistry, Philipps University, Marburg,Germany�2Center for Anatomy& Cell Biology,Medical University of Vienna,Vienna,Austria3BiologyII,Ulmuniversity,Ulm,Germany�4InstituteofGeneralMicrobiology,Christian-Albrechts-UniversitätzuKiel,Kiel,Germany*Correspondingauthor:waeber@staff.uni-marburg.deRNasePistheessentialtRNA5'-endmaturationendonuclease.Recently,anewformofRNA-freeRNasePwasdiscoveredinthehyperthermophilicbacteriumAquifexaeolicus,termedAaRP(AquifexaeolicusRNaseP)[1].Incontrasttotheknown protein-only RNases P from Eukarya (PRORPs), AaRP is only a smallprotein(~23kDa).Wewereabletodetecthomologs,termedHARPs(Homologsof Aquifex RNase P), in some bacteria andmany archaea. In a few of thesebacteriaandinallofthosearchaea,theHARPcoexistswiththeclassicalRNasePconsistingofacatalyticRNAand thecorrespondingprotein subunit(s).WehavebeguntocharacterizetheHARPproteinsofselectedarchaealspeciesthatareamenabletogeneticstudies.OurstudiesincludeinvitroexperimentswithrecombinantHARPs(cleavageofvariousmodelsubstratestoanalyzesubstraterecognition, kinetics, crystallization, biophysical analyses etc.) as well as invivostudies (complementation analyses on solid and in liquid media,physiologicalcharacterizationofHARPknockoutstrains).[1]NickelA.I.etal.,PNAS,2017Oct17;114:11121

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Characterization of RNA post-transcriptional modifications inStaphylococcusaureusduringinfectionandantibioticstressesL.Antoine,P.Wolff,I.Caldelari,P.RombyandS.MarziInstitutdebiologieMoléculaire,UPR9002Architectureetréacivitédel’ARN15RueRenéDescartes,67084Strasbourg-FranceS.aureusisaGram-positivemajorhumanpathogeninvolvedinawiderangeofhuman infectious diseases. The treatment of S. aureus infection is verychallengingduetotheemergenceofmultipleantibioticresistantisolates.Thehigh diversity of clinical symptoms caused by S. aureus, as well as its drugresistance mechanisms depend on the precise expression of numerousvirulencefactorsandstressresponsepathways,whicharetightlyregulatedatseverallevels(transcription,translation,mRNAdecay).Duringthelasttwodecades,ithasbecomeevidentthatsmallregulatoryRNAs(sRNAs)playamajorroleintheseadaptiveresponses,mainlytargetingmRNAtranslation.Besides,tRNAsandcodonbiasareimpactingtranslationelongationand protein folding during stress responses and adaptation. In these RNAmediated regulatory mechanisms, RNA post- transcriptional modificationswhich can be modulated in response to adaptive processes could add anadditional layer of control. Altering the chemical and physical properties ofnucleotides,theyaffectRNAbase-pairingformation,proteinrecognition,RNAstructureandstability,mRNAtranslationandthewobble-basetRNAdecodingproperties.Theirimpactinpathogenicbacteriaduringgrowthadaptationandinfectionhasjuststartedtobeappreciated.UsingmassspectrometryandRNA-Seqmethods,wearecharacterizingtheepitranscriptomeofS.aureusinvariousconditions encountered during infection and by mutagenesis analysis themachineriesinvolvedintheirsynthesisandregulations.ThemethodsusedandsomepreliminarydatashowingdifferencesintRNAsmodificationsindifferent

stresseswillbediscussed.

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P2.2

Time-resolved NMRmonitoring of RNA maturation in cellularextractsrevealedcomplexcircuitsofmodificationsintRNAs�P.Barraud,A.Gato,M.Catala,C.Tisné�Institut de biologie physico-chimique, UMR 8261, CNRS, Université ParisDiderot,FranceDecades of exploration in the field of RNAmodification have revealedmorethan90differentribonucleosidestructuresincorporatedaspost-transcriptionalmodifications in tRNAs, which not only display the largest variety of post-transcriptionaldecorationamongRNAmolecules,butalsothehighestdensityofmodificationperRNAtranscript.Forinstanceinyeast,13modificationsaretypically found per tRNA molecule, which corresponds to ~12-20% of itsnucleotidesbearingachemicalmodification.Manyofthesemodificationsareintroduced at positions in the neighbourhood of others, enabling potentialinterplay intheir incorporation.Althoughthesecircuitsofmodificationshavebeen investigatedand identified in somecasesof rather strongdependency,theirstudyremainsdifficultsincemonitoringthematurationoftRNA inreal-timeatasinglenucleotidelevelistechnicallychallenging.Here,usinganoriginalmethodology,weshowthatnuclearmagneticresonancespectroscopy(NMR)isapowerfultooltomonitortRNAmaturationeventsinanon-disruptiveandcontinuous fashion. Time-resolved NMR measurements of differentmodification events in complex environments such as yeast cell extractsrevealed a defined chronology in the incorporation of tRNA modifications.Furthermore, a detailed analysis of snapshots measured along the tRNAmaturation route under various conditions revealed a complex circuitry ofmodifications where most modifications are seen to have positive and/ornegativeimpactontheincorporationofotherones.WebelievethattheNMR-basedmethodologypresentedherecanbeadaptedtoinvestigatemanyaspectsoftRNAmaturationandRNAmodificationsingeneral.

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P2.3LossoftRNAmodificationi6A37leadstomitochondriopathyandincreased+1frame-shiftinginmiceS.Bohleber,N.FradejasVillar,W.Zhao,U.Schweizer�InsitutfürBiochemieundMolekularbiologie,Bonn,GermanyIn mammals, cytoplasmic tRNAs carrying N6-isopentenyl adenosine (i6A) aretRNASEr(UCN)andtRNASer(UGA)MitochondrialisopentenylatedtRNAs,Tyr,Trp,Phe,and Ser(UCN), are further thiomethylated to ms2i6A (2-methylthio-N6-isopentenyl adenosine). While i6A is among the tRNA modifications longestknown, its significance in animals remained unclear. We have conditionallyinactivated Trit1 (tRNA:dimethylallyl-isopentenyltransferase 1) in mousehepatocytes and neurons accordingly abrogating tRNA isopentenylation intargeted cells. Ribosomal profiling of nuclear transcripts in liver revealsincreased+1frame-shifting.A-siteoccupancyofcodonsrequiringwobblebasepairs is changed in mitochondria. Expression of mitochondrial electrontransport chain subunits is impaired and themitochondrial integrated stressresponse pathway is mounted. Neuron-specific Trit1 knockout mice exhibitmicrocephaly and seizures and upregulate markers of the unfolded proteinresponsepathway.InactivationoftRNAisopentenylationismoreseverethaninactivationoftRNAthiomethylationunderliningthefundamentalroleofi6Ainmammalianbiology.

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P2.4

Queuosineand5-methylcytosinetRNAmodifications:ImpactoncellularprocessesandinvivoconsequencesCansuCirzi1,2,FrancescaTuorto1,FrankLyko1

�1DivisionofEpigenetics,DKFZ-ZMBHAlliance,GermanCancerResearchCenter,Heidelberg, Germany 2Faculty of Biosciences, University of Heidelberg,Heidelberg,GermanyTo date,many conserved tRNAmodifications have been catalogued. Recentstudies have provided novel insights into how these modifications and theresponsibleenzymesinfluencevariousbiologicalprocesses.However,verylittleis known about their contribution to cell fate decisions. To comprehensivelyunderstandtheinfluencesofthesemodificationsonvariouscellularprocesses,we aim to functionally characterize Q and m5C38 modifications, which arefound in the anticodon loop. We recently generated Qtrt1, Qtrtd1, Qtrt1-Dnmt2, andQtrtd1-Dnmt2 single and double knockoutmESCs using CRISPR-Cas9technology.PreliminaryexperimentshavebeenperformedtostudythefunctionalrelevanceofQandm5C38modificationsandtorevealtheeffectofthese modifications on proliferation, protein translation, and proteinhomeostasis. Firstly, the influenceofmicronutrientQonmammalianDnmt2activityhasbeenanalyzedinbothmouseandhumancell linesusingbisulfiteconversionbasedhigh-throughputsequencing.Secondly,dynamicregulationof protein synthesis is currently being analyzed by SuNSET and polysomeprofiling. Furthermore, using ribosome footprinting and computational dataanalysis, the impact of Q deficiency on protein translation fidelity at single-codon resolution will be determined. Lastly, to provide insights into theconsequencesofspecificmodificationsinvivo,Qtrt1andQtrtd1knockoutmicearebeinggeneratedandwillbecrossedwithDnmt2knockoutmicetofurtheranalyzethebiologicalfunctionsofthesemodificationsinvivo.Takentogether,thisprojectwillhavesignificantimplicationsforconnectingtRNAmodificationswithkeycellularprocessesand,inthelongterm,withhumandiseases.

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P2.5tRNAmodifyingenzymes:studyofexpressionchangesduringanepithelial-mesenchymal - like transition in a breast cancer cellmodel,byribosomeprofilingT. Fernández 1,2,M.Davyt 1, K. Perelmuter 3, C. Chalar 1,M. Bollati-Fogolín3,R.Ehrlich1,4,M.Marín11-Biochemistry-MolecularBiologySection,FacultyofSciences,Universidaddela República, Iguá 4225, Montevideo, Uruguay. 2- Bioinformatics Unit, 3-Cellular Biology Unit & 4- Institut Pasteur Montevideo, Mataojo 2020,Montevideo,Uruguay.MultiplespeciesandsubspeciesofisoacceptorandisodecodertRNAsconstitutethetRNApopulationofaparticularcellinadefinedstate.ThediversityofthetRNA population results from the differential expression of tRNA genes andtheir diverse state of post-transcriptionalmodification.Modificationof tRNAbases involvesa largesetofenzymesexhibitinghighspecificity (targettRNA,specific base and precise location in the structure). Given the emergingevidencethatlinkstRNApopulationwithcellfate,ouraimistounderstandifparticularcellsstateshaveadefineddistributionofitstRNAvariants,adaptedtotheongoingcellularprogramandtometabolicconditions.Asanapproachtobetter understand adaptive changes in tRNA populations, we studied thevariationsintheexpressionoftheenzymesinvolvedintRNAmodificationsatthetranscriptionalandtranslationallevels,duringanepithelial-mesenchymal-liketransitioninabreastcancercellmodel(MCF7derived).Enzymesexpressionlevels were obtained by ribosome profiling. The results are discussedconsideringthestateofthemainmetabolicpathwaysthatprovidesubstratesfortRNAmodificationenzymes.

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P2.6

DissectingtheroleofthetRNAmodifiersADAT3&WDR4inthewellness/illnessofthedevelopingcerebralcortex�JordiDelPozo,EfilBayam,PeggyTilly,MartinMarek,ElizabethRamos,ChristopheRomier,JulietteGodinInstitut de génétique et de biologie moléculaire et cellulaire (IGBMC),Strasbourg,FranceDevelopment and functioning of the cerebral cortex depends on thecoordinated production, migration and differentiation of excitatory andinhibitoryneurons.Disruptionoftheseprocessescancausesocially-devastatingneurodevelopmentaldisorderssuchepilepsyorseverelearningandintellectualdisabilities.MutationsingenesencodingtRNAmodificationenzymeshavebeenincreasinglylinkedtohumanneurologicaldisorders.Weaimatunderstandingtherole incerebralcorticogenesisofADAT3andWDR4,twotRNAmodifiers,whichneedtoformactiveheterodimerswithADAT2andMETTL1,respectively,to modify tRNAs. Mutations in both genes have been linked toneurodevelopmentaldisorders. InmiceembryoswehaveshownthatADAT3andWDR4aswellastheirpartnersshowadynamicexpressionduringcorticaldevelopment.ByusinginuteroelectroporationofmiRNAsinmouseembryosduringcorticaldevelopmentwehaveshownthatdepletionofbothADAT3andWDR4 delays radial migration of cortical neurons. Furthermore, we havedemonstratedthatalthoughmutantADAT3andWDR4stillformcomplexwiththeirpartners,thestabilityandfoldingofthemutantcomplexesareseverelyimpaired.

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P2.7

Queuine links translational control in eukaryotes to amicronutrientfrombacteriaMartin Müller1, Carine Legrand2, Francesca Tuorto2, Vincent Kelly3,YaserAtlasi4,FrankLyko2andAnnE.Ehrenhofer-Murray1

1Institut fürBiologie,Humboldt-Universität zuBerlin, Philippstr. 13,Haus22,10099Berlin�2DKFZ, ImNeuenheimerFeld580,69120Heidelberg�3SchoolofBiochemistry & Immunology, Trinity Biomedical Sciences Institute, 152-160Pearse Street, Trinity College Dublin, Ireland�4Department of MolecularBiology, Faculty of Science, Radboud Institute for Molecular Life Sciences,RadboudUniversityNijmegen,Nijmegen,NetherlandsIneukaryotes,thewobblepositionoftRNAwithaGUNanticodonismodifiedtothe7-deaza-guanosinederivativequeuosine(Q34),buttheoriginalsourceof Q is bacterial, since Q is synthesized by eubacteria and salvaged byeukaryotes for incorporation into tRNA. Q34 modification stimulatesDnmt2/Pmt1-dependent C38 methylation (m5C38) in the tRNAAsp anticodonloopinSchizosaccharomycespombe.Here,weshowbyribosomeprofilinginS.pombethatQmodificationenhancesthetranslationalspeedoftheC-endingcodonsforaspartate(GAC)andhistidine(CAC)andreducesthatofU-endingcodons for asparagine (AAU) and tyrosine (UAU), thus equilibrating thegenome-widetranslationofsynonymousQcodons.Furthermore,Qpreventstranslation errors by suppressing second-position misreading of the glycinecodonGGC,butnotofwobblemisreading.TheabsenceofQcausesreducedtranslationofmRNAsinvolvedinmitochondrialfunctions,andaccordingly,lackofQmodificationcausesamitochondrialdefectinS.pombe.WealsoshowthatQ-dependentstimulationofDnmt2isconservedinmice.Ourfindingsrevealadirect mechanism for the regulation of translational speed and fidelity ineukaryotesbyanutrientoriginatingfrombacteria.

129

P2.8Codon-specificeffectsoftRNAanticodonloopmodificationsontranslational misreading errors in the yeast Saccharomycescerevisiae�K.Joshi,M.J.Bhatt,P.J.FarabaughDepartmentofBiological Sciences,UniversityofMarylandBaltimoreCounty,Baltimore,Maryland,USAPost-transcriptionalmodificationoftRNAshaverolesinstabilizingthethreedimensionalstructureof the tRNAand supporting codon-anticodon recognitionduringaminoacyl-tRNArecruitment.ThroughtheserolestheystabilizethetRNAto degradation and improve decoding.Much of the information about their roles indecodinghavederivedfromstudiesofcognatedecodingbutarguablyequallyimportantis their roles inmodulating erroneous, near-cognatedecoding.Wehaveused in vivoreportersystemstodeterminethefrequenciesofmisreadingeventsbyindividualtRNAs.ThesystemsexploitessentialaminoacidsinthereporterproteinsE.colib-galactosidaseandPhotinuspyralis(firefly)luciferase.WehaveusedthesesystemstotesttheroleoftRNA modifications on near-cognate decoding by tRNAGlu

UUC and tRNALys

UUU in the

bacteriumE.coliandtheyeastS.cerevisiae.Wefinddifferentrolesforx5m2sU34inthetwospecies.InE.coli,mnm5s2U34eitherincreasesordecreasesmisreadingregardlessofthe codon recognized while in S. cerevisiae the effect of mcm5s2U34 the samemodificationonaparticulartRNAcaneitherincreaseordecreasemisreadingdependingon the codon being decoded. In E.coli, therefore, themodification affects a generalproperty of the tRNA while in S. cerevisiae the modification modulates the codon-anticodoninteractioncontextspecifically.Wealsofoundthatt6A37modificationinyeasthasdistincteffectsonmisreadingdependingonthepositionofthebasemismatch. Itstronglydecreasesmisreading involving firstpositionU1-U36mismatchesbut increasemisreadinginvolvingsecondorwobblepositionmismatches.Thiscontrastswithitsroleincognatedecoding inwhich itstabilizes theweakA1-U36pair;whereast

6Astabilizescognatedecodingbyacross-strandstackinginteractionwiththefirstcodonnucleotide,itsunkowninteractionwiththeextremelyweakU1-U36interfereswithitsformationaspartofrecruitmentofthenear-cognatetRNA.TheseresultssuggestthatpredictionofthefunctionoftRNAmodificationsduringmisreadingcannotbepredictedbytheirrolein cognatedecodingand thateven closely relatedmodificationsmayhavedrasticallydifferentfunctionsinmodulatingmisreading.

130

P2.9

Structuralandbiochemicalanalysisofthedual-specificityTrm10enzyme from Thermococcus kodakaraensis promptsreconsiderationofitscatalyticmechanismRanjanKumarSingh1,2,a,AndréFeller3,a,MartineRoovers4,a,DanyVanElder3,LinaWauters1,2,5,LouisDroogmans3,WimVersées1,21StructuralBiologyBrussels,VrijeUniversiteitBrussel,Pleinlaan2,1050Brussel,Belgium.� 2VIB-VUB Center For Structural Biology, Pleinlaan 2, 1050 Brussel,Belgium.�3LaboratoiredeMicrobiologie,UniversitélibredeBruxelles(ULB),12RuedesProfesseursJeeneretBrachet,6041Gosselies,Belgium.�4InstitutdeRecherchesMicrobiologiquesJean-MarieWiame-Labiris,AvenueE.Gryson1,1070 Bruxelles, Belgium. 5Department of Cell Biochemistry, University ofGroningen,Nijenborgh7,Groningen9747AG,Netherlands.�aTheseauthorscontributedequallytothisworkN1methylation of purines at position 9 of tRNA is catalyzed by the SPOUTmethyltransferase (MTase) Trm10. While certain Trm10 orthologues arespecificforeitherGorA,othersshowadualspecificity.WereportthestructureandbiochemicalanalysisofthedualspecificityenzymefromT.kodakaraensis(TKTrm10). We solved the crystal structure of a construct of this enzyme,consistingof theN-terminaldomainandthecatalyticSPOUTdomain.CrystalstructuresoftheSPOUTdomain,eitherintheapoformorboundtoSAMorSAHrevealconformationalplasticityoftwoactivesiteloopsuponsubstratebinding.KineticanalysisshowsthatTKTrm10hasahighaffinityforitstRNAsubstrates.Mutation of either of two active site residues (D206, D245) results in onlymodest effects on the methylation reaction, with a small shift toward apreferenceform1Goverm1Aformation.OnlyadoubleD206A/D245Amutationseverelyimpairsactivity.Theseresultsareinlinewiththerecentfindingthatthe single active-site D was dispensable for activity in the G-specific yeastTrm10,andsuggestthatalsodualspecificityTrm10useamechanismwithoutresiduesactingasgeneralbasecatalysts.

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P2.10Unexpected effect on selenoprotein expression by lack of i6AmodificationintRNA[Ser]SecN.FradejasVillar,S.Bohleber,W.Zhao,U.Schweizer�InstitutfürBiochemieundMolekularbiologie,Bonn,GermanySelenoproteinscontaintheaminoacidselenocysteine(Sec)encodedbyUGA.RecodingofUGAasSecrequiresa3’stemloopinselenoproteinmRNAs,proteinfactors, and tRNA[Ser]Sec(UGA). This tRNA is isopentenylated at adenosine 37(i6A37)bytheenzymeTrit1.Several linesofevidencesuggestedthat i6A37intRNA[Ser]Sec is essential for efficient recodingofUGA: 1) Lovastatin treatmentreduced selenoprotein expression in cultured cells. 2) Mice expressingtRNA[Ser]SeccarryingA37Gdonotexpressall selenoproteins.3)KnockdownofTRIT1 inHepG2 cells showed decreased selenoprotein expression under lowseleniumconditions.InordertoaddresstheroleofisopentenylatedtRNA[Ser]Sec,wehavegeneratedTrit1-knockoutmiceandanalysedselenoproteintranslationbywestern-blotandribosomeprofiling.Therewasnogeneraldownregulationof selenoprotein expression in Trit1-knockout mice. Ribosome profilingrevealed unchanged UGA recoding efficiency. Selenoprotein expression inpatient fibroblasts carrying an inactivating TRIT1 mutation was also notaffected.Wespeculatethataroleofi6A37modificationoftRNA[Ser]Secmayonlyberevealedunderconditionsofseleniumdeficiency.

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Cytosine-5 tRNA methylation links protein synthesis to cellmetabolism�N. A. Gkatza1, C. Castro2, M. C. Popis1, S. Blanco3, S. Bornelöv4, J.Gleeson5,J.L.Griffin2,J.A.West2,S.Kellner6,S.Dietmann4,M.Frye1,7�1UniversityofCambridge,DepartmentofGenetics,DowningStreet,CambridgeCB2 3EH, UK, 2University of Cambridge, Department of Biochemistry, TennisCourtRoad,CambridgeCB21QW,UK;3CancerCellSignalingandMetabolismLab,ProteomicsUnitCICbioGUNE,Bizkaiatechnologypark,801building48160,Derio,Spain;4UniversityofCambridge,Wellcome–MedicalResearchCouncilCambridgeStemCellInstitute,TennisCourtRoad,CambridgeCB21QR,UK;5UCSan Diego School of Medicine, Department of Neurosciences, 9500 GilmanDrive, La Jolla, CA 92093-0662, USA; 6Department of Chemistry, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, Haus F, 81377 Munich,Germany; 7German Cancer Center (DKFZ), ImNeuenheimer Feld 280, 69120Heidelberg,GermanyChemicalmodificationsintRNAareoftencriticalfornormaldevelopmentastheyadaptprotein synthesis to adynamically changingmicroenvironment.However, theprecisecellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modificationpathway remain unclear. Here, we identified the cytosine-5 RNA methyltransferaseNSUN2as a sensor for external stress stimuli. Exposure tooxidative stress efficientlyrepressed NSUN2 causing a reduction of methylation at specific tRNA sites. Usingmetabolicprofiling,weshowedthatlossoftRNAmethylationcapturedcellsinadistinctcatabolicstate.Mechanistically, lossofNSUN2alteredthebiogenesisoftRNA-derivednon-coding fragments (tRFs) in response to stress leading to impaired regulation ofprotein synthesis. The intracellular accumulation of specific tRFs was sufficient todynamically repress global protein synthesis. Finally, NSUN2-driven RNAmethylationwasfunctionallyrequiredtoadaptcellcycleprogressiontotheearlystressresponse.Insummary, we revealed that changes in tRNAmethylation profiles were sufficient tospecify cellular metabolic states and efficiently adapt protein synthesis rates to cellstress.

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ReprogrammingtRNAmodificationstocombatcancer�PavlínaGregorová,L.PeterSarin�Molecular and Integrative Biosciences Research Programme, Faculty ofBiologicalandEnvironmentalSciences,UniversityofHelsinki,Helsinki,FinlandTransfer RNAs (tRNAs) are richly decoratedwith various post-transcriptionalmodifications that influence the rate and accuracy of translation. Althoughperturbations in tRNAmodificationunbalance translation, their exact roleasmediators in tumour formation and proliferation is only starting to emerge.Hence, we aim to (i) identify tRNA modifications with significantly alteredabundance and (ii) investigate tRNA modification-mediated translationalchangesinvariouscancers.WehypothesizethatcancercellscanberescuedbypinpointingtRNAmodificationsthatmaintaintumorigenicgrowthandcorrecttheirmodificationlevelstothatofhealthy,non-oncogeniccells.Tothisend,weuse mass spectrometry and ribosome profiling to quantitatively determinetRNA modification levels and monitor translational changes in healthy vs.cancerouscells.Thefollow-upcorrelationofRNAmodificationandtranslationchangeswillcreateadetailedcharacterizationoftRNA-mediatedtranslationalresponsesand identifykeyregulatorymodificationsonspecifictRNAspecies.This study will highlight the regulatory function of tRNA modifications andexpandourunderstandingofcancerformation.

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P2.13

A hypertension-associated mitochondrial DNA mutationintroduces an m1G37 modification into tRNAMet, altering itsstructureandfunction1,2Mi Zhou, 3Ling Xue, 3Yaru Chen, 4Haiying Li, 2Qiufen He, 3BibinWang,1,2FeilongMeng,1,2MengWangand1,2,Min-XinGuan*1DivisionofMedicalGeneticsandGenomics,TheChildren’sHospital,ZhejiangUniversitySchoolofMedicine,Hangzhou,310058,Zhejiang,China�2InstituteofGenetics,ZhejiangUniversitySchoolofMedicine,Hangzhou,310058,Zhejiang,China 3Attardi Institute of Mitochondrial Biomedicine, Wenzhou MedicalUniversity,Wenzhou,325035,Zhejiang,ChinaDefectivenucleotidemodificationsofmitochondrial tRNAshavebeenassociatedwithseveralhumandiseases,buttheirpathophysiologyremainspoorlyunderstood. Inthisreport, we investigated the pathogenic molecular mechanism underlying ahypertension-associated4435A>GmutationinmitochondrialtRNAMet.Them.4435A>Gmutationaffectedahighlyconservedadenosineatposition37,3’adjacenttothetRNA’santicodon,whichisimportantforthefidelityofcodonrecognitionandstabilization.Wehypothesized that the m.4435A>G mutation introduced an m1G37 modification oftRNAMet,alteringitsstructureandfunction.Primerextensionandmethylationactivityassays indeed confirmed that the m.4435A>G mutation created a tRNAmethyltransferase 5 (TRMT5)–catalyzedm1G37modification of tRNAMet.We foundthat thismutation altered the tRNAMet structure, indicated by an increasedmeltingtemperatureandelectrophoreticmobilityofthemutatedtRNAcomparedwiththewild-typemolecule.Wedemonstratedthatcybridcelllinescarryingthem.4435A>Gmutationexhibitedsignificantlydecreasedefficiencyinaminoacylationandsteady-statelevelsoftRNAMet,ascomparedwiththoseofcontrolcybrids.TheaberranttRNAMetmetabolismresultedinvariabledecreasesinmitochondrialDNA(mtDNA)-encodedpolypeptidesinthe mutant cybrids. Furthermore, we found that the m.4435A>G mutation causedrespiratory deficiency,markedly diminishedmitochondrial ATP levels andmembranepotential,and increasedtheproductionof reactiveoxygenspecies inmutantcybrids.These results demonstrated that an aberrant m1G37 modification of mitochondrialtRNAMet affected the structure and function of its tRNA and consequently alteredmitochondrialfunction.Ourfindingsprovidecriticalinsightsintothepathophysiologyofmaternallyinheritedhypertension,whichismanifestedbythedeficienttRNAnucleotidemodification.

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P2.14

Investigating Geobacillus kaustophilus TilS dynamics usingmoleculardynamicsimulationsGuinto,F.andAlexander,R.�WakeForestUniversity,Winston-Salem,NorthCarolinaNucleotidemodificationscontributetothestability,structure,andfunctionoftRNAmolecules.Somemodificationsareessentialfortranslationalfidelityandtheir loss scan affect cell viability. One such modification proposed to beessentialinprokaryotesisthelysidineinstalledatthetRNAIle2wobblepositionby tRNA isoleucine lysidine synthetase (TilS). Addition of lysine at the C2positionisessentialfordecodingtherareisoleucineAUAcodoninprokaryotes.Because TilS is thought to be essential for specific and accurate proteinsynthesis,itisusefultoinvestigatethedynamicsandmechanismoftheenzymetogainabetterunderstandingof itsabilitytorecognizeandbinditscognatesubstrate, including both the anticodon in the enzyme’s active site and theacceptorendatadistance~80Åaway.Throughtheuseofmoleculardynamics,theflexibilityandmobilityofTilScanbemonitored.Weperformedsimulationsusing the 3.65 Å Geobacillus kaustiphilus TilS:tRNA Ile2 crystal structure,including protein-only and tRNA-bound systems, to better understand thechangesinflexibilityuponcomplexformation.

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RoleoftRNAbindingproteinKti12forfunctionoftheElongatorcomplexAlexander Hammermeister1, Rościsław Krutyhołowa2, Rene Zabel3,MarkHelm4,KarinD.Breunig3,SebastianGlatt2,andRaffaelSchaffrath11InstitutfürBiologie,UniversitätKassel,Germany�2MaxPlanckResearchGroup,Malopolska Centre of Biotechnology, JagiellonianUniversity, Krakow, Poland3Institut für Biologie, Martin- Luther-Universität, Halle-Wittenberg,Germany�4InstitutfürPharmazieundBiochemie,UniversitätMainz,GermanyCertain tRNA species contain Elongator complex dependentmodifications atanticodon wobble uridines (U34). These are targets for zymocin, a fungalribotoxinproducedbyKluyveromyceslactis.ZymocincleavesintheanticodonofmodifiedtRNAsandtherebyresultsincelldeath.MutationsintheElongatorcomplexresultinlossofU34modificationandresistancetowardszymocin.Here,wedemonstratethatsimilarU34modificationdefectsareobservableifKTI12,agene coding for an Elongator partner protein, is inactivated or mutated.Interestingly,theU34modificationsdependonanNTPbindingP-loopdomaininKti12, which is conserved with a tRNA kinase (PSTK) and whose mutationabolishes interactionwith Elongator in yeast. This strengthensour view thatphysical contact between Kti12 and Elongator is required for anticodonmodification.Indeed,LC-MS/MSmeasurementsperformedontRNAsextractedfromP-loopmutantsverifylossofU34modification.Inaddition,weshowthatKti12,likePSTK,isabletobindtotRNAsinafashionthatappearstobeP-loopindependentinvitro.ProjectsupportbyDFG,Germany,toRS(SCHA750/18;SCHA750/20)

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NucleartRNAexportinTrypanosomabruceiE. Hegedűsová1, A. C. Kessler3, S. Kulkarni1,2, B. Burgman1,4, J. D.Alfonzo3andZ.Paris1,21Institute of Parasitology, Biology Centre CAS, České Budějovice, CzechRepublic�2FacultyofScience,UniversityofSouthBohemia,ČeskéBudějovice,CzechRepublic�3DepartmentofMicrobiologyandTheCenterforRNABiology,TheOhioStateUniversity,Columbus,OH43210,USA4TheUniversityofArizona,Tucson,Arizona,USAProtein and RNA transport across the nuclear envelope occurs through thenuclear pore complex (NPC) and requires proteins of the karyopherin family(exportins).Xpo-tinvertebratesandLos1andMsn5inyeastspecificallyexporttRNAsfromthenucleus.T.bruceiisasingle-cellparasite,that,incontrastwithother eukaryotes, lackmost of the transcriptional control; the bulk of geneexpression regulation occurring post-transcriptionally. Nuclear tRNA exportmightprovideanadditional levelofregulationofgeneexpressionduringthecomplexlifecycleoftheseparasites.However,onlyalimitedsetoftheexportfactors,conservedinotherorganisms,canbeeasilyidentifiedintheT.bruceigenome.Ourresults indicate thatsimilar toothereukaryotes,TbXpo-t isnotessentialforgrowthoftrypanosomes.Moreover,RNAisilencingofTbXpo-tdidnot result either in disruption of the tRNA translocation, or in intron-tRNAaccumulation in the nucleus, both phenotypes described for yeastmutants.These observations are discussed in the context of the possible roles of themRNAexportpathwayinthetranslocationofmaturetRNAsthroughtheNPCanditsroleontRNAprocessing.

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P2.17

Longandbranchedpolyaminesarerequiredformaintenanceoftheribosome,tRNAHis,andtRNATyrinThermusthermophiluscellsathightemperaturesMisa Nakashima1, Ryota Yamagami1, Chie Tomikawa1, Yuki Ochi1,Toshiyuki Moriya2, Haruichi Asahara3, Dominique Fourmy4, SatokoYoshizawa4,TairoOshima2andHiroyukiHori11Grad. Sch. of Sci. and Eng., Ehime Univ., Matsuyama, Japan�2Inst. ofEnvironmentalMicrobiol.,KyowaKako,Machida,Japan�3NewEnglandBiolabs,Ipswich, MA, USA�4Inst. for I2BC, CNRS, Université Paris-Sud, Gif-sur-Yvettecedex,Francehori.hiroyuki.my@ehime-u.ac.jpThermusthermophilusisanextremelythermophiliceubacteriumthatproducesvarious polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAMdecarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis ofpolyamines. The speB and speD1 gene-deleted strains (DpeB and DpeD1,respectively) cannot synthesize long and branched polyamines. Althoughneitherstraingrewathightemperatures(>75oC),bothstrainssurvivedat80oCwhentheywereculturedat70oCuntilthemid-logphaseandthenshiftedto80oC.WepreparedtheDpeBandDpeD1cellsusingthismethod.Microscopicanalysisshowedthatbothstrainscansurvive for10hat80oC.Althoughthemodificationlevelsof2’-O-methylguanosineatposition18,7-methylguanosineat position 46, 5-methyluridine at position 54 and 1-methyladenosine atposition58intRNAfrombothstrainswerenormal,amountsoftRNATyr,tRNAHis,rRNAs, and 70S ribosomes were decreased. Furthermore, in vivo proteinsynthesisinbothstrainswascompletelylost10hafterthetemperatureshift.Thus,longandbranchedpolyaminesarerequiredforatleastthemaintenanceof70SribosomeandsometRNAspeciesathightemperatures.

139

P2.18SpecificityintheBiosynthesisoftheUniversaltRNANucleosideThreonylcarbamoylAdenosine(t6A)William Swinehart, 1

Christopher Deutsch, 1

Amit Luthra, 2

Manal

Swairjo,2andDirkIwata-Reuyl1

1DepartmentofChemistry,PortlandStateUniversity,Portland,OR�2DepartmentofChemistryandBiochemistry, SanDiegoStateUniversity, SanDiego,CAN6-threonylcarbamoyl adenosine (t6A) is a universal nucleosidemodificationfoundatposition37oftRNAsdecodingANNcodons.InBacteriatheproteinsTsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatusresponsiblefort6Aformation.TsaCcatalyzestheformationoftheintermediatethreonylcarbamoyladenosinemonophosphate(TC-AMP)fromATP,threonine,andCO2,andTsaDcatalyzesthetransferofthethreonylcarbamoylmoietyfromTC-AMP to A37 of substrate tRNAs. Several related modified nucleosides,including hydroxynorvalyl carbamoyl adenosine (hn6A), havebeen identified,butnothingisknownabouttheirbiosynthesis.Tobetterunderstandstructuralconstraintsont6Aformation,andtodetermineifrelatedmodifiednucleosidesare formed via parallel biosynthetic pathways or the t6A pathway, weinvestigatedthespecificityofthet6AsystemsfromE.coliandT.maritima.OurdatademonstratethatTsaC/C2exhibitrelaxedsubstratespecificity,producingavarietyofTC-AMPanalogsthatcandiffer inboththe identityoftheaminoacidandnucleotidecomponent,whileTsaDdisplayshighspecificity inE.coliandrelaxedspecificityinT.maritima.

140

P2.19

Leber's hereditary optic neuropathy is potentially associatedwithanovelm.5587T>CmutationinChinesepedigreesYanchunJi1,2,Min-XinGuan1,2*1DivisionofMedicalGeneticsandGenomics,theChildren'sHospital,ZhejiangUniversitySchoolofMedicine,Hangzhou,Zhejiang,China.2InstituteofGenetics,ZhejiangUniversitySchoolofMedicine,Hangzhou,Zhejiang,China

Mitochondrial (mt)DNAmutations have been revealed to be associatedwith Leber'shereditary optic neuropathy (LHON). Our previous investigation has shown themutationalincidenceandspectrumoftheND1,ND4andND6geneinalargecohortof1281ChinesesubjectswithLHON.Resultantvariantswereevaluatedforevolutionaryconservation,allelicfrequencies,andstructuralandfunctionalconsequences.However,themutationalspectrumandincidenceinthetRNAgenearepoorlyunderstoodintheLHONpatients.Furthermore,itisanticipatedthatadditionalmutationscanbefoundinthetRNAgene.Inthisinvestigation,atotalof4(3menand1female)outof14matrilinealrelativesintwoChinesefamiliesexhibitedvisualimpairmentwithvariableseverityandage of onset. The average age of onset of visual loss was 20.5 years old.Molecularanalysisofthecompletemitochondrialgenomeinthesepedigreesdemonstratedthatthe threeprimarymutations associatedwith LHONwerenotdetected;however, thehomoplasmicm.5587T>Cmutationwasidentified,whichwaslocalizedattheendofthemitochondrially encoded transfer (t)RNA alanine gene and may alter the tertiarystructure of this tRNA. Subsequently, this structural alteration may result in tRNAmetabolism failure. In addition, distinct sets of mtDNA polymorphisms belonging tohaplogroupF1weredetectedinbothfamiliestested.Thefindingsofthepresentstudysuggestedthatthem.5587T>Cmutationmaybeinvolvedinthepathogenesisofvisualimpairment.Inaddition,themtDNAvariantm.15024G>A(p.C93H)inthemitochondriallyencodedcytochromeBgenewasdetectedinbothfamilies,whichexhibitedevolutionaryconservation,indicatingitmayserveapotentialmodifyingroleinthedevelopmentofvisualimpairmentassociatedwithm.5587T>Cmutationinthesefamilies.Furthermore,other modifying factors, including nuclear modifier genes, and environmental andpersonalfactorsmayalsocontributetothedevelopmentofLHONinsubjectscarryingthis mutation. Our findings may provide valuable information for pathophysiology,management,andgeneticcounselingofLHON.

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P2.20

MitochondrialtRNA(Glu)14693A>GmutationisapotentiallyriskfactorinthephenotypicmanifestationofLeber'shereditaryopticneuropathyinacohortofChinesefamilies�Yanchun Ji1,2, Meng Wang1,2,Ye Chen1,2, Pingping Jiang1,2, Min-XinGuan1,2

1DivisionofMedicalGeneticsandGenomics,theChildren'sHospital,ZhejiangUniversity School of Medicine, Hangzhou, Zhejiang, China.�2Institute ofGenetics,ZhejiangUniversitySchoolofMedicine,Hangzhou,Zhejiang,China�Mitochondrial DNA (mtDNA) mutations have been associated with Leber’s hereditary opticneuropathy(LHON).Inthisstudy,Acohortof1741HanChineseprobandsand485controlsubjectsunderwent sequence analysis ofmitochondrial (mt)DNA. Resultant variants were evaluated forevolutionary conservation, allelic frequencies, and structural and functional consequences. weinvestigatedthepathophysiologyofaLHONsusceptibilityallele(m.14693A>G)inthemitochondrialtRNAgene.Them.14693A>Gmutationaffectedahighlyconservedatposition54oftheTΨCloopoftRNA(Glu).Thedestabilizationofbase-pairing(54A-U58)causedbythem.14693A>GmutationperturbedtheconformationandstabilityoftRNA(Glu).Theincidenceofm.14693A>Gmutationwas1.8%, either isolation in 15 pedigrees or together with the m.11778G>A in 12 pedigrees, andm.14484T>C mutations in 3 pedigrees, and m.3460G>A mutations in one pedigree in theseprobands with LHON. The occurrence of m.14693A>G mutation in these genetically unrelatedpedigreesaffectedbyLHONanddifferingconsiderablyintheirmtDNAhaplotypes(M7,N9,D4,D5,Y) strongly indicated that this mutation is involved in the pathogenesis of this disorder.Furthermore, the average penetrance of LHON among 12 Han Chinese pedigrees carryingm.14693A>G and m.11778G>A mutations, and 3 pedigrees bearing both m.14693A>G andm.14484T>Cmutations,andonepedigreebearingbothm.14693A>Gandm.3460G>Amutationswere significantly higher than those carrying only m.14693A>G, m.11778G>A, m.14484T>C orm.3460G>Amutation(p<0.0001).Inparticular,theaveragepenetrancesofopticneuropathyof26familiescarryingonlym.3394T>Cmutationwere12.27%,whiletheseamong21pedigreesharboringbothm.3394T>Candm.11778G>Aorm.14484T>Cmutationswere39.47%,46.28%and42.85%,respectively. Therfore, we hypothesized that the biochemical consequences caused by them.14693A>Gmutation may deteriorate the mitochondrial dysfunction associated with primarymutation,therebyincreasingthepenetranceandriskofLHON.OurfindingsprovidenewinsightsintothepathophysiologyofLHONarisingfromthesynergybetweenmitochondrialND1andND4mutations.

142

P2.21

Post-transcriptional modification and cellular energy levelscontroldegradosome-dependenttRNAclearanceSatoshiKimuraandMatthewWaldor�BrighamandWomen’sHospital,Boston,UnitedStatesHarvardMedicalSchool,Boston,UnitedStatesHHMI,Boston,UnitedStatesThefactorsandmechanismsthatmediateandcontroltRNAstabilityinbacteriaare not well understood. Here, we studied the roles of post-transcriptionalmodification in bacterial tRNAs (tRNA modification) in tRNA stability. Wefocused on 4-thiouridine (s4U), amodified nucleoside found in bacterial andarchaeal tRNAs. Comprehensive tRNA quantification revealed that theabundanceofasubsetoftRNAspeciesdecreasedinas4Udeficient(ΔthiI)strainofVibriocholeraeinastationaryphasespecificfashion.Multiplemechanisms,including rapiddegradationofhypomodified tRNAs,account for the reducedlevel of tRNAs in the absence of thiI. We identified additional tRNAmodifications that promote tRNA stability and bacterial growth. Geneticanalysisof suppressormutantsaswellasbiochemicalanalyses revealed thatdegradation of hypomodified tRNAs is mediated by the RNA degradosome.Finally,wefoundthatlowcellularNTPlevelstriggerthedecayofhypomodifiedtRNAs.Together,theseobservationssupporttheexistenceofacellularenergyresponsive bacterial tRNA quality control mechanism in whichhypomodificationsensitizestRNAstoRNAdegradosomemediateddecay.

143

P2.22

NSUN2 introduces 5-methylcytidines in mammalianmitochondrialtRNAs�Sho Kitagawa1, Saori Nakano2, Shinichi Nakagawa3, Kimi Araki4,MasatakeAraki4,TakeoSuzuki1,andTsutomuSuzuki1�1DeptofChem.andBiotech.,GraduateSchoolofEngineering,TheUniversityofTokyo,� 2Dept of Biochem. andMol. Biol., Graduate school ofMedicine andFaculty of Medicine, The University of Tokyo,� 3Faculty of PharmaceuticalSciences, Hokkaido University,� 4Div. of Dev. Genet., Institute of ResourceDevelopmentandAnalysis,KumamotoUniversityHumanmitochondrial(mt-)tRNAscontain16speciesofmodifiednucleosidesat135positions.Thesemodificationsplayacriticalroleinprecisedecodingofgenetic informationandstabilityof tRNAstructure.However,biogenesisandfunctional rolesof thesemodificationsarenot fullyunderstood.Wemappedeigjht5-methylcytidine(m5C)atpositions48-50intheextraloopofsixmt-tRNAspecies. The cytidinemethyltransferase NSUN2 introducesm5C at the samepositions of cytoplasmic tRNAs. Lack of m5C reduces tRNA stability andaccumulates stress-induced tRNA fragments. Loss-of-function mutations inNSUN2 gene cause autosomal-recessive intellectual disability and Dubowitzsyndrome. Super-resolution microscopy using structured illuminationmicroscopy (SIM) revealed that NSUN2 partially localizes in mitochondria,thoughitispredominantlyinnucleus.Then,weconductedmassspecanalysisof mt-tRNAs isolated from Nsun2 null mouse liver as well as from NSUN2knockoutHEK293Tcells,andfoundcompletelossofm5Cinanyofmt-tRNAs,demonstratingthatNSUN2isalsoresponsibleform5Cformationinmt-tRNAs.Wearetryingtoidentifymitochondria-relatedphenotypeofNSUN2KOcells.

144

P2.23

Combined tRNA modification defects activate multipletranscriptionalstarvationresponsesA. Bruch1, T. Laguna2, N. Casas Vila2, F. Butter2, R. Schaffrath1, R.Klassen1�1UniversitätKassel,Germany;2InstituteofMolecularBiology,Mainz,GermanySimultaneous removal of critical tRNA anticodon loopmodifications inducessynthetic negative phenotypes in yeast. We characterized proteomic andtranscriptomic changes in mutant combinations previously shown to affecteithertRNALysUUUortRNAGlnUUG.WhiletranslationalinefficienciesofmRNAshighlyenrichedforeitherofthecognatecodoncouldbedemonstrated,mostsignificant changes occurred at the transcriptional level. We observed atranscriptionalupregulationofdifferentstarvationresponses,includinggeneralaminoacidcontrol,carbohydratecataboliteandnitrogencataboliterepressedpathwaysindependentofwhethertRNALysUUUortRNAGlnUUGwereaffected.In addition, all combined modification mutants inadequately inducedautophagyundernutrientrepleteconditions,suggestingthatthemodificationstatus of different tRNAs is crucial for sensing nutrient availability. SincetranscriptionalstarvationresponsesweresuppressedbyelevatedlevelsofthehypomodifiedtRNAs,thesearelikelyaconsequenceoftranslationalinefficiencyratherthanlossofthemodificationsitself.SupportedbyDFGPriorityProgrammeSPP1784(KL2937/1andSCHA750/20).

145

P2.24Structural insights into regulatory mechanisms of ElongatorcomplexRościsław Krutyhołowa1,2, Alexander Hammermeister3, Rene Zabel4,KarinBreunig4,RaffaelSchaffrath3andSebastianGlatt11MaxPlanckResearchGrouphostedbytheMalopolskaCentreofBiotechnologyof the Jagiellonian University, Krakow, Poland - 2Department of CellBiochemistry, Faculty of Biochemistry, Biophysics and Biotechnology,Jagiellonian University, Poland - 3Department of Microbiology, University ofKassel, Germany�4Institute of Biology, Dept. of Genetics, Martin- Luther-University,Halle-Wittenberg,GermanyThecm5modification,whichisprovidedbytheElongatorcomplexatthewobbleU34 position of certain tRNA species, is a major mechanism of translationalcontrol.1 The structure of the Elongator complex was recently solved usingelectron microscopy,2 however, regulatory mechanisms, which enable cm5

modification, remain elusive. Here we provide structural insights into thefunctionofKti12,a transientlyassociated regulatory factorofElongator.Wedemonstrate that Kti12 is an tRNA-induced ATPase and solved the crystalstructureofKti12’sATPasedomainboundtoa transitionstatemimicofATPhydrolysis.Additionally,wemappedtheinteractionsiteoftheKti12proteinontheElp1protein,a“dockinghub”oftheElongatorcomplex.Understandingthebiogenesisofcm5modificationmaypavethewayfornoveltreatmentstrategiesof Elongator associated pathophysiologies, like familial dysautonomia,intellectualdisability,androlandicepilepsy.3

fundedbyNCNUMO-2015/19/B/NZ1/00343.References:1Bauer,F.etal.TranslationalControlofCellDivisionbyElongator.CellRep.1,424–433(2012).�2 Dauden, M. I. et al. Architecture of the yeast Elongator complex. EMBO Rep.e201643353(2016).doi:10.15252/embr�3Kojic,M.&Wainwright,B.TheManyFacesofElongator inNeurodevelopmentandDisease.Front.Mol.Neurosci.9,115(2016).�

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Queuosine: The role of an essential tRNA modification inparasiticprotistTrypanosomabrucei.S.Kulkarni1,2,H.Stanzl1,2,A.C.Kessler3,E.Hegedűsová1,J.D.Alfonzo3

&Z.Paris1,21Institute of Parasitology, Biology Centre CAS, České Budějovice, CzechRepublic�2FacultyofScience,UniversityofSouthBohemia,ČeskéBudějovice,CzechRepublic�3DepartmentofMicrobiologyandTheCenterforRNABiology,TheOhioStateUniversity,Columbus,OH,USAQueuosine (Q), is a modified base, found at the wobble position of theanticodonofseveraltRNAs.Despiteitsomni-presenceinalldomainsoflife,itsfunction isnotclear. Inthisstudy,weusedprotozoanparasiteT.bruceiasamodelforcomprehensiveanalysisoftRNAguaninetransglycosylase(TGT),theenzymecatalyzingtheQ-modification.Unlikeitsbacterialcounterpart,inmosteukaryotesTGTpredominantlyfunctionsasaheterodimer.WeidentifiedtwoTGTsubunitsinT.brucei,TbTGT1andTbTGT2.Bothsubunitsarenecessaryforthecatalyticactivity.Interestingly,unlikehighereukaryotes,TbTGTheteromerislocalizedtothenucleus.InordertoexaminethephysiologicalroleofQ,wegeneratedaknock-outofTbTGT2andperformed in vivo characterization, tosimulate natural parasite infection in itsmammalian host. Upon infection ofmice, the mutant parasites showed significantly delayed parasitemia, andconse-quently,prolongedsurvivalofmice,ascomparedtoWTparasites.Ourdata suggests thatQplaysan important roleduring survivalof theparasitesinsidethemammalianhostandmaybeattheheartofvirulence.

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MitochondrialtRNAsepitranscriptomeinplants�S. Lalande, C. Villette, J. Zumsteg, T. Salinas, D. Heintz, P. Giegé, L.Maréchal-DrouardInstitutdebiologiemoléculairedesplantes,CNRS,Strasbourg,FranceTransferRNAsareuniversallyconservedmoleculesessentiallyknownfortheirimplication in translation, andmore andmore non- canonical functions arecurrently described. They are also themost post-transcriptionnallymodifiedRNAs, with an average of 17% of modified residues. However, while thesemodificationsareofgreatimportancetounderstandtRNAsstructure,stabilityand functions,data regarding them inplantsarestillveryscarce. Inorder toresolvethislackofknowledge,wewanttogetacompletemappingoftheplanttRNAs epitranscriptome. A focus is made on mitochondrial tRNAs, with themodelplantsArabidopsisthalianaandSolanumtuberosum.Asfirstexperiment,total S. tuberosum mitochondrial tRNAs have been digested intomononucleosides and analysed by HPLC-MS. In parallel, some specificmitochondrialtRNAsfromA.thalianahavebeenpurifiedwithacomplementaryoligonucleotideand1)digestedintomononucleosides inordertoknowwhatmodificationsarecarriedbythesespecifictRNAs.2)partiallydigestedinordertomapthesemodificationsonthetRNAssequences.ThesepreliminaryresultsareafirststepbeforegettingacompletemappingplantmitochondrialtRNAsmodifications.

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P2.27tRNAModificationMappingusingMassSpectrometry�ChristineM. Leufken1,2, Johannes Leufken1,2, SandraD. Kienast1,2andSebastianA.Leidel1,21Max Planck Research Group for RNA Biology, Max Planck Institute forMolecular Biomedicine, Von-Esmarch- Str. 54, Muenster, 48149, Germany;2Cells-in-Motion Cluster of Excellence, University of Muenster, Muenster,48149,GermanytRNAs are the key for mRNA translation by connecting the amino acids tospecific codon sequences. Importantly, they carry chemically modifiednucleosides. Hypomodification can disturb protein homeostasis, which is ahallmarkofseveralhumandiseases.However,theroleoftRNAmodificationsinsuch pathologies remains largely enigmatic and it will be crucial to quantifytRNAmodifications in the sequence context to change this. Currently, usingsequence-specificRNAsesfollowedbyLC-MS/MSistheonlyapplicablemethod.Twochallengesofthismethodexist:1)RNAsesaretoxictotheexpressionhost,2)duetotheshortlengthofthedigestionproductsofcommerciallyavailableRNAses, sequencecoverage is low.Hence, it iscritical tocombineRNAsesofdifferentspecificities.Weexpressthreebase-specificRNAsesinthecytoplasmof E. coli,which combinedwill improve the sequence coverage by 20%.WecurrentlydevelopaLC-MS/MSmethodandatailoredsoftwarefordataanalysis.ThiswillallowustoquantifychemicalRNAmodificationsandtolocalizethemwithintRNAsequences,enablingustoshedlightontheirbiologicalroleduringstressandintheetiologyofhumandiseases.

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tRNAmethyltransferaseTrmXpromotebreastcancermetastasisthroughtranscriptionalregulationRu-JuanLiu1*HaoLi1,andEn-DuoWang1*1Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy ofSciences;320YueyangRoad,Shanghai200031,P.R.China*Correspondence to: Ru-Juan Liu (liurj@sibcb.ac.cn) and En-duo Wang(edwang@sibcb.ac.cn).tRNA undergoes numerous chemical modifications which may affect itsstructureandfunction.AberranttRNAmodificationswerefoundinnumerouscancers,whilethemechanismremainslargelyunclear.Inthecurrentstudy,wefound out that the gene TrmX is extremely high-expressed in some tumortissues versus adjacent from breast cancer patients. The tRNAmethyltransferaseenzymaticactivitiesofTrmXwereconfirmedbyinvitroandin vivo analyses, and the RNA substrate and RNA recognitionmechanism ofTrmXwereidentified.ThecorrespondingtRNAmodificationisupregulatedastheexpressionofTrmXintumortissues.Resultsfromloss-orgain-offunctionassays in cell line and inmouse show that TrmXpromote cellmigrationandtumor metastasis. Interestingly, TrmX is located in both the nucleus andcytoplasm.Subsequently,wefoundoutthatTrmXregulatestheexpressionofoncogenes through directly binding to DNA and further modulate genetranscriptioninnucleus.Chip-seqwasperformedtoidentifythedown-streamtarget genes of TrmX, and the result is highly consistent with the globaltranscriptome analyses. Collectively, our results show that TrmX is a humantRNA modification enzyme that regulates breast cancer metastasis throughtranscriptionalregulation.

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A totally unexpected mechanism of the bacterial t6A tRNA-modificationSophia Missoury, Dominique Liger, Bruno Collinet and Herman vanTilbeurgh1InstituteforIntegrativeBiologyoftheCell(I2BC),CEA,CNRSUMR9198,Univ.Paris-Sud,UniversitéParis-Saclay,91198GifsurYvetteCedex,FranceThe universal N6-threonylcarbamoyladenosine (t6A) modification at positionA37ofANN-decodingtRNAsisessentialfortranslationalfidelity.InbacteriatheTsaC enzyme first synthesizes an L- threonylcarbamoyladenylate (TC-AMP)intermediate. In cooperationwith TsaB and TsaE, TsaD then transfers the L-threonylcarbamoyl-moietyfromTC-AMPontotRNA.WewillpresentthecrystalstructureoftheTsaB-TsaE-TsaD(TsaBDE)complexofThermotogamaritimainpresenceofanon-hydrolysableAMPCPP.TsaEispositionedattheentranceoftheactivesitepocketofTsaD,contactingboththeTsaBandTsaDsubunitsandprohibitingsimultaneoustRNAbinding.AMPCPPoccupiestheATPbindingsiteofTsaEandissandwichedbetweenTsaEandTsaD.Unexpectedly,thebindingofTsaEpartiallydenaturestheactivesiteofTsaDcausing lossof itsessentialmetalbindingsites.TsaEinterferesinapre-orpost-catalyticstepanditsbindingtoTsaBDisregulatedbyATPhydrolysis.Thisnovelbindingmodeandactivationmechanism of TsaE offers good opportunities for antimicrobial drugdevelopment.

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Acid/BaseResiduesin4-ThiouridineSynthetase�Kelly L. Sullivan, Brittany L. Sims, Kenneth Andrew Pavy, RussellWilliams,andEugeneG.Mueller*DepartmentofChemistry,UniversityofLouisville,Louisville,KYUSA4-Thiouridineatposition8intRNAservesasaphotosensorinbacteriabycross-linking to cytidine-13 upon exposure to UV light, which triggers controlledgrowth arrest. Conserved acid/base residues in 4-thiouridine synthetae (alsocalledThiI)weremutatedtotestfortheirpossibleparticipationintRNAbindingorcatalysis.TheeffectofsubstitutionatthosepositionsintheE.colienzymewasexaminedbyinvivoscreeningandinvitro4-thiouridinegenerationassays.The results will be presented and interpreted in light of a homologymodelbasedonthecrystalstructureoftheshorter4-thiouridinesynthetasefromT.maritima.

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Characterizationoffitness-enhancingBurkholderiacenocepaciatRNAIle2variantsMuraskiM.,CooperV.,AlexanderR.�WakeForestUniversity,Winston-Salem,NC,USABacterialtRNAIle2isinitiallytranscribedwithaCAUanticodonthatismodifiedtoLAUbytheenzymetRNAisoleucinelysidinesynthetase(TilS).InstallationoflysidineatthewobblepositionrestrictstRNAIle2pairingattheribosometotherare AUA isoleucine codon. Single nucleotide polymorphisms (SNPs) in theBurkholderia cenocepacia TilS and tRNA Ile2 genes were identified followingforward evolution experiments in a nutrient-limited environment. Selectedstrains exhibited fitness increases of up to 25%. We have shown that theenzyme variants display significant functional loss, and we are nowcharacterizingthetRNAIle2variantsU15CandU63GassubstratesforBcTilS.Theefficacy of tRNA Ile2 variants as substrates of TilS is quantified in vitro usingradiolabeledlysineandafilterbindingassay.Invivolysidinelevelsareidentifiedby LC/MS of cellular tRNA. The TilS:tRNA binding affinity is evaluated by anelectrophoreticmobilitygelshiftassay.

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Comparisonofnon-essentialBurkholderiacenocepaciaTilStoitsvitalEscherichiacolihomologEmilNilsson1,VaughnCooper2,RebeccaAlexander1�1WakeForestUniversity,Winston-Salem,UnitedStatesofAmerica�2UniversityofPittsburghMedicalSchool,Pittsburgh,UnitedStatesofAmericatRNA isoleucine lysidine synthetase (TilS) is proposed to be an essentialprokaryotic gene product, as demonstrated using temperature sensitive TilS(TilSTS)variantsexpressedinEscherichiacoli.DuringgrowthatTilSTSpermissivetemperatures,cellsreplicateinanormalfashioncomparedtocellsharboringthe WT TilS; while at elevated temperatures that inactivate TilTS, AUAdependent translational deficiencies lead to cell death. We have previouslyshown that mutations causing severe functional loss in Burkholderia

cenocepaciaTilS(BcTilS)areassociatedwithagain-of-fitnessphenotypeunderamino acid and trace metal starvation conditions. Here we identify keydifferencesinsubstraterecognitionbetweenE.coliTilS(EcTilS)andBcTilSandpresent a comparative kinetic analysis of these enzymes. EcTilS enzymaticefficiency is substantially higher than that of its B. cenocepacia homolog.FurthermoreweshowthatBcTilSusesauniquesetrecognitionelementswithinits tRNAIle2 substratecomparedto thosepreviously reported fromEcTilS.Wealso demonstrate that the loss-of-function substitutions identified in B.cenocepacia have vastly different effects on enzymatic function whentransplantedintotheE.colienzyme.

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P2.33Functionalanalysisofpre-tRNAcappinginbuddingyeast�T.Ohira,I.Kikuchi,T.Nakatsuka,T.Suzuki�DepartmentofChemistryandBiotechnology,GraduateSchoolofEngineering,TheUniversityofTokyo,Tokyo,JapanIneukaryote,5’methylguanosinecapstructureisinstalledco-transcriptionallyon RNA pol II transcripts, and play pivotal roles in RNA stability, export andtranslation.Previously,wehappenedtofindthattRNAprecursors(pre-tRNAs)bearing 5’ leader sequences had methylguanosine cap structures inSaccharomycescerevisiae,nerverthelesstRNAgenesaretranscribedbyRNApolIII.Wenamedthisphenomenon"pre-tRNAcapping"(OhiraandSuzuki,2016).Geneticstudyrevealedthat5’cappingstabilizespre-tRNAsbyprotectingfrom5’exonucleolyticdegradation.However,thebiologicalsignificanceofpre-tRNAcapping remains to be investigated. To identify pre-tRNA species with 5’capping, pre-tRNAs accumulated in RNase P-repressed cells wereimmunoprecipitated with anti-methylguanosine antibody and subjected todeep sequencing, elucidating a subset of pre-tRNAs 5’ capped efficiently. Inaddition,wefoundthateIF-4Einteractswith5’cappedpre-tRNAsinthecell,indicatingthat5’cappedpre-tRNAshaveafunctionalroleinsequesteringeIF-4Etomodulatetranslationinitiation.

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P2.34StructuralandfunctionalstudyoftwotRNAthiolation[4Fe-4S]clusterdependentenzymesOrnellaBimai1,SimonArragain1,PierreLegrand2,Jean-LucRavanat3,LaurentBinet4,5,MarcFontecave1*,BéatriceGolinelli-Pimpaneau1*1LaboratoiredeChimiedesProcessusBiologiques,UMR8229CNRS,CollègedeFrance,UniversitéPierre etMarie Curie, 11 PlaceMarcelin Berthelot, 75231 Paris cedex 05, France�2SynchrotronSOLEIL, L'Orme des Merisiers, Saint Aubin BP48, Gif-sur-Yvette 91198, France�3University ofGrenobleAlpes,CEA,iNAC,SyMMES,F-38054Grenoble,France4IRCNRSRenard,Chimie-ParisTech,11 rue Pierre et Marie Curie, 75005 Paris, France�5PSL Research University, Chimie-ParisTech,InstitutdeRecherchedeChimie-Paris,11 ruePierreetMarieCurie,75005Paris,France�e-mail:beatrice.golinellli@college-de-france.frPost-transcriptionalmodifications of transfer RNA (tRNA) are essential for translational fidelity.Sulfur ispresent inseveralnucleosideswithintRNAs.Thethiolationoftheuniversallyconservedmethyl-uridine at position 54, catalyzed by the enzyme called TtuA, stabilizes tRNAs fromthermophilic bacteria and hyperthermophilic archaea and is required for growth at hightemperature. Moreover, the thiolation of uridine 34 in the anticodon loop of tRNAs, which isrequiredfornormalgrowthandstressresistanceinyeast,iscarriedoutbytwocompletelydifferentsystems:thewell-studiedMnmAprotein(presentinbacteriaandintheeukaryoticmitochondrion)andtheNsc6/NcsA/Ctu1proteinsinallotherorganisms,includingtheeukaryoticcytoplasm.Spectroscopic and enzymatic studies demonstrate that TtuA andNcsA catalyze the simple non-redoxsubstitutionoftheC2-uridinecarbonyloxygenbysulfurusinga[4Fe-4S]clusterandthusarerepresentativesofanewenzymessuperfamily.AseriesofcrystalstructuresofTtuAandNcsAshowthat:(i)the[4Fe-4S]clusterisligatedbythreecysteinesonlythatarefullyconserved,allowingthefourthuniqueirontobindanexogenoussulfide,whichlikelyactsasthesulfuratingagent;(ii)theATP-bindingsiteisadjacenttothecluster[1].AnewmechanismfortRNAsulfurationisproposed,in which the unique iron of the catalytic cluster functions as a sulfur carrier, opening newperspectivesregardingfunctionsofiron-sulfurproteinsinbiology.Interestingly,TtuAandNcsAshowthehigheststructuralsimilaritywithlysidinesynthetase(TilS),anN- type ATP pyrophosphatase that targets position 34 in tRNA, whose structure has beendetermined in complex with either ATP or the tRNA substrate. Mdels of the TtuA/tRNA andNcsA/tRNA complexes suggest that a flipped adenylated uridine intermediate is formed duringcatalysisbythetRNAthiolasesandthattwotRNAmoleculesarelikelyboundoneachsideofoneTtuA/NcsAdimer,withthetwozincfingerdomainsbelongingtodifferentpolypeptidechainsbeingusedtoclamptheloopcontainingthetargeturidineonoppositesides.[1].Nonredox thiolation in tRNAoccurringvia sulfuractivationbya [4Fe-4S]cluster.ArragainS,BimaiO,LegrandP,CaillatS,RavanatJL,TouatiN,BinetL,AttaM,FontecaveM,Golinelli-PimpaneauB.ProcNatlAcadSciUSA.2017,114(28):7355-7360

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Nuclear-cytoplasmic tRNA dynamics in required for tRNAmaturationinTrypanosomabruceiE. Hegedűsová1, A. C. Kessler3, S. Kulkarni1,2, B. Burgman1,4, J. D.Alfonzo3andZ.Paris1,21Institute of Parasitology, Biology Centre CAS, České Budějovice, CzechRepublic�2FacultyofScience,UniversityofSouthBohemia,ČeskéBudějovice,CzechRepublic�3DepartmentofMicrobiologyandTheCenterforRNABiology,The Ohio State University, Columbus, OH, USA 4The University of Arizona,Tucson,Arizona,USAAlingeringquestioninretrogradetRNAtransportiswhetheritisrelegatedtoS.cerevisiae and multicellular eukaryotes or alternatively, is a pathway withdeeper evolutionary roots. In the early branching eukaryote T. brucei, tRNAsplicing, like in yeast, occurs in the cytoplasm. In this report, we used acombination of cell fractionation and molecular approaches that show thepresenceofsignificantamountsofsplicedtRNATyrinthenucleusofT.brucei.Notably, the modification enzyme tRNA-guanine transglycosylase (TGT)localizestothenucleusand,asshownhere,isnotabletoaddqueuosine(Q)toanintron-containingtRNA.Wesuggestthatretrogradetransportispartlytheresult of the differential intracellular localization of the splicing machinery(cytoplasmic) and a modification enzyme, TGT (nuclear). In addition, RNAisilencing of a novel tRNA exporter led to accumulation of mature splicedtRNATyrsupportingtheexistenceofthetRNAretrogradepathway.Moreover,thelevelofQmodifiedtRNAisinfluencedbytheefficiencyofthetRNAnuclearexport. These findings expand the evolutionary distribution of retrogradetransport to include early diverging eukaryotes, while highlighting itsimportanceforQbiosynthesis.

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P2.36THG1LcatalysestheadditionoftheaminoacylationdeterminantG-1onhumanmitochondrialtRNAHis�CA.Powell,M.Minczuk�MRCMitochondrialBiologyUnit,UniversityofCambridge,UKAlmost all tRNAHis molecules analysed to date deviate from the canonicalstructureoftRNAsbythepresenceofanadditional5’guanineresidue(G-1).Thesource of this additional nucleotide has been well characterised inSaccharomycescerevisiae,whereithasbeenshowntobepost-transcriptionallyadded by Thg1 in the cytosol, and genomically encoded and retained aftercleavage in mitochondria. Here, we demonstrate that human mitochondriadiffer from yeast through the utilisation of the ‘cytosolic pathway’ of G-1inclusion, namely, post-transcriptional addition by Thg1-like 1 (THG1L). Wefurther show that the presence of G-1 is critically required for theaminoacylation of mitochondrial tRNAHis and therefore mitochondrialtranslationasawhole.

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P2.37CCA-addition in the cold: characterizationof the enzyme fromPlanococcushalocryophilus�Raphaël de Wijn1, Felix Ernst2, Oliver Hennig2, Heike Betat2, MarioMörl2&ClaudeSauter11Architecture et Réactivité de l’ARN, Université de Strasbourg, CNRS, IBMC,67084 Strasbourg, France 2Institute for Biochemistry, Leipzig University,Brüderstr.34,04103Leipzig,GermanyEnzymes from psychrophilic organisms usually achieve catalysis at lowtemperaturesbyareductionofsecondarystructureelementstoincreasetheirflexibility.ForCCA-addingenzymes,thereisadiscrepancybetweentheneedofatightlycontrolledflexibilityduringpolymerizationandanincreasedflexibilityasastrategyforcoldadaptation.Usingbiochemicalandstructuralanalyses,weinvestigated the properties of the CCA-adding enzyme (48 kDa) fromPlanococcus halocryophilus, a bacterium from the Arctic permafrost whichgrows at -15 °C. This enzyme is strongly cold-adapted and catalyzes CCA-additiondownto0°Cinvitro.Crystal structures of the apo enzyme and of the complex with CTP weredeterminedathighresolution.Thenucleotidebindingpocketislocatedinthegrooveoftheneckdomain,nexttothebodydomaindisplaying10%lessalpha-helicesthanthatofGeobacillusstearothermophilusenzyme.Detailed mutational analysis revealed that residues in a flexible elementbetweenheadandneckdomain–responsibletomodulatedomainmovementsduring CCA addition – contribute to cold- adaptation. Furthermore, deepsequencingofthetRNApoolofP.halocryophilus indicatesthattheincreasedflexibilityoftheenzymehasanimpactonthepolymerizationfidelity.Thus,CCA-additioninthecoldisachievedattheexpenseofanincreasederrorrateduringCCAsynthesis.

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Regulation of Elongator’s U34 modification function by tRNAbindingproteinKti12Alexander Hammermeister1, Rościsław Krutyhołowa2, Rene Zabel3,Mark Helm4, Karin D. Breunig3, Mike Stark5, Sebastian Glatt2, andRaffaelSchaffrath11InstitutfürBiologie,UniversitätKassel,Germany�2MaxPlanckResearchGroup,Malopolska Centre of Biotechnology, Krakow, Poland 3Institut für Biologie,UniversitätHalle,Germany�4InstitutfürPharmazieundBiochemie,UniversitätMainz,Germany�5SchoolofLifeSciences,UniversityofDundee,UKZymocin is an anticodon nuclease that attacks tRNA species which carryElongator dependent wobble uridine (U34) modifications. Hence, Elongatormutantshelp yeast cells to survive zymocin.Here,we show that deletionofKTI12,agenecodingforanElongatorinteractor,causeszymocinresistance,too.Inaddition,Kti12contributestotRNAnonsenseandmissensesuppression.BothdependonanNTPbindingP-loopdomaininKti12thatisconservedwithatRNAkinase(PSTK),andwhenmutated,abolishesbindingtotheElongatorcomplex.This reinforces our view that Kti12 association to Elongator promotes U34modification.InsupportofthisnotionaredatashowingthatElongatorbindingP-loopmutantshaveU34modificationdefects.Intriguingly,likePSTK,Kti12isable to bind tRNAs and NTPs. Together with NTP dependent effects on thecapacity of Kti12 to bind Elongator, our data imply a potential cofactorrequirement for the regulation of Elongator’s U34 modification function byKti12.SupporbyBBSRC(UK)toMS(BB/F0191629/1)andRS(BB/F019106/1)andDFG(Germany)toMH(HE3397/13)andRS(SCHA750/18;SCHA750/20)

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P2.39HierarchicalmodificationoftRNAbyTrmA,TruB,andTrmB�SarahSchultzandUteKothe�AlbertaRNAResearchandTrainingInstitute(ARRTI),DepartmentofChemistryandBiochemistry,UniversityofLethbridge,Lethbridge,AB,CanadaAbout 10% of nucleosides within tRNA are post-transcriptionally modified.TrmAandTruBaretheonlytwoenzymesthatmodifyeverytRNAintroducing5-methyluridine (m5U) 54 and pseudouridine (Ψ) 55, respectively. TrmBgenerates7-methylguanosine(m7G)46inmanytRNAs.DespitetheabundanceoftRNAmodifications,itremainsunclearhowthetRNAmodificationsbymanyenzymesareconcerted.TrmA,TruBandTrmBcaneachactonunmodifiedtRNA;however,invivotheycannotinteractsimultaneouslywithnascent,unmodifiedtRNAdue to steric hindrance. Thus,we hypothesize that these enzymes arelikely to modify tRNA in a preferential, hierarchical fashion rather than astochastic order. To test this, we used in vitro transcribed, single-modifiedtRNAsinbindingandmodificationassays.Thereby,weshowthatTruBpreferstobindandactuponunmodifiedtRNA,whereasTrmAbindstRNAcontainingΨ55moststrongly.BothTrmAandTruBprefertobindandmodifytRNAlackingm7G46. Our results suggest that TrmA and TruB act early during tRNAmaturation,whereasTrmBislikelytoactlater.ThisresearchlaysthefoundationtouncoverhowtRNAsaresohighlymodifiedinthecell.

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Characterization of decoding of the CGN codon family in C.merolaeorganellesA.Soma1andH.Sano1�1GraduateSchoolofHorticulture,ChibaUniversity,Matsudo,JapanAsorganellegenomesofvariousorganismsdonotcodeforthecompletesetoftRNAgenes,theirtranslationoftenrequiresimportofnuclear-encodedtRNAsfromcytoplasm.Ontheotherhand,tRNAimportdoesnotnecessarilyexplainall casesof incomplete tRNAsets,andno importof cytosolic tRNAhasbeensuggested in chloroplasts or in humanmitochondria. As for the CGN codonfamily of arginine, most eubacteria and organelles contain two species ofanticodons;tRNAArg(ICG)ofwhichthewobblepositionhasadeaminatedformof adenosine (I, inosine) recognizes CGU, CGC, and CGA codons, whereastRNAArg (CCG)recognizestheCGGcodon.Organellegenomesofsomeplants,includingP.patens,encodeforbothtRNAArg(A/ICG)andtRNAArg(CCG),whilemostofothers, includingA.thalianaandC.merolae,encodeonlyfortRNAArg(A/ICG),andthedecodingmechanimofthelatterisnotclarified.ToseehowtheplantorganelleswithtRNAArg(A/ICG)decodetheCGNcodons,weanalysedtheinosinemodificationatthewobblepositionoftRNAArg(A/ICG)andthetRNAimportfromcytoplasminC.merolae,aunicellularredalga.

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Metabolic and chemical regulation of tRNA modificationassociatedwithtaurinedeficiencyandhumandiseaseTakeo Suzuki1, Kana Asano1, Ayaka Saito1, Fan-Yan Wei2, KazuhitoTomizawa2,YurikoSakaguchi1,TsutomuSuzuki11DepartmentofChemistryandBiotechnology,GraduateSchoolofEngineering,TheUniversityofTokyo,2DepartmentofMolecularPhysiology,FacultyofLifeSciences,KumamotoUniversityModifieduridinecontainingtaurine,5-taurinomethyluridine(τm5U),isfoundatthewobblepositionofmitochondrial(mt-)tRNAs.Wepreviouslyreportedthatτm5U isabsent inmt-tRNAswithpathogenicpointmutationsassociatedwithmitochondrial diseases. However, biogenesis and physiological role of τm5Uremainelusive.Here, we successfully reconstituted τm5U on mt-tRNA in vitro catalyzed byMTO1 and GTPBP3 compelx in the presence of 5,10-methylene-tetrahydrofolate and taurine as substrates. GTPBP3-knockout cells exhibitedrespiratoryimpairmentandreducedmitochondrialtranslation.Verylittleτm5Uwasdetectedinpatient'scellswiththeGTPBP3mutation,showingthatthelackofτm5Uresultsinpathologicalconsequences.Taurinedepletiondownreguledτm5U frequency in cultured cells and animal tissues (cat liver and flatfishmuscle), suggesting that mitochondrial protein synthesis is dynamicallyregulatedbysensingintracellularmetabolicstatus.Moreover,wehappenedtodetect 5- carboxymethylaminomethyluridine (cmnm5U), in which taurinemoietyofτm5U is replacedwithglycine, inmt-tRNAsunder taurine-depletedconditions.ThisisthefirstinstanceofchemicalswitchingofRNAmodificationunderphysiologicalconditionsinhumanmitochondria.

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Pseudouridines in tRNA play a role in maintaining genomicmutationfrequencyinPseuduomonasspecies�MariTagel,HeiliIlves,MargusLeppik,MaiaKivisaar,JaanusRemmeUniversityofTartu,InstituteofMolecularandCellBiology,EstoniatRNAs are the most abundantly modified nucleic acids in the cell. tRNAmolecules have numerous interaction partners that are crucial for theirfunctionalityandthismaybethereasonforthecomplexsetofmodifications.Still, the function of many modifications is not clear. Modifications in theanticodonloopcanfacilitateanticodon-codonrecognitionandhelptomaintaintranslation fidelity.We are studying the role of pseudouridines (Ψ) at tRNApositions 32 or 38-40 in Escherichia coli MG1655, Pseudomonas aeruginosaPAO1andP.putidaPaW85.InourpreviousstudiesweidentifiedthetRNAmodificationenzymeTruAasafactorinmutationfrequency,asitsdisruptionsignificantlyincreasesmutationrateinP.putida.TruAcatalyzestheisomerizationofuridinetoΨinpositions38-40.Ontheothersideoftheanticodon,position32ispseudouridylatedbytheenzymeRluA.Hereweshowthatthelackofeitherofthesemodificationenzymescausesa3-5-foldriseinmutationfrequencyinP.putida,whereasinP.aeruginosa the effect on mutation frequency is less relevant. This effect isspecifically caused by the lack of pseudouridylation activity, asmutating thecatalyticasparticacidofeitherenzymecausedthesamephenotypeinP.putidaas deletion of truA. Contrary to the effect onmutations the truA and rluA-deficient strains display different non- mutation related phenotypes in P.putida, P. aeruginosa and E. coli (e. g. growth rate, stress tolerance). TheseintriguingresultsindicatethattRNAmodificationscanaffectDNAmutationsviayetunknownmechanism.

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TheroleoftRNAmodificationonproteomehomeostasis�J.Tavares1,S.Kellner2,G.Moura1,P.C.Dedon2,3andM.A.S.Santos11DepartmentofMedicalSciences,InstituteforBiomedicine-iBiMED,Universityof Aveiro, Portugal�2Department of Biological Engineering, MassachusettsInstitute of Technology – MIT, Cambridge, US 3Singapore-MIT Alliance forResearch and Technology, Campus for Research Excellence and TechnicalEnterprise–CREATE,SingaporeProtein synthesis is central to life and is being intensively studied at variouslevels. The exception is mRNA translation fidelity whose study has beenhamperedbytechnicaldifficultiesindetectingaminoacidmisincorporationsinproteins.WeareinvestigatingtheroleofRNAmodificationbyRNAmodifyingenzymes(RNAmods)inproteinsynthesisefficiencyandaccuracy.Thus,wehavesetupayeastgeneticscreentoidentifytRNAmodsthatplayaroleonproteinsolubility and proteome homeostasis and identified a subgroup of yeasttRNAmodsthatplayroles inproteinsynthesisfidelityandfolding,preventingprotein aggregation. The data indicate that some tRNAmods influence tRNAstability and decoding efficiency while others have direct impact on mRNAdecodingfidelity.WeidentifiedtheaggregatedproteinsintRNAmodsKOsandfoundthatgenesencodinginsolubleproteinswereenrichedincodonsdecodedby thehypomodified tRNAs.Aminoacidmisincorporationsdetectedat thesecodonsitesshowedthattRNAU34modificationsplayanimportantroleinthecontroltranslationalaccuracy.ThisworkwassupportedbyFCTprojectsUID/BIM/04501/2013andPTDC/BEX-BCM/2121/2014.

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ThecrystalstructureoftheenterohemorrhagicE.coliAtaT-AtaRtoxin-antitoxincomplexYukaYashiroandKozoTomita*�DepartmentofComputationalBiologyandMedicalSciences,GraduateSchoolofFrontierSciences,TheUniversityofTokyo,Kashiwa,Chiba277-8562,Japan.E-mail:kozo_tomita@cbms.k.u-tokyo.ac.jpBacterialtoxin-antitoxin(TA)systemscontrolkeycellularprocessestofacilitatecellsurvivalduringstressconditionsbycausingcelldormancyandpersistence.InthecaseoftypeIITAsystems,toxinsareboundandinactivatedbyantitoxinsunder normal growth conditions, whereas stress conditions induce specificdegradationofantitoxinsandleadtoxinactivation.AtaT(toxin)-AtaR(antitoxin)isatypeIITAsystemfoundinenterohemorrhagicE.coliO157:H7.RecentstudyshowedthatAtaTisaGNATfamilyenzymeandadimericformofAtaTtransfersacetyl group from acetyl-CoA to the amine group of methionyl aminoacylmoiety of initiator tRNAfMet, thereby AtaT inhibits the initiation of proteinsynthesis.HerewepresentthecrystalstructureofE.coliO157:H7AtaT-AtaRcomplex in the presence or absence of acetyl-CoA. AtaT-AtaR is aheterohexamericcomplex(AtaT2-AtaR4),wheretwodimericAtaRsbridgetwomonomericAtaTsandpreventAtaTsfromforminganactivehomodimer.SinceAtaTaloneformsanactivehomodimer,itislikelythatAtaTdimerisdissociated,when it is complexedwith dimeric AtaRs or that AtaT is prevented from itsdimerizationinthepresenceofAtaR.

166

P2.45

Directnutritionalcontrolofproteintranslationbyqueuosine�FrancescaTuorto,CarineLegrand,CansuCirzi&FrankLyko�DivisionofEpigenetics,DKFZ-ZMBHAlliance,GermanCancerResearchCenter,Heidelberg,GermanyGlobal protein translation as well as translation at the codon level can beregulatedbytRNAmodifications.Ineukaryotes,levelsoftRNAqueuosinylationreflectthebioavailabilityoftheprecursorqueuine,whichissalvagedfromthedietandgutmicrobiota.WehaveshownthatnutritionallydeterminedQ-tRNAlevels promote Dnmt2 mediated methylation of tRNA- Asp and controltranslational speedofQ-decoded codons aswell as at near-cognate codons.Deregulationoftranslationuponqueuinedepletionresultinunfoldedproteinsthattriggerendoplasmicreticulumstressandactivationoftheunfoldedproteinresponse,both inculturedhumancell linesand ingerm-freemicefedwithaqueuosine-deficientdiet.Takentogether,ourfindingscomprehensivelyresolvethe roleof thisanticodon tRNAmodification in thecontextofnativeproteintranslationanddescribeanovelmechanismthatlinksnutritionallydeterminedmodificationlevelstoeffectivepolypeptidesynthesisandcellularhomeostasis.

167

P2.46

A totally unexpected mechanism of the bacterial t6A tRNA-modificationSophiaMissoury,DominiqueLiger,BrunoCollinetandHermanvanTilbeurghInstituteforIntegrativeBiologyoftheCell(I2BC),CEA,CNRSUMR9198,Univ.Paris-Sud,UniversitéParis-Saclay,91198GifsurYvetteCedex,FranceThe universal N6-threonylcarbamoyladenosine (t6A) modification at positionA37ofANN-decodingtRNAsisessentialfortranslationalfidelity.InbacteriatheTsaC enzyme first synthesizes an L- threonylcarbamoyladenylate (TC-AMP)intermediate. In cooperationwith TsaB and TsaE, TsaD then transfers the L-threonylcarbamoyl-moietyfromTC-AMPontotRNA.WewillpresentthecrystalstructureoftheTsaB-TsaE-TsaD(TsaBDE)complexofThermotogamaritimainpresenceofanon-hydrolysableAMPCPP.TsaEispositionedattheentranceoftheactivesitepocketofTsaD,contactingboththeTsaBandTsaDsubunitsandprohibitingsimultaneoustRNAbinding.AMPCPPoccupiestheATPbindingsiteofTsaEandissandwichedbetweenTsaEandTsaD.Unexpectedly,thebindingofTsaEpartiallydenaturestheactivesiteofTsaDcausing lossof itsessentialmetalbindingsites.TsaEinterferesinapre-orpost-catalyticstepanditsbindingtoTsaBDisregulatedbyATPhydrolysis.Thisnovelbindingmodeandactivationmechanism of TsaE offers good opportunities for antimicrobial drugdevelopment.

168

P2.47

Characterization of the human TRM10 family of tRNAmethyltransferasesElisaVilardo1,FabianAmman2,UrsulaToth1,WalterRossmanith11Medical University Vienna�2Department of Theoretical Chemistry of theUniversityofViennaTo date, more than 100 RNA modifications have been described, mostly intRNAs. However, the synthesis pathways of many modifications, and theirimpact on the RNAmolecule remain poorly understood. TRM10 is a widelyconserved family of methyltransferases found in Archaea and Eukaryotes,responsibleforthemethylationofnitrogen1ofpurine-9oftRNAs.Thehumangenome encodes three forms of TRM10 methyltransferase; however, thespecific role of the different forms remains unclear. The methyl groupintroduced interferes with canonical base pairing, and causes errors duringreversetranscription.Themodificationcanthusbeidentifiedasahyper-errorsignatureinNGSdata.HereweusedNGStoidentifythefullrepertoireoftRNAsmethylated at position-9 in a human cell line. Using CRISPR/Cas we havegeneratedcelllinesdeletedforthemembersoftheTRM10familytoinvestigatetheir role in tRNAmodification.Wefoundthat thedifferentmembersof thefamily have evolved distinct substrate specificity. We aim to elucidate thefunction of the expanded TRM10 family in vertebrates, their role in tRNAmodification,andtheimportanceofR9methylationfortranslation.

169

P2.48ThecrystalstructureofNanoarchaeumequitanstRNA(uracil-54,C5)-methyltransferase–RNA–cofactorternarycomplexprovidesinsightsintosubstrateselectivityandenzymaticfunctionAuxilienS1. , RoseS2. , LegrandP3. ,GrosjeanH.1 ,Douthwaite S.2 ,FourmyD.1andWalbottH.11InstituteforIntegrativeBiologyoftheCell(I2BC),CEA,CNRS,Univ.Paris-Sud,Université Paris-Saclay, 91198 Gif-sur- Yvette cedex, France�2Department ofBiochemistry&MolecularBiology,UniversityofSouthernDenmark,Campusvej55,DK-5230OdenseM,Denmark3SynchrotronSOLEIL,L'OrmedesMerisiers,Saint-AubinBP48,91192Gif-sur-Yvettecedex,FranceThe5-methyluridine(m5U,alsotermedrTforribothymidine)isinvariablyfoundat position 54 in almost all tRNAs from Bacteria and Eukarya but rarely inArchaea. Ribothymidine is additionally present in rRNAs of all Bacteria andcertainArchaea.Wehaverecentlyshownthatnom5UisfoundinrRNAsfromthe archaeal N. equitans. However, its genome displays one gene possiblycoding for a S-adenosyl-L-methionine-dependent RNA (uracil, C5)-methyltransferase(MTase).Here,wedemonstratethatthisenzyme(renamedNeqTrmU54) catalyzes in vitro the formation of m5U54 in tRNA. The crystalstructureofNeqTrmU54incomplexwithitsRNAsubstrateandcofactorat2.3Åresolution reveals strong similarity to its closest homolog PabTrmU54 fromP.abyssi. The protein is organized into three domains like E. coli RlmD, whichcatalyzes the same reaction at position 1939 of 23S rRNA. Interestingly, thepresenceoftwoadditionalflexibleloopsinthecentraldomainofNeqTrmU54,involvedinRNAbinding,mayexplainthedifferenceinsubstrateselectivityofbothenzymes.ThestructureoftheNeqTrmU54–RNA–cofactorternarycomplexalsoconfirmstheproposedcatalyticmechanismofRNAm5UMTases.

170

P2.49

DeletionofTrmuinZebrafishrevealedtheessentialroleoftRNAmodificationinmitochondrialbiogenesisandhearingfunctionQinghaiZhang1,2,#,LuwenZhang1,2,#,YeChen1,2,*

,andMin-XinGuan1,2,*

1DivsionofMedicalGeneticsandGenomics,TheChildren'sHospital,ZhejiangUniversitySchoolofMedicine,Hangzhou,Zhejiang310058,China�2InstituteofGenetics,ZhejiangUniversityandDepartmentofGenetics,ZhejiangUniversitySchoolofMedicine,Hangzhou,Zhejiang310058,ChinaTrmu is a highly conserved tRNA modifying enzyme responsible for thebiosynthesisofτm5s2UatthewobblepositionoftRNAGln,tRNAGluandtRNALys.Our previous investigations showed that TRMU mutation modulated thephenotypic manifestation of deafness-associated mitochondrial 12S rRNAmutation.However, thepathophysiologyof TRMUdeficiency remainspoorlyunderstood. Using the trmu knock-out zebrafish generated by CRISPR/Cas9system,wedemonstratedtheabolished2-thiouridinemodificationofU34ofmitochondrialtRNALys,tRNAGluandtRNAGlninthetrmuknock-outzebrafish.Theelimination of this post-transcriptional modification mediated mitochondrialtRNAmetabolisms,causingtheglobaldecreasesinthelevelsofmitochondrialtRNAs.TheaberrantmitochondrialtRNAmetabolismsledtotheimpairmentofmitochondrial translation, respiratory deficiencies and reductions ofmitochondrial ATP production. These mitochondria dysfunctions caused thedefects in hearing organs. Strikingly, trmu-/- mutant zebrafish displayed theabnormalstartleresponseandswimmingbehaviors,significantdecreasesinthesizesofsaccularotolithandnumbersofhaircellsintheauditoryandvestibularorgans. Furthermore, trmu-/- mutant zebrafish exhibited the significantreductionsinthehairbundledensitiesinutricle,sacculeandlagena.Therefore,our findingsmayprovidenew insights into thepathophysiologyofdeafness,which was manifested by the deficient modifications at wobble position ofmitochondrialtRNAs.

171

P2.50

Gtpbp3deficiencycausedmitochondrialtRNAmobilityshiftandleadstohypertrophiccardiomyopathyinzebrafishDanniChen, ZengmingZhang, ShihaoYao,QingxianYang, Feng Li,XiaoHe,ChengAi,MengWang,Min-XinGuanInstituteofGenetics,ZhejiangUniversityandDepartmentofGenetics,ZhejiangUniversitySchoolofMedicine,Hangzhou,Zhejiang,ChinaWobble uridinemodificaitons of somemt-tRNAs are carried out by a set ofnuclear-encoded mitochondrial genes GTPBP3, MTO1 and TRMU. It wasreported mutations in GTPBP3 are associated with hypertrophiccardiomyopathy(HCM).However,thefunctionofGTPBP3inmyocardiumandcardiacdevelopment isnot fullyunderstood.Here,wegeneratedazebrafishgtpbp3mutantlineusingtheCRISPR/Cas9system.Comparedtocontrol,gtpbp3mutant exhibited delayed embryonic heart development, impaired heartfunctionanddevelopedHCM in adult. For the first time,weusednative gelelectrophoresis to show mobility shift in related mt-tRNAs, indicating tRNAstructurechangeduetolackofmodificationandprovidingmoredirectevidenceforGtpbp3mutationtocauseatranslationdefect.Interestingly,wealsofoundahugeincreaseintheaminoacylationlevelofmttRNAs,andwededucedthisasaresultofthedelayedtranslationprocess.ThisimbalancebetweenfreemttRNAsandaminoacylatedmttRNAsmightacttogetherwithtranslationdefecttobreakmitochondrialhomeostasisandleadtodysfunctionalmitochondria.

173

P3.1

Shortening of tRNAs as amechanismof translational silencingduringstressMarina Cristodero, Roger Fricker, Rebecca Brogli, Shikha Schikha,AndréSchneiderandNorbertPolacekDepartmentofChemistryandBiochemistry,UniversityofBern,SwitzerlandRegulation of gene expression is essential for cell survival, especially underadversegrowthconditions.�ThepathogenicprotozoanparasiteTrypanosomabruceilargelylackstheabilitytoregulatetranscriptionofproteincodinggenesandallprotein-codinggenesare transcribedpolycistronically.Therefore theyrely on posttranscriptional means to regulate gene expression. We areparticularlyinterestedinunderstandinghowtranslationisregulated,focusingontheroleoftRNAinthisprocess.�Oneofourdiscoverieswastheshorteningof the bulk of the tRNAs during nutritional stress at the expense of theconserved 3’-CCA tail, which renders tRNAs unsuitable substrates fortranslation.TrimmedtRNAscanberepairedbytheCCAaddingenzymeandarethereforerecycledfortranslationoncenormalgrowthconditionsarerestored.Despite the strong decrease in polysomes triggered by starvation the tRNAslackingtheCCA-tailareenrichedinthepolysomefraction,suggestingafunctionforthesetRNAspeciesontheribosomesintranslationcontrol.Weareatthemoment working on the identification of the nuclease involved in CCA-tailshortening.

174

P3.2

Systematic detection of amino acid substitutions in proteomerevealsamechanisticbasisofribosomeerrors.Ernest Mordret1, Orna Dahan1, Avia Yehonadav1, Georgina DBarnabas2,JürgenCox3,TamarGeiger2,ArielBLindner4,YitzhakPilpel11DepartmentofMolecularGenetics,WeizmannInstituteofScience,Israel�2Department of Human Molecular Genetics and Biochemistry, Tel AvivUniversity,Israel3ComputationalSystemsBiochemistry,MaxPlanck Institute forBiochemistry,Martinsried,Germany�4French InstituteofHealthandMedicalResearch,ParisDescartesUniversity,FranceErrorsintranslationcanresulteitherfromthechargingofatRNAwithanon-cognate amino acid or from the ribosome failing to discriminate againstimperfect codon-anticodon complexes. Currently, our understanding of thecausesofaminoacidmis-incorporationsishinderedbythelackofmethodstoobserve errors at the full proteome. In this project, we have developed apipelineaimingtosystematicallydetecterrorsinentireproteomesusingmassspectrometrydata.WeusedthispipelineinordertocharacterizethespectrumofaminoacidsubstitutionsinbothE.coliandS.cerevisiae.Wefoundthatmostsubstitutions occur due to codon-anticodonmispairing rather than chargingerrors.Wefurthershowedthaterrorspatternsseemtobesimilarinbacteriaand yeast. By analyzing the set of substations obtained, we found thattranslation errors tend to occur at positions that are less evolutionarilyconserved,minimallyaffectproteinstabilityandarefastlytranslated.Finally,weexploredhowenvironmentalperturbationsaffecttranslationerrorpatternbytreatingE.colicellswithadrugknowntoaffectribosomalproofreadingaswellasstarvingthemtoseveralaminoacids.

175

P3.3Oxidative stress response and adaptation by tRNAsmismethionylation and methionine misincorporation in theyeastS.cerevisiaeSylvainDebard,MarionWendenbaumandHubertD.BeckerUMR7156,GMGM,UniversityofStrasbourg,67083Strasbourg,FranceTheoxidativestressresultsinanexcessofelectronscarriedbyoxidantssuchasReactive Oxygen Species. Misincorporation of methionine (Met) has beenproposed to constitute an important antioxidant defensemechanism. It hasbeenshownbytRNAmicroarraysthatmethionylationofnon-cognatetRNAsbythemethionyl-tRNAsynthetase(MetRS) increasedunderoxidativestressandthatsometRNAsaremore likelymischargedbyMetthanothers.Toconfirmtheseresultsandgofurther,wedevelopedabifluorescentreportertocomparemisincoporationofMetforallcodonsintheyeastS.cerevisiae.Resultsshowapreference for Met incorporation at all Leu codons in the absence of anyoxidative stress (fermentation growth condition) in a wild type strain. Aftertreatment with H2O2, misincorporation of Met at these codons increasedsubstantially. Using an arc1Δ strain that mimics the diauxic shift andmitochondrial oxidative phosphorylation activity, the level of MetmisincorporationatLeucodonsincreasesandvariousnewothercodonstriggerMetmisincorporation.AllthesedatasuggestaglobalmethioninemischargingduringoxidativestressinvolvingmismethionylationofunexpectedtRNAs.

176

P3.4Microfluidichigh-throughputscreeningofcodon-anticodonpairsaccepted by the ribosome reveals the critical role of basemodificationsintRNAsKetty PERNOD1, Laure SCHAEFFER1, Johana CHICHER2, Eveline HOK3,Christian RICK1, Renaud GESLAIN3, Gilbert ERIANI1, Eric WESTHOF1,MichaelRYCKELYNCK1,FranckMARTIN11 Institut deBiologieMoléculaire et Cellulaire, “ArchitectureetRéactivitédel’ARN” CNRS UPR9002, Université de Strasbourg, 15, rue René Descartes, F-67084 Strasbourg (France) 2 Institut de Biologie Moléculaire et Cellulaire,PlateformeProtéomiqueStrasbourg–Esplanade,CNRSFRC1589,UniversitédeStrasbourg,15,rueRenéDescartes,F-67084Strasbourg(France)3LaboratoryoftRNABiology,DepartmentofBiology,RitaLiddyHollingsScienceCenter,58ComingStreet,Charleston,SouthCarolina(USA)Inordertodeterminethecodon-anticodonpairingcombinationsefficientlyacceptedbytheeukaryoticribosomeintheabsenceofmodificationintheanticodonloopandbiasfromcompetitionbetweentRNAsorproteinaminoacidsequence,wetookadvantageoftheIRESfromtheintergenicregion(IGR)oftheCricketParalysisVirus.ItcontainsanessentialpseudoknotPKIthatstructurallyandfunctionallymimicsacodon-anticodonhelix.With a reporter containing the IGR upstream of the GFP coding sequence,wescreenedtheentiresetof4,096possiblecombinationsusinghigh-throughputscreeningscombining in vitro coupled transcription/translation and droplet-based microfluidics.Only97combinationsareefficientlyacceptedandaccommodatedfortranslocationandfurtherelongation:38combinations involvecognaterecognitionand59 involvenear-cognate recognition. A/U-rich codon-anticodon combinations did not promote GFPtranslation and themajority ofmissing combinations have aU at the first anticodonposition.Thesedatacomfortthecontributionofnucleotidemodificationstothestabilityoftripletformation.Mostofthenear-cognatecombinations(51)containaGatthefirstpositionoftheanticodon(numbered34oftRNA).Inshort,onthebasisoftheacceptedcombinations,weshowthat(1)intheabsenceofcompetitionbetweentRNAsfromthetRNA pool, as well as between tRNAs and release factors, the stability of thecodon/anticodontripletisasufficientandmajorfactorforproductivetranslation;(2)intheabsenceoftRNAmodificationsintheanticodonloop,tRNAbindingtotheribosomaldecodingcentreiseithernotproductive(inA/Urichcodon/anticodontriplets)orleadtoextensivemiscoding,especiallywithG34-containingtRNAs.

177

P3.5

IntronremovalfromtRNAgenesinS.cerevisiaeS.Hayashi,T.Yoshihisa�GraduateSchoolofLifeScience,UniversityofHyogo,JapanInyeast,total59tRNAgenesgenerateintron-containingpre-tRNAssubjectingtRNAsplicingonthemitochondrialsurface.FordecadesmechanisticprocessesoftRNAsplicinghavebeenstudied,whereasthebiologicalrolesoftRNAintronsremain to be elucidated. We eliminated yeast tRNA introns from differentisodecodertRNAgenestoevaluatetheirimpactoncellularmechanismsmainlyfocused on protein synthesis. Here, we report that all intronless mutantsmaintainedtRNAaminoacylationstatusderivedfromintronlesstRNAgenesandhadlittleeffectonglobaltranslationmonitoringbypolysomeprofiles.Wealsoexamined the nucleolar morphology by immunofluorescence for nucleolarmarkerNop1duetosomemutantspossessingcoldsensitivityand5.8SrRNAreduction. However, all mutants displayed typical round nucleolar shape,suggesting intron removal from certain tRNA genes could affect rRNAbiogenesis but that may be independent of general nucleolus functionsaffecting its morphology. Together, tRNA introns in yeast genes could haveminorrolesonproteinsynthesisundernon-stressedconditions,whiletheymayhaveadvantagesforothercellularprocessesindifferentconditions.

178

P3.6LeuRS-UrzymeMinihelixAminoacylation�JessicaJ.Hobson,CharlesW.Carter,Jr.�UniversityofNorthCarolinaatChapelHill,ChapelHill,NC27599The origin of codon-dependent translation remains one of science’squintessentialmysteries. Aminoacyl-tRNA synthetases (aaRSs) likely played acentralrole intheseevents,astheyenforcetherulesofthegeneticcodebyactivating amino acids and attaching them to the corresponding tRNAs. Ourlong-term goal is to recapitulate as much of translation as possible withplausibleancestralcomponents.WehypothesizethatextantaaRSsandtRNAsarelikelyelaborationsonmuchsimplerprebiotictranslationalapparatuses.Totest this hypothesis, we are reconstituting and testing the aminoacylationactivityofan“ancestral”LeuRS,LeuRS-Urzyme,onasetofdesigned“ancestral”tRNAminihelices.Previously,wedemonstratedaminoacylationactivityfortwoother“ancestral”aaRSs,theTrpRS-andHisRS-Urzyme,onfulllengthtRNAs,buthaveyettodemonstratethisactivityfortheLeuRS-Urzyme.Moreover,wehavenot tested whether any Urzyme aminoacylatesminihelices. As Urzymes caninteractonlywiththe3’CCAend,weexpectthattheLeuRS-Urzymewillacylateminihelices.CognateUrzyme-minihelixpairswillprovideanewparadigm forstudiesoftheoriginofcodon-dependenttranslation.

179

P3.7

PhysiologicalconsequencesofadecreasedtRNAGlyactivityunderoxidativestressinEscherichiacoliL.E. Leiva1, A. Pincheira1, S. Elgamal2, S.D. Kienast3,4, V. Bravo5, J.Leufken3,4,S.Leidel3,4,O.Orellana1,M.Ibba2,A.Katz11Programa de Biología Celular y Molecular, ICBM, Facultad de Medicina,UniversidaddeChile,Santiago8380453,Chile2Department of Microbiology and The Center for RNA Biology, Ohio StateUniversity,Columbus,Ohio43210,USA.�3Max Planck Research Group for RNA Biology, Max Planck Institute forMolecularBiomedicine,Von-Esmarch-Strasse54,48149Muenster,Germany.4Cells-in-Motion Cluster of Excellence and Faculty ofMedicine, University ofMuenster,Albert-Schweitzer-Campus1,48149Muenster,Germany.�5InstitutodeCienciasBiomédicas,FacultaddeCienciasdelaSalud,UniversidadAutónomadeChile.Santiago8910060,Chile.Escherichiacoliisamodelorganismusedtoanalyzethebacterialresponsetooxidativestress.Thereisplentyknowledgeabouttranscriptionalregulationoftheresponse,butlittleisknownabouttheroleofthetranslationmachineryintheadaptationtothiscondition.WeperformedascreeningforchangesintRNAlevels and found an inactivation of tRNAGly and the consequent decrease ofglycineaminoacylationunderoxidativestress.Thesechangesprobablyexplainanalteredefficiencyoftranslationofglycinecodonswehaveobservedinthiscondition.Interestingly,thistranslationaleffectdependsontheavailabilityofthataminoacid in theculturemedia.Modifying the in vivo concentrationoftRNAGlyalteredtheresponsetooxidativestresssuggestingtheexistenceofamechanism whereby bacteria modulate the translational machinery as aresponsetoenvironmentalalterationsandthemetabolicstateof thecells inordertoregulateitsresponsetoenvironmentalchanges.

180

P3.8OnthefunctionofcryptictRNAarraysinprokaryotes�JavierSantamaría-Gómez1,MiguelÁngelRubio1,2,AntoniaHerrero1,MichaelIbba2andIgnacioLuque11InstitutodeBioquímicaVegetal yFotosíntesis.CSICandUniversidaddeSevilla.AvdaAméricoVespucio49.E-41092Seville,Spain.2DepartmentofMicrobiology,OhioStateUniversity,ColumbusOH43210,USAThenumberof tRNAgenesvariesdramatically, from less than100 tomore than104,amongthespeciesofthetreeoflife(1).However,inBacteriathenumberoftRNAgenesisrelativelylowandconsistent,withanaverageof40-50genesencodingnon-redundantisoacceptortRNAspergenome(2).InthevastmajorityofbacterialphylaindividualtRNAgenesarefoundscatteredinthegenomeoroccasionallyformingshortclustersoftwotothreegenes.However,inadditiontothiscomplementoftRNAgenes,somespeciessprinkledinthebacterialradiationcontainoneortwolongtRNAgenearraysprobablyacquired by horizontal gene transfer (HGT) from a donor of the Firmicutes phylum,where such gene arrays are common (2-4). The function of these arrays in speciesoutsideoftheFirmicutesphylumisintriguingandhasbeenlittleinvestigated.Previousworkshowedthatinadditiontoasetof48genesscatteredinthechromosomeencodingthe housekeeping tRNA complement, the cyanobacteriun Anabaena sp PCC 7120contains a tandemarray of 23 genes (hereafter trn operon) in a plasmid. The tRNAsencodedinthisarraycanbeprocessedandchargedbuttheirlevelinthecellisextremelylow(ordersofmagnitudebelowthelevelofthehousekeepingtRNAs).Deletionofthearraydidnotimpaircellgrowthindiverseconditions(5),whichsuggeststhatthisarray,probablyacquiredbyHGT,constitutesanon-functionalrelicinAnabaena(5).TogaininsightsintothepossiblefunctionoftRNAgenearraysinbacteriaoutsideoftheFirmicutesweanalyzedtheexpressionoftheAnabaenatrnoperoninawidevarietyofconditions and found that it is induced up to 103-fold in diverse stress conditions.Furthermore, most of the tRNAs encoded in this array are charged under inductionconditionsandsomearefoundinthepolysomalfraction.ThisindicatesthatthesetRNAsparticipate inproteinsynthesis instressconditionsandsuggeststhattheymayplayarole inpreserving cell viabilityunder stress. Interestingly, someobservations indicatethatafewofthetRNAsencodedinthetrnoperonaremisacylated(i.e.chargedwithanon-canonicalaminoacid).Furtherconsequencesoftheexpressionofthetrnoperonarestillunderinvestigation.Bibliography:�1.Bermudez-SantanaC,AttoliniCS,KirstenT,EngelhardtJ,ProhaskaSJ,SteigeleS,StadlerPF.2010.GenomicorganizationofeukaryotictRNAs.BMCGenomics11:270.�2.MorgadoSM,VicenteACP.2018.BeyondtheLimits:tRNAArrayUnitsinMycobacteriumGenomes.FrontMicrobiol9:1042.�3.TranTT,BelahbibH,BonnefoyV,TallaE.2015.AComprehensivetRNAGenomicSurveyUnravelstheEvolutionaryHistoryoftRNAArraysinProkaryotes.GenomeBiolEvol8:282-95.�4.BustamanteP,CovarrubiasPC,LevicanG,KatzA,TapiaP,HolmesD,QuatriniR,OrellanaO.2012.ICEAfe1,anactivelyexcisinggeneticelementfromthebiominingbacteriumAcidithiobacillusferrooxidans.JMolMicrobiolBiotechnol22:399-407.�5.Puerto-GalanL,VioqueA.2012.ExpressionandprocessingofanunusualtRNAgeneclusterinthecyanobacteriumAnabaenasp.PCC7120.FEMSMicrobiolLett337:10-7.

181

P3.9DNA damage-induced cell death relies on SLFN11-dependentcleavageofdistincttypeIItRNAsManqingLi1*#,DaneMalone1#,ElaineKao1#,XiaGao1,JeanY.J.Wang1,2,3

andMichaelDavid1,2*1Section of Molecular Biology, Division of Biological Sciences, University ofCalifornia, SanDiego, La Jolla,CA92093 2MooresCancerCenter, La Jolla,CA92093�3DivisionofHematology/Oncology,DepartmentofMedicine,UniversityofCaliornia,SanDiego,LaJolla,CA92093#TheseauthorscontributedequallytothisworkDNA-damagingagents(DDAs)representthelargestgroupofcancerdrugs,butprimaryorsecondaryresistanceseverelylimitstheireffectiveness.Reportsoftwolarge-scalescreeningeffortsrevealedthathumanSchlafen11(SLFN11)–aproteinwepreviouslyfoundtoinhibittranslationofHIVencodedproteinsduetoatypicalcodon-usageintheviralRNA–sensitizescancercellstoDDAs.WenowshowherethatSLFN11alsoinhibitstranslationofATRuponDNAdamageandtherebycausessensitivitytoDDAswithoutdisruptingearlyeventsoftheDNAdamageresponse.TypeIItRNAs,whichhavealongvariablestemof13-19bases instead of a short variable loop (4-5 bases), are cleaved by a SLFN11-dependent mechanism in response to DDAs. Type II tRNAs accounts for allserineandleucinecodons,andwedemonstratethatgenesmakingfrequentuseofTTA(Leu)codonlikeATRaresubjecttoselectivetranslationalsuppressionbySLFN11. Gapmer-mediated down-regulation of tRNA-Leu-TTA resulted inablatedATRproteinexpressionandre-sensitizationofSLFN11-deficientcellstoDDAs.Ourstudyuncoveredanovelmechanismofcodon-specifictranslationalinhibitionbySLFN11-dependenttRNAcleavageintheDNAdamageresponse,and suggest that SLFN11-deficient cancer cells can be re- sensitized to DDAtherapybytargetingATRortRNA-Leu-TAA.

182

P3.10Cell cycle protein cdc13 and growth of SchizosaccharomycespombeareaffecteduponoverexpressionoftRNA.1,2AriasL.,1MoreiraS.,1KatzA.and1OrellanaO.1Programa de Biología Celular y Molecular, ICBM, Facultad de Medicina,Universidad de Chile. 2Facultad de Ciencias Químicas y Farmacéuticas,UniversidaddeChile.Santiago,ChileLevelsandchemicalmodificationsoftRNAcanaffecttheirfunctions,includingtranslationrate.ThepurposeofthisworkwastodeterminewhetherincreasingthetRNAlevelsaffectstheexpressionandfoldingofproteinsand/orthecellgrowthinS.pombe.WeinducedoxidativestressonS.pombecellsculturedinliquidmediumbyH2O2andanalyzedthetRNAlevels.UndertheseconditionstRNAGly

UCCandtRNAArg

UCUincreasedtheirlevelswhiletRNAThr

UGUdidnotchange.WestudiedtheeffectoftheoverexpressionofthesetRNAsintheexpressionandsolubilityofcdc13,acyclinrequiredfortheG2/Mtransition.Cdc13mRNApossesses rare codons recognized by these tRNAs. Overexpression of tRNAgenesinS.pombeincreasedaggregationofcdc13,butdidnotaffectthemRNAand protein levels. Cells harboring increased tRNA levels showed a reducedcolony size. Syncronized cells showed an elongated phenotype revealing adefectincelldivision.Inconclusion,overexpressionofthesetRNAsaffectedthesolubilityofcdc13andthecellcycleofS.pombe.Whetheraggregationofcdc13and/orothercyclinsisresponsibleforthephenotypesisunderinvestigation.SupportedbyFondecyt,Chile1150834toOO,Fondecyt,Chile,Postdoctorado3150366toSMandBecaCONICYT,Chilemagister22151224toLA.

183

P3.11

InvestigatingbiasesintRNAsequencinglibrariesThomasE.Mulroney1,2TuijaA.A.Pöyry1andAnneE.Willis11MRCToxicologyUnit,UniversityofCambridge,UnitedKingdom�2AstraZenecaDrugSafetyandMetabolism,CambridgeSciencePark,UnitedKingdomRecent developments in RNA sequencing techniques have broadened ourunderstanding of many small RNAs, such as microRNAs (miRNAs), smallnucleolar RNAs (snoRNAs) andpiwi-interactingRNAs (piRNAs).However, theapplicationofsmallRNAsequencingtechnologies tobetterunderstandtRNAbiologyremainschallenging.Thisismainlyduetoabundantpost-transcriptionalmodifications in tRNAs that inhibit typical approaches to librarypreparation,including cDNA synthesis and adapter ligation.Multiple techniques that relyupon high-throughput sequencing have been developed to reduce theseproblemsandtoascertaintRNAexpressioninaquantitativemanner.However,relativelylittlevalidationofthesequencingdatahasbeenconductedsofar,andnocomparisonbetweenthesemethodshasbeenreported.WehaveinvestigatedthevalidityofseveralmethodstopreparecDNAlibrariesof mature tRNAs from human cells. In particular, we have assessed theefficiency of reverse transcription and adapter ligation for individualdemethylatedtRNAsandinvestigatedsourcesofsequencebiasintRNA-derivedsequencinglibraries.Basedontheseexperiments,weofferrecommendationsforpreparingcDNAlibrariesfortRNAs,andsmallRNAsingeneral,whichcouldassistamorereliableinterpretationofresultsfromhigh-throughputsequencingexperiments.

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P3.12DifferenteffectsoftRNApseudouridinesontranslationalfidelityinE.coliandPseudomonasspeciesMariTagel,KarlJürgenstein,MargusLeppik,HeiliIlves,MaiaKivisaar,JaanusRemmeIMCBUniversityofTartu,EstoniaModified nucleosides play several roles inmodulating tRNA structure and functions.Modifications in the anticodon loop direct codon recognition and facilitate codon-anticodonrecognition.Modificationsaroundtheanticodonare important foroptimaltranslationalfidelity.Importanceofpseudouridinesoutsideanticodonisstillenigmatic.Translational accuracy has been studied mostly in E. coli. Moreover, molecularmechanismsofaatRNAselectionandcontroloferrorfrequencyhavebeenelucidatedusinginvitrotranslationderivedofE.colisystem.Weareinterestedtoelucidatetheroleofpseudouridinesintranslationalfidelityindiversebacteria.Herewestudytranslationalfidelity of wt and pseudouridine deficient strains using three bacterial species:Escherichiacoli,Pseudomonasaeruginosa,andPs.putida.Pseudouridinesareconservedaroundtheanticodonstematthepositions32,38,39,and40ofmanytRNAspecies.ThesepsiresiduesaremadebyenzymesRluA(pos32)andTruA(38-40).Physiologicalroleofthesepseudouridinesisnotknown.Wehavedevisedabroadrangeplasmidderiveddual luciferasemRNAtranslationfidelityassaysystem.The E. coli MG1655, Pseudomonas aeruginosa PAO1, and Ps. putida PaW85 strainslackingeitherTruA,RluAorbothweretestedforframeshiftfrequencyat-1(-1FS)and+1(+1FS)sitesandreadthroughatUGAstopcodon.DeletionofRluAdoesnothaveeffectontheerrorfrequencyinanyspecies.Incontrast,deletionofTruAleadstoincreased-1FSatspecificcontextinE.coliandPsputidabutnotinPsaeruginosa.Similareffectwasfoundwith UAG reathrough. Frequency of +1FS is hardly affected by absence of psiresidues. The results point to the species specific effect of the conserved tRNAmodificationtotranslationalfidelity.Whenwild-typebacteriaarecompared,frameshiftismorefrequentinPseudomonasspeciesascomparedtoE.coli.ThedifferencebetweenE. coli and Pseudomonas can be partially due to the E. coli derived programmedframeshift signal used.On the other hand, UGA readthrough occurswith the similarfrequencyinallthreebacteriaspecies.TheresultsopennewaspectsofmRNAcontextspecificeffectsonthetranslationandnewphysiologicalroleofpseudouridineintRNA.

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P3.13InvestigatingtheroleofMetRSonpersisterformation.RubioMA1,BackesN2,PhillipsGJ2,IbbaM11DepartmentofMicrobiology,AronoffLab,OhioStateUniversity,Columbus,OH43210,USA.�Email:rubiogomez.1@osu.edu,ibba.1@osu.edu�2DepartmentofVeterinary Microbiology, 1907 ISU C-Drive; Veterinary Medical ResearchInstitute,Building#6;Ames,IA50011,USAE-mail:gregory@iastate.eduPersisterscellsenteradormantstateinwhichtheybecomehighlytoleranttoantibiotics. After antibiotic treatment, persisters are able to reactivate andresume infection, leadingtorelapsesandchronicdisease.Themechanismofpersister formation remains poorly understood. Recent studies identifiedEscherichia coli metG mutants (encoding methionyl-tRNA synthetase) withincreased persister formation rates, establishing a link between aminoacyl-tRNA synthetases and persister formation. Characterization of these metGmutants revealed striking differences in the KM of the substrates as well asvariationinkcat.BecauseoftheprominentroleaaRSsplayintranslation,wealsoexplored effects of themetGmutants on cellular protein synthesis and ATPhomeostasis in the presence of both cognate and non-cognate amino acids.Intriguingly,theMetRSmutantsaresensitivetothenon-cognatehomocysteine,andwealsohaveevidence that tRNAMet ismischargedwith this amino acid.Under some stress conditions a certain degree of mistranslation may bebeneficialasameanstoexpandphenotypicheterogeneity,suggestinganovellinkbetweenMetRSmistranslationandpersisterformation.

186

P3.14

The dual role of the 2’-OH group of A76 tRNATyr in thediscriminationofD-Tyrosineduringthefirststepsoftranslation�M.Rybak,O.Kovalenko,M.Tukalo�InstituteofMolecularBiologyandGeneticsoftheNASofUkraine,Kyiv,UkraineTyrosyl-tRNA-synthetase (TyrRS) is exceptional in its biochemical context: itweaklydistinguishesenantiomersofTyranddemonstratesdualaminoacylationon2’-and3’-OHgroupsofA76tRNATyrTocorrectthemistakesinrecognitionofTyrisomers,anadditionalenzyme,D-aminoacyl-tRNA-deacylase(DTD),exists.However,thecatalyticroleofOHgroupsofthetRNATyrA76inthecatalysisbyTyrRSandDTDremainedunknown.Toaddressthisissue,[32P]-labelledtRNATyr

substrates (A76, 2’d, 3’d) have been tested in vitro in aminoacylation anddeacylation assays with Thermus thermophilus TyrRS and DTD. TheaminoacylationactivityofL-Tyrissimilaronboth2’and3’-OHgroupsoftRNATyr.D-Tyrattachestothe2’-OHgroupofA762800-foldfastercomparingtothe3’-OH.Thus,TyrRSdemonstratessignificantpreferencetoward2’-OHinchargingwithD-Tyr.DTDcatalysesthehydrolysisofD-Tyr-tRNATyrspecificallyfromthe3’-OHgroupwhilethe2’-OHassistsinthishydrolysis.Itisworthtomentionthatthetransacylation,occurringfromtheprimarysite(2’-OH)totheneighborOH-group(3’-OH)isdramaticallyessentialforfurtherhydrolysis,similartoIleRS.

187

P3.15ImplicationsofdiscriminatorbasedoperationalcodeforchiralitybasedproofreadingsystemDTDRajanSankaranarayananCSIR-CentreforCellularandMolecularBiology,Hyderabad,INDIAD-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged ontRNAs and therefore are implicated in enforcing homochirality in proteins.DTD’smechanisticdesignprincipleisbasedonlyonL-aminoacidrejectionandhenceitactsonachiralglycineattachedtotRNA.Theactivityonglycyl-tRNAisadvantageous as DTD clears glycine mischarged on tRNA(Ala). We haveidentifiedATD(Animalia-specifictRNAdeacylase)asanewvariantofDTDwhichrectifies a unique tRNA mis-selection problem of eukaryotic AlaRS. ThestructuralbasisofATDfunctionanditssubstratespecificitywillbediscussedindetail.OurrecentstudieshaveledustoidentifykeyelementsintRNAwhichinfluence DTD’s enzymatic activity. The study has revealed the role ofdiscriminatorbaseinensuringfaithfulglycinedeliveryforproteinbiosynthesis.It throws light on the fundamental and underappreciated problem ofdiscriminator base bias in all three branches of life and the evolutionaryselectionpressuresthatactonit.References:�1.Routhetal.PLoSBiology(2016)14:e1002465:1-22.�2.Pawaretal. Elife (2017) 6:e24001, 1-19.�3. Kuncha, Mazeed et al. Nature commun.(2018)9:1-13.4.Kunchaetal.bioRxiv(2018)doi.org/10.1101/322289.

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P3.16KRASG12Cmutationdifferentiallyrewiresproteinsynthesisviac-MYCmodulatedtranslationinitiationmachineryrecruitmentinNSCLCGeorge Kyriakopoulos#, Vicky Katopodi#, Katerina Grafanaki, IliasSkeparniasandConstantinosStathopoulosDepartmentofBiochemistry,SchoolofMedicine,UniversityofPatras,GreeceKRAS mutants affect translation initiation by activating the MAPK andPI3K/AKT/mTOR signaling pathways, which functionally converge withcomponentsofthetranslationalmachinery,alteringproteinsynthesisratesofseveral oncoproteins, including c-MYC. Concomitantly, c-MYC is recruited tocontroltranscriptionbyallRNApolymerasesthusrewiringproteinsynthesisviamodulationofribosomebiogenesis,tRNAtranscriptionandformationofeIF4Fcomplex.TodeciphertheeffectofKRASG12ContranslationinitiationinNSCLC,weconstructedTetON-inducibleKRASG12CstableA549andCL1-5celllinesusingTALENs.WeobservedthateIF4Fformationisdifferentiallyregulatedinac-MYC-depictive manner. The 43S PIC formation along with the tRNAiMet levelsindicatedvariationsinglobalproteinsynthesisrates.SeveralmitochondrialtRFswerefoundmodulatedandcorelatedwithapossibletranslocationofKRASG12C

towards mitochondrial membrane. In conclusion, we report a divergentresponseamongtwoepithelial-derivedNSCLCcelllinesuponinductionofthesame KRAS mutant, which affects the rewiring of protein synthesis, tRNAfragmentationandpossiblymitochondrialintegrity.#equalcontribution

189

P3.17tRNAstructuresintheribosomeMasahito Kawazoe1, Chie Takemoto2, Fumikazu Konishi3, TakashiYokogawa4,ChiekoNaoe5,YuriTomabechi2,Takashi Itagaki6,MikakoShirouzu2,ShigeyukiYokoyama11RIKEN Structural Biology Laboratory, Japan. 2RIKEN Center for BiosystemsDynamicsResearch,Japan.3TokyoInstituteofTechnology,EducationAcademyof Computational Life Sciences, Japan.� 4Dpt. of Chemistry andBiomolecularScience, Faculty of Engineering, Gifu University, Japan.� 5School ofPharmaceuticalSciences,ShowaUniversity,Japan.6RIKENYokohamaInstitute,SystemsandStructuralBiologyCenter,Japan.ThetRNAL-shapestructurerationallydemonstratesitsfunctions,base-pairingwithmRNAandtransferringthecognateaminoacidtothepeptidyltransferasecenter.The3DstructureisorganizedmainlybyinteractionsbetweenDandTyloops togetherwith participation of the variable region. Recently, structuraldynamicsoftRNAisexplored,butthereislittlestructuralinformationaboutaclass II tRNA. Here, we have determined the crystal structures of the 70SribosomefromT.thermophilusHB8complexedwithE.colitRNASercognateforAGYcodons,SerV(93nt).Inobtainedstructures,theelectrondensityfortRNAin the P-site was unambiguously visible and the structure of all nucleotidesincludingalongvariableregion(21nt)wasdeterminedconfidently.ThevariableregionformseightbasepairsstartingfromA45-U47p,whichinteractswithaninsertionresidueofDloop,G20b.Thetipresidueintheloopregion(V-loop),A47finteractswiththeedgeoftheA-sitefinger,helix38inthe23SrRNA(H38).Inaddition,theedgeofH38,A887andA47ginV-loopareclosetoribosomalproteinS13.Meanwhile,variableregionsofE-siteandA-sitetRNAsexhibitpoorelectron density, suggesting that there is no significant interactionwith anyribosomal components. Structural changes in the vicinity of V-loop duringtranslocationwillbediscussed.

190

P3.18tRNA-dependentmechanismoftheerrorseditingintranslationqualitycontrol�M. Tukalo, O. Kovalenko, M. Rybak, A. Rayevskiy, M. Il’chenko, O.Gudzera,I.Kriklivyi,G.Yaremchuk,I.Dubey�InstituteofMolecularBiologyandGeneticsNASofUkraine,Kyiv,UkraineFidelity of translation is controlled by several mechanisms including theproofreadingmechanisms by aminoacyl- tRNA-synthetases and trans-editingfactors.We have studiedmolecularmechanisms of editing by two differentediting enzymes, leucyl-tRNA synthetase (LeuRS) and D-aminoacyl-tRNA-deacylase (DTD) fromThermusthermophiles,usinganumberofapproaches,includingmolecularmodeling,quantum-mechanicalcalculations,site-directedmutagenesisenzymeandmodificationoftRNA.Ourintensivealaninescanningmutagenesis of LeuRS and DTD editing sites has failed to identify catalyticresiduesforhydrolysiswithintheactivesite.Ontheotherhands,modification

oftRNATyratthe2’-OHofA76andtRNALeuatthe3’-OHofA76byreplacingthemeachwith a proton, revealed an essential function for these groups inhydrolysis. On the basis of obtained experimental results and our QMcalculationswesuggesttRNA-dependentmechanismofpost-transfereditingbyLeuRS and DTD. The most important element of that mechanism is theformationofintramolecularhydrogenbondbetweenthe2'-OHor3’-OHofA76and carbonyl group of the substrate,which strongly facilitate the hydrolysisreaction.

191

P3.19Mechanism of EF-P(Arg32) rhamnosylation revealed by thecrystal structure of glycosyltransferase EarP·translation factorEF-PcomplexToru Sengoku1, Takehiro Suzuki2, Naoshi Dohmae2, ChiduruWatanabe3, Teruki Honma3, Yasushi Hikida1, Yoshiki Yamaguchi4,HideyukiTakahashi5,ShigeyukiYokoyama1,TatsuoYanagisawa1:1RIKEN Struct. Biol. Lab., 2RIKEN CSRS, 3RIKEN CLST, 4RIKEN Global Cluster,5NationalInstituteofInfectiousDiseasesTranslation elongation factor P (EF-P), a ubiquitous protein over the entire range ofbacterial species, rescues ribosomal stalling at consecutive prolines in proteins. InEscherichia coli and Salmonella enterica, theb-lysyl modification of Lys34 in EF-P isimportantfortheEF-Pactivity.Theb-lysylationpathwayisconservedamonglessthan30%of bacteria. Recent reports found that theShewanella oneidensis,Pseudomonas

aeruginosa, and Neisseria meningitidis EF-P proteins, containing an Arg residue atposition 32 (corresponding to Lys34 in E. coli EF-P), are modified with the sugarrhamnosebytheglycosyltransferaseEarP.Inadditiontotheb-lysylationofEF-P(Lys34),the rhamnosylation of EF-P(Arg32) is important for the EF-P activity, cell growth,pathogenicity,andantibioticresistance.EarPspecificallytransferstherhamnosylmoietyfromdTDP-b-L-rhamnosetoArg32ofEF-P.ToelucidatethedetailedreactionmechanismsofEF-P(Arg32)rhamnosylation,wesolvedthecrystalstructuresoftheN.meningitidisEarPapoform,andtheEarP/dTDP-rhamnoseandEarP/EF-Pcomplexes.TheEarPstructurecontainstwotandemRossmann-fold domains, thus classifying EarP in glycosyltransferase superfamily B. EarP formsnumerous interactions that specifically recognize theconserved residuesofEF-P,andbindstheentireb-sheetstructureofEF-PdomainI.Thereby,Arg32ofEF-PisproperlypositionedattheEarPactivesite,andcausesastructuralchangeinaconserveddTDP-b-L-rhamnose-binding loop of EarP. Furthermore, Asp20 of EarP was identified as thegeneralbaseresidue intheSN2reaction.WeproposethattheArg32bindingandtheaccompanying structural change of EarP convert the rhamnose-ring conformation,therebyfacilitatingtherhamnosylationofEF-P(Arg32).UnderstandingtheEarPstructureand its unique reaction mechanism will enable the structure-based design of drugcandidates against a specific group of pathogenic bacteria, includingN.meningitidis,Neisseriagonorrhoeae,andBordetellapertussis.

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Examining the relation between tRNA expression and cellularproliferationstateNoaAharon-Hefetz,IdanFrumkin,OrnaDahan,RoniRak*andYitzhakPilpel*DepartmentofMolecularGenetics,TheWeizmannInstituteofScience,Rehovot76100,IsraelWe have previously shown that proliferating and differentiated mammaliancellsexpressdistinctsetsoftRNAmolecules,thatarecompatiblewiththecodonusage of either pro-proliferation genes or pro-differentiation genes,respectively (Gingold et al., Cell 2014). This study aimed to assess theessentialityofthecoordinatedchangesinthetRNApoolandtheproliferativestateofthecell.WeusedCRISPR-iCas9techniquetotargetandmanipulatethepro-proliferation and pro- differentiation tRNA levels in HeLa cell line usingsgRNA library we designed, in which each sgRNA is targeting one tRNAisoacceptorfamily.ToexploretheeffectoftRNAknock-outonHeLacells,weactivated iCas9 inaheterogeneouscellpopulation, inwhicheachcellcarriesoneofthesgRNAtypes,andsequencedthesgRNAs.Theresultsindicatethatreductioninthepro-proliferationtRNAlevelsreducedthegrowthrateofthecells,whilepro-differentiationtRNAknock-outsshowedlittleeffectoncellulargrowth.WefurtherexploredtheimpactofeditingonthetRNAgenesequenceandexpressionbytargetingsingletRNAisoacceptorfamily,followingactivationof iCas9.We then sequenced the targeted tRNAs,both in theDNAandRNAlevel.OurresultssuggestthatHeLacellsdonottolerateIndelmutationinpro-proliferationtRNAgenes,asindicatedbytherapideliminationofsuchcellsfromthe population. In contrast, cells carrying Indel mutation in the pro-differentiation tRNA genes didn’t show any growth defect and continued topropagate.Theseresultsindicateanactiveroleofpro-proliferationtRNAsinthecellularproliferationstate.

195

P4.2

AnimprovedworkflowforquantitativetRNAtranscriptomics.D.Behrens,D.Nedialkova�MaxPlanckInstituteforBiochemistry,Martinsried,GermanyAccurate,globaltRNAquantificationisnecessaryforunderstandinghowtRNApools impactcellphysiology.High-throughputtRNAtranscriptomicshasbeenimpeded experimentally and computationally by tRNA modifications andstructure, as well as sequence similarity between multi-copy isoacceptorfamilies.We present improvements to transcriptome-wide tRNA abundancemeasurements inbothof theseregards.WecomparedrecentapproachestoovercomingreversetranscriptionroadblockstofindastrikingvariationintheirabilitytomitigatetRNAsequencingbias.Byusingahighlythermostablereversetranscriptase under conditions that favor misincorporation at modifiedpositions, we achieve a uniform tRNA coverage without biases towardsparticulartRNAspecies.Accountingformisincorporationsatmodifiedresiduesin our computational analysis drastically increases read alignment sensitivityand specificity, further improving tRNA quantification. We have used ouroptimized workflow for quantitative tRNA transcriptomics to define howmodificationdefectsimpacttranslationallyavailabletRNApools.

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P4.3NewtyrosineidentitysetinPlasmodiumfalciparumapicoplast�Cela,M.,Paulus,C.,Santos,M.A.S.,Moura,G.,Frugier,M.*,Rudinger-Thirion,J.�UniversitédeStrasbourg,CNRS,ArchitectureetRéactivitédel’ARN,UPR9002,InstitutdeBiologieMoléculaireetCellulaire,15rueRenéDescartes,�F-67084StrasbourgCedex,FranceAminoacylationisahighlyspecificprocessthatreliesonthepresenceofidentityelements,usuallyconservedthroughoutevolution.However,thetyrosylationsystem escapes this rule: tRNATyr and tyrosyl-tRNA synthetases (TyrRS) havedevelopedfeaturesthatpreventcross-tyrosylationbetweendifferentphylae.OnthetRNAside,thisbarrierconsistsinauniqueelement:the1-72basepair(G-Cinprokaryotes,C-Gineukaryotes/archaea).Surprisingly,inarelictplastid(apicoplast) in the human malaria parasite P. falciparum, tRNATyr share anuncommonA1-U72pair.Giventheimportanceoftheaminoacylationprocessandtheapicoplastfortheparasitesurvival,weaimedtodeciphertheidentityrulesthatgovernapicoplastictyrosylation.Weproducedan“harmonizedversion”oftheapicoplasticTyrRS(apiTyrRS);andthe tRNATyr sequences were in vitro transcribed, both WT and mutated atstrategic positions. Kinetic tyrosylation parameters were determined andcomparedtotheWTmolecule.ResultsareinfavorofanunusualrecognitionpatternbetweentheapicoplastictRNATyrbyitscognateTyrRS.Thesepeculiarproperties make apiTyrRS a promising candidate for antimalarial drugdevelopment.

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P4.4tRNAswithMultipleIntronsAreWidelySpreadinFungi�PatriciaP.ChanandToddM.Lowe�DepartmentofBiomolecular Engineering,UniversityofCalifornia SantaCruz,USAA portion of precursor transfer RNAs contain introns thatmust be removedduringmaturation.MosttRNAintronsarelocatedonenucleotidedownstreamoftheanticodon,althoughnumeroustRNAsintronsinarchaeahavebeenfoundin other, seemingly random “noncanonical” positions. In eukaryotes,noncanonical tRNA introns were believed to be rare, given they have beenidentifiedonlyintwoalgae.UsingtRNAscan-SEandcomparativeanalyses,weshowthatthesenoncanonicalintronsarespreadacrossatleastninespeciesinPezizomycotinaof Fungi,withPenicillium rubenshaving themost (20outof217)amongthestudiedgenomes.Theintronsarelocatedindifferentpositionswithinthegenes,similartothosepreviouslyfoundinarchaeaandalgae.Unlikewell-studied fungi such as Saccharomyces cerevisiae, many species inPezizomycotinaalsohaveamajorityoftRNAsthatcontainintrons.Interestingly,mostofthenewnoncanonicalintronswerefoundintRNAgenesthatalsohaveacanonicalintron.Althoughtheacquistionmechanismoftheseintronsisstillunknown, our findings provide a new perspective on eukaryotic tRNA geneevolution.

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P4.5

Looking at the end: Sequencing tRNA 3’-termini provides newinformationaboutmaturationandintegrity�AndreasCzech�Institute of Biochemistry and Molecular biology, Prof. Z. Ignatova group,UniversityofHamburg,GermanyAnticodonand3’-terminusarecrucialparts forcanonical tRNA function.Theessential mature 3’-CCA-tail is created by ribonucleases during 3’-trailerprocessing andmaintained by the CCA-adding enzyme. Furthermore, the 3-terminuscanbeelongatedbypoly(A)polymeraseortheCCA-addingenzymetogeneratea tag fordegradation.Weemployedanewsequencing strategybypreparinglibrariesfromthe3’-terminalpartofE.colitRNAsonly.Byusingthisshort hypo- modified segment we avoid sequencing biases from secondarystructureormodifications.ThisallowedustonotonlyquantifymaturetRNA,but alsoprecursorsof different lengths, shortenedandelongated tRNA.Ourresults revealed that 3’-trailer processing efficiency varies greatly amongdifferenttRNAspecies. Inaddition, incontrasttostationarygrowthphase, intheexponentialphaseasignificantamountof tRNAshasshortenedCCA-tailsand is translation-incompetent. Integrityvaried largelyamongdifferent tRNAspecies and seemed to depend on the identity of discriminator base. Wehypothesize, that the CCA-tail of certain tRNAs is damaged during 3’-trailerprocessingandneedstoberepairedbytheCCA-addingenzyme.

199

P4.6

Murine aminoacyl-tRNA synthetases: current knowledgerepresentationDrabkin,HJ1,Wu,C2,andBlake,JA1.1TheJacksonLaboratory,BarHarbor,ME04609,USA.2UniversityofDelaware,Newark,DE19711,USAAminoacyl-tRNA synthetases are well studied as part of research efforts tounderstandthemechanismsoftranslation.Dataaboutmurineaminoacyl-tRNAsynthetasescomefromthepeer-reviewed literatureor frominferencesfromcomparativecontextoforthologsorphylogenetics.ComprehensiveintegrationofthesedataisessentialfordataanalysisTheMouseGenomeInformatics(MGI,http://www.informatics.jax.org,) istheinternationaldatabaseresourceforthelaboratorymouse,providingintegratedgenetic,genomic,andbiologicaldatatofacilitatethestudyofhumanhealthanddisease.MGIusestheGeneOntology(GO,http://www.geneontology.org)forfunctionalannotationofmousegenes.GOdefinesconceptsusedtodescribegeneproductfunctioning,location,andparticipationinbiologicalprocesses,aswellasrelationshipsbetweentheseconcepts.Becauseasingleeukaryoticgenecan encode multiple protein isoforms, MGI uses Protein Ontology (PRO(http://proconsortium.org/pro) which supplies unique identifiers to specificproteoforms. These forms are organized in an ontological framework thatexplicitly describes how these entities relate to each other. These resourcesprovidesanoverviewofthegenomicandproteomicdataassociatedwithusageof alternate promoters or polyadenylation sites, alternative splicing of theprimarytranscript togeneratedifferentmRNAs,selectionofalternativestartsitesduringtranslationofanmRNA,and/orsingleormultiplepost-translationalprocessing events including proteolytic cleavage as well as amino acidmodifications. An overview of genomic and proteomic data associated withmurineaminoacyl-tRNAsynthetasesisoutlinedhere,usingGarsasanexample.SupportedbyNIHGrantsHG000330,HG002273,andGM080646.

200

P4.7

InsightsintoGenomeRecodingfromtheMechanismofaClassicQuadruplet-CodonReaderHowardGamperThomasJeffersonUniversity,Philadelphia,USAWhilequadrupletcodonsareattractiveforincorporatingnon-canonicalaminoacids toexpand thegeneticcode, little isknownofhowtheyare translated.Hereweprovidemechanisticinsightintotranslationofquadrupletcodonsbyaclassicquadruplet-readingtRNA,isolatedasSufB2withGGG-Gastheexpandedanticodon.Incontrasttotheconventionalperception,weshowthatSufB2isatriplet reader of the proline CCC codon at the ribosomal A-site but that ittransformstoaquadruplet-readerofCCC-CduringtranslocationtotheP-site.This transformation, however, is suppressed upon inhibition of quadrupletpairingoruponpost-transcriptionalmodificationsthatoccuratpurine37onthe 3'-side of the anticodon. This study establishes a framework for how toimprove quadruplet reading in new strategies of genome expansion andhighlightsthepotentialoftRNAtranslocationacrosstheribosomeasaregulatorofquadrupletreading.

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P4.8CoLoC-seq,anewgenome-wideapproach toprofileorganellarRNAimportomes�D.Jeandard,I.Tarassov,N.Entelis,A.Smirnov�UMR7156(UNISTRA-CNRS)GénétiqueMoléculaire,Génomique,Microbiologie(GMGM),28rueGoethe67000Strasbourg,FranceMitochondriaareessentialorganellesofeukaryoticcellsinvolvedinseveralkeyprocesses, including cellular respiration, apoptosis and iron-sulfur clustersformation. Despite the presence of their own small genome, mitochondrialfunctions critically depend on the import of proteins and noncoding RNAs(tRNAs, 5S rRNA,miRNAs...) encoded by the nucleus. However, detection ofpartially imported RNAs is often confounded by the persistence of cytosoliccontaminant RNAs even after most thorough purification of organelles. Todistinguish between RNAs genuinely present inside mitochondria and merecontaminant, our laboratory developed a conceptually novel methodology,ControlledLevelofContaminationcoupledwithsequencing(CoLoC-seq).UsingCoLoC-seqprocedurewecomprehensivelylandscapedtheRNAimportomeofhuman HEK293T cells, confirmed the mitochondrial import status of somenuclear-encodedRNAsandidentifiedafewnovelimportedRNAs.OuranalysesalsoclarifiedtheunresolvedimportstatusofRNAseMRPandRNAsePRNAs,suggestedtobeinvolvedinmitochondrialDNAreplicationandtRNAprocessing.ThisworkissupportedbytheLabexMitoCross(PIAProgram,IdexUNISTRA)

202

P4.9

Proglambile misincoporation of amino acids increasesprobabilityofproteinevolutionToshihikoEnomotoand*DaisukeKiga�WasedaUniversity,Tokyo,JapanConsideringthathighfidelityinthepresenttranslationisachievedbyevolvedmachinerysuchaseditinginaaRS,earlyevolutionarystagesoftranslationhadlowfidelitypolymerizationsystem.Toexplorepropertiesofproteinsevolvedinearlylifewithlowfidelity,wecreatedgeneticcodeswithadjustablelowfidelitybyengineeringofcell-freetranslation.Fidelityinourcodewasdecreasedbythe

additionoftRNAServariantswithanticodonreplacementscorrespondingtoThrcodons.Byadose-dependentmannerof tRNAvariantsaddition,GFP lost itsfluorescencedespitenochangeinamountofproteinproduction.Wecreatedothermisincorporationseasily,becauseofSerRSandAlaRSacceptanticodonreplacementsintheirtRNA.Our engineered code is expected to produce amore smooth protein fitnesslandscapeonDNAsequencespace,becausenotonlyproteinwiththeencodedaminoacidsequence,butproteinvariantswithpointmutationsareproducedfromoneDNAsequence.ThermostabilityanalysisofGFPvariantsshowedthatthesmoothingeffectofourlowfidelitycodediminishedlocaloptimumpointsand thus expanded the number of evolutionary pathways to the globaloptimum.

203

P4.10

Integrative tRNAs, tRFs and miRNAs profiling in lung cancerbiopsies�DimitriosΑnastasakis1,AnthonyMarchand2,ChristosKontos3,KaterinaGrafanaki1, Ilias Skeparnias1, Panagiotis Alexopoulos4, DimitriosDougenis4, Fabrice Jossinet2, Andreas Scorilas3 and ConstantinosStathopoulos1�1Department of Biochemistry, School of Medicine, University of Patras,Greece�2IBMC-CNRS, Architecture et Réactivité de l'ARN, Université deStrasbourg, Strasbourg, France�3Department of Biochemistry and MolecularBiology,UniversityofAthens,Greece�4DepartmentofCardiothoracicSurgery,SchoolofMedicine,UniversityHospitalofPatras,Patras,GreeceSeveral cancer types exhibit tRNAs overexpression, tRFs production andalteredmiRNAlevelsthatdrivecellproliferation. Inthepresentstudyweanalyzed the differential expression of tRNAs, tRFs, and miRNAs in lungcancerbiopsieswith similar classification in comparison tohealthy tissuesamples from the same patients. Analysis of miRNAs revealed amongothers,upregulationofmiR-210anddownregulationofmiR-126.AmodifiedprotocolforNGSwasusedtodiscirminatethetRNAsandtRFspoolsandthedatawereanalyzedusingtREFLtool.ThedifferentiallyexpressedtRNAsandtRFs were validated via RT-qPCR analysis. Interestingly, tRF-5003 issignificantlyupregulatedwhereas tRF-3021 isdownregulated in tumours.FilteringofdatausingCLASHtoolpredictedpossibletargetsforbothtRFsviaAGO-mediatedloadingandsilencing.EffectivetransfectionofA549cellline using siRNAs corresponding to both tRFs showed that tRF-3021effectively downregulates ARIH1, whereas tRF-5003 effectivelydownregulates PAQR4 thus, verifying the targets prediction. These areamong the fewexamples of tumor-derived tRFs that affect regulation ofessentialgenescontrollingtumorprogression.

204

P4.11

AnalysisoftRNAGeneCopyNumberVariationandCodonUsagePatternsin460FungalSpeciesForPredictiveModelingofHighlyExpressedGenesRhondeneWint1,2,AsafSalamov2,IgorV.Grigoriev2,31Department of Quantitative and Systems Biology, University of California-Merced, USA, 2 US Department of Energy Joint Genome Institute, USA, 3

DepartmentofPlantandMicrobialBiology,UniversityofCalifornia–Berkeley,USANumerousstudiesshowtRNAabundance,proxiedbytRNAgenecopynumber,asamajorcontributorofcodonusagebiaswhichhasbeenshowntocorrelatewith gene expression level (Ikemura 1982, Plotkin et al 2011). We havecharacterized the copy number variation of tRNA genes, obtained fromtRNAscan-SE(Loweetal2016),andthecodonusagepatternsinover6millionproteincodingsequences,acrossseveralhundredsoffungalgenomesthatweresequenced at the Joint Genome Institute for projects like the 1000 FungalGenomesProject,makingitthemostextensiveandcomprehensivestudyofitskindwithintheKingdomFungi.Wehaveanalyzedhowcodonusagecorrelateswith tRNA gene content between species. We have identified both highlyconservedandraretRNA.Finally,armedwiththegeneexpressiondataofeachgenome,weproposetodevelopapredictivemachinelearningbasedmodeltoidentify highly expressed genes basedon features of codonusage and tRNAgenecontent.

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P4.12Characterization of the Arabidopsis tRNAome in PatternTriggeredImmunityP.Zwack,G.Greene,X.Dong�Howard Hughes Medical Institute, Department of Biology, Duke University,Durham,NC,USAPreviousfindingshave indicatedthatplantsrapidlyaccumulatehigh levelsofbothchargedandunchargedformsofspecifictRNAsinresponsetopathogenrecognitionbycellsurfacelocalizedPatternRecognitionReceptors.Inaddition,wehaveidentifiedapatternofdifferentialcodonusageinpathogeninducedtranscripts. Recently, novel approaches applied in yeast and mammaliansystemshavesuccessfullyovercomeearlierproblemsassociatedwithNext-gensequencing of highly structured tRNAs. These advances presented theopportunitytoadaptmethodsinordertoperformthefirsteverhighresolutionanalysisofaplant“tRNAome”asameansoffurtherexploringthefunctionofdifferentialaccumulationofspecifictRNAsinplantimmuneresponses.Weareexamining the abundance and chargingof tRNAduring the induced immuneresponse in Arabidopsis through a combination of biochemical and next-generationsequencingtechniques.Thisisbeingdoneunderconditionsidenticaltogenome-widetranscriptionalandtranslationalstudiesfromourlaboratory,allowingforcomparativeanalysisofoutputfrommultiplegeneticmechanisms.Preliminary results suggest that changes in codon optimality resulting fromcoordinatedmodificationoftRNAsupply(isoacceptorabundance)anddemand(codonusage),mayfacilitaterapidglobalchangesingeneexpression.ThisworkisanticipatedtoprovideacomprehensiveanalysisofthedynamicsandfunctionoftRNAinrapidplantimmuneresponses.

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P5.1A tRNA half modulates translation as stress response inTrypanosomabrucei�R. Brogli1, R. Fricker1, H. Luidalepp1, M. Zywicki2, A. Schneider1, M.Cristodero1,andN.Polacek11DepartmentofChemistryandBiochemistry,UniversityofBern,Switzerland�2InstituteofMolecularBiologyandBiotechnology,AdamMickiewiczUniversity,PolandThe pathogenic parasite Trypanosoma brucei lacks extensive transcriptionalcontrol of protein coding genes and thus is crucially dependent on post-transcriptional mechanisms tomodulate gene expression. Despite this, howparasitesregulatetranslationhasbeensofarunexplored.Analysesofribosome-associated small ncRNAs allowed the identification of several life stagedependent and stress-induced tRNA-halves in T. brucei. One of the mostabundant ones, the tRNAThrAGU 3’ half is predominantly present duringnutritional stress. Interestingly, this half associates with ribosomes andpolysomes in vivo and stimulates protein synthesis during nutritional stressrecovery in vivo and in vitro. This stimulatory effect is conserved in otherorganisms, such as mammals and archaea. Depletion or blocking of theendogenous tRNA half showed a relaxation of the translation stimulation invitro and in vivo. Additionally, the tRNAThr half preferentially interacts withstressed ribosomes in ribosome binding studies. Our results suggest a novelbiologicalfunctionofatRNAfragment,namelyglobalstimulationoftranslationduringtherecoveryfromnutritionalstress.

209

P5.2

A functional genomics approach to investigate ArabidopsisnuclearRNasePinteractionsM.Bruggeman,A.Gobert,P.Giegé�Institutdebiologiemoléculairedesplantes,Strasbourg,FranceMaturation of the 5’ end of tRNA-precursors is catalysed by Ribonuclease P(RNaseP)inalldomainsoflife.RNaseP,whichcanbearibonucleoproteinsorProtein-onlyRNaseP (PRORP)enzymeshavebeen shown toplaya varieyofadditionalroles,includingthecleavageoftRNA-likestructures(TLS)inmRNA,in both prokaryotes and eukaryotes.�Using functional genomics, we aim tounravel Arabidopsis PRORP2 functional interactions at both the protein andRNAlevels.TransgeniclinesweregeneratedwithataggedversionofPRORP2toidentifybyco-immunoprecipitationbothitsproteinpartnersandRNAtargetsatthetranscriptomewidelevel.Additionaly,themRNAoftheRNApolymeraseIII transcriptional repressor MAF1 was identified as a putative target for aregulation mediated by PRORP2. In vitro assays showed that MAF1 mRNAprecursor is a PRORP2 substrate. Further work, using i.e. 5’ RACE and virusinduced gene silencing, is currently ongoing to investigate the effects andbiologicalsignificanceofthiscleavageinvivo.

210

P5.3

tRNAGln derived fragments mediate pancreatic β-cellsdysfunctionandapoptosisinTRMT10AdiabetesCristina Cosentino1, Sanna Toivonen1, Stéphane Demine1, AndreaSchiavo1,DécioL.Eizirik1,MiriamCnop1,2,MarianaIgoillo-Esteve11ULBCenter forDiabetesResearch,UniversitéLibredeBruxelles; 2DivisionofEndocrinology,ErasmusHospital,UniversitéLibredeBruxellesLoss-of-functionmutationsinTRMT10A,atRNAmethyltransferase,causeearlyonset diabetes and microcephaly. Here we investigated the molecularmechanisms underlying β-cell demise in TRMT10A deficiency.�iPSCs fromcontrolsandTRMT10Apatientsweresuccessfullydifferentiatedintoβ-likecells.TRMT10AexpressionwassilencedinEndoC-βH1cells.Inpatient-derivedβ-likecellsandTRMT10A-depletedEndoC-βH1cellsm1G

9wasreducedinasubsetofcytosolic tRNAs, including tRNAGln. Hypomethylation of tRNAGln resulted infragmentation with accumulation of 5’-tRNAGln fragments in patient-derivedcells. Transfection of TRMT10A-competent EndoC-βH1 cells with synthetictRNAGln fragments induced apoptosis. Conversely, transfection of antisenseoligonucleotidestargetingtRNAGln fragments inhibitβ-cellapoptosis inducedbyTRMT10A-deficiency.TRMT10Asilencing ledtomitochondrialdysfunction,increasedoxidativestressandactivationoftheintrinsicpathwayofapoptosis.These findings provide unequivocal evidence for the importance of tRNAmodificationsinhumanpancreaticβ-cellsandidentifytRNAhypomethylationandfragmentationasanovelmechanismofβ-celldemiseindiabetes.

211

P5.4

EffectsofthetRNAProhalfontranslationinmammaliancellsY.Gonskikh1,J.Grillari2,N.Polacek21DepartmentofChemistryandBiochemistry,UniversityofBern�2DepartmentofBiotechnology,UniversityofNaturalResourcesandLifeSciencesViennaRecentdatarevealedtRNA-derivedRNAasnewclassofncRNAsthatareableofregulating gene expression post- transcriptionally. The tRNAPro 5’ half wasdetectedinseveralmammaliancell lines.Northernblotanalysisrevealedthepresence of the tRNAPro 5’ half in the polysomal fractions. The ribosomeassociationwasconfirmedviafilterbindingassaysinvitro.AdditionofsynthetictRNAProhalfininvitrotranslationreactionshoweduniqueeffectsontranslationinseveralspeciesincludingyeast,CHO,HEK,rabbitreticulocytes,andHeLacells.Addition of the synthetic tRNAPro half inhibits global translation and causesupregulation of a specific translational product consisting of both RNA andaminoacids.TransfectionofthesynthetictRNAProhalf intoHeLacells leadtoformationofthesameproductinvivo.Themigrationoftheproductinacidicgels,insensitivitytocoppersulfateandto3’polyadenylation,andassociationwith80Smonosomessuggestthattheaccumulatedproductispeptidyl-tRNA.We speculate that binding of the tRNAPro 5’ half to the ribosome leads toribosomestalling,consequenttranslationinhibitionandformationofpeptidyltRNA.

212

P5.5

Theoretical and experimental investigation of tRNA-proteininteraction in Cytochrome C and tRNA nucleotidyltransferasemodelsA.Graczyk,R.Pawlowska,D.Jedrzejczyk,A.Chworos

CentreofMolecularandMacromolecularStudies,PolishAcademyofSciences,Lodz,PolandTheRNA-proteincommunicationisfundamentalformanybiologicalprocesses.RNA and proteins are critical in every biological system so the in-depthinvestigationoftheirinteractionisvitaltobroadenourknowledgeaboutthoseprocesses. Two pathways can be followed to study such arrangements.Experimental investigation of RNA-protein complexes with techniques likeelectrophoretic mobility shift assay (EMSA) or microscale thermophoresis(MST). Theexperimentally collecteddatahowever, still calls tobe refined inorder to reach a high resolution, which may be achieved following thetheoretical path - computational prediction of the patterns governing theformation of the RNA-protein complex. Among RNAs, the tRNA deservesparticular attention. It holds a highly conserved structure, serves as a coreelement in protein biosynthesis – translation and partake in apoptosis.Therefore,thecytochromeCandtRNAnucleotidyltransferase(TRNT)servedasproteinmodelsforinvestigationoftRNA-proteincomplexes.AsetofsynthetictRNAmimicswasintroducedtoeachoftheproteinmodelsfortheexperimentalanalysisandweresubjectedtothecomputationalcomplexanalysis.

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P5.6Angiogeninmodulates the levels of tRNA fragments in cardiacmicrovascularendothelialcellsLiapiE1,VerhesenW1,DerksK2,vanLeeuwenREW1,SchroenB11Department of Cardiology, Cardiovascular Research Institute Maastricht(CARIM), Maastricht University, The Netherlands 2Department of ClinicalGenetics Maastricht University Medical Centre+ | azM, Maastricht, TheNetherlands e.liapi@maastrichtuniversity.nlThe diabetes-induced hyperglycemic environment is an important contributor tomicrovascular endothelial dysfunction and the progression of heart failure withpreservedejectionfraction(HFpEF).HFpEFisadiseasewithouttreatmentoptions,anda better understanding of the underlyingmolecularmechanisms is urgently needed.Non-coding RNAs are important regulators of gene expression in the cardiovascularsystem, of which transfer RNA fragments (tRFs) comprise a novel group with greatpotential. Although the function and biogenesis of tRFs remains largely unknown,specificribonucleaseshavebeenassociatedwithtRFgeneration,includingAngiogenin(ANG).To identifycardiacdifferentiallyexpressedtRFs inHFpEFand investigatewhether tRFlevels are affected by knock-down of the ribonuclease enzyme Angiogenin (ANG) inHuman cardiac Microvascular Endothelial Cells (HMVECs).�Methods andResults�CardiacsmallRNA-seqreadsfrom20weeks-oldleanZSF1controlratsandobeseZSF1HFpEFratsweremappedtotheR.norvegicustRNAdatabaseusingan inhouse-designedprotocol,andprocessedtRNAsweredetected.HMVECswereexposedtobasaland high glucose to induce endothelial dysfunction and siRNA-mediated ANG knock-down.tRFlevelsweredeterminedviaNorthernblotinthesesamples.IntheobeseZSF1leftventricle,therewasasignificantinductionof~30nttRFs(tRNA-halves)comparedtotheleanZSF1LV.5’tRNA-halfG98*wasupregulatedbothintheobeseLVandinhigh-glucosetreatedHMVECsandconfirmedviaNorthernblot.ANGknock-downinHMVECsresulted in a profound alteration of the profile of tRFs in multiple tRNA anticodonfamilies.GlucotoxicstimulusaffectstheprocessingofspecifictRNAsintheLVofobeseZSF1ratsandinglucose-challengedcardiacHMVECs.ANGknock-downalteredtheprofilesoftRFsderiving frommultipletRNAs,suggestiveofan importantroleofANG indifferentiallyregulatingthecleavageofspecifictRNAtemplates.Ourcurrentinvestigationfocusesonthe effect of ANG knock-down and the functional role of 5’tRNA-half G98* inmicrovascularendothelialdysfunction.*arbitrarygenename;unpublisheddata

214

P5.7

Tetra-molecular5'tiRNAsinhibit48SribosomescanningthroughdirectinhibitionofeIF4FShawnM.Lyons1,2PavelIvanov1,2,PaulAnderson1,21DepartmentofMedicine,HarvardMedicalSchool,Boston,MA,USA,2Divisionof Rheumatology, Immunology and Allergy, Brigham andWomen’s Hospital,Boston,MA,USAIn response to stressful environments, cells react by generating small non-codingRNAsthatwehavetermedtRNA-derivedstress-inducedRNAs(tiRNA).ThesesmallRNAsfunctiontomodulatetranslationinanefforttoconserveandredirectcellular resourceswith theaimofpromotingsurvival.Weshowthattwo tiRNAs, derived from the 5’ halves of tRNAAla and tRNACys are potentinhibitors of translation (5’tiRNAAla/Cys). These tiRNAs are unique in that theycontain a conserved cis element called a terminal oligoguanine (TOG)motif,whichweshowisrequiredforactivity.Wefurthershowthatthismotif foldsintoparallelRNAG-quadruplex(G4)andthatthisstructure,ratherthansolelyTOGsequence, is required forbioactivity.Bypreventing the formationofG4through ionic equilibration or throughuse ofmodified nucleic acids,we canprevent the activity of TOG tiRNAs. Mechanistically, we show that thesetetramerictiRNAsdirectlyinteractwithandinactivatestheheterotrimericeIF4Fcomplex composed of eIF4E, eIF4G and eIF4A. Consistent with eIF4Finactivation, these tiRNAs inhibit 48S ribosome scanning to block translationinitiation.

215

P5.8

Characterization of aminoacyl-tRNA transferases related tobacterialMprFinAspergillusfumigatusNassiraMahmoudi1, Nathaniel Yakobov1, Hervé Roy2, Yusuke Saga3,TetsuoKushiro3,FrédéricFischer1,a,HubertBecker1,a1UniversitédeStrasbourg,CNRS,InstitutdeBotanique,GMGMUMR7156,28,rue Goethe, 67083 Strasbourg cedex, France 2Burnett School of BiomedicalSciences,CollegeofMedicine,UniversityofCentralFlorida,Orlando,FL32826,USA3GraduateSchoolofAgriculture,MeijiUniversity,1-1-1Higashimita,Tama-ku,Kawasaki,Kanagawa214-8571,Japan�aCorrespondingauthors:Prof.HubertBecker,h.becker@unistra.fr,Dr.FrédéricFISCHER,frfischer@unistra.frAspergillus fumigatus is the most common mold pathogen inimmunocompromised patients. Themortality, despite advances in diagnosisand treatment remains unacceptably high at 50- 80% of patient. Numerousvirulence determinants have been characterized in A. fumigates but otherremaintobeidentifiedinordertodecreasecomplicationsandmortality.Weidentified in fungi, and especially in A. fumigatus, two MprF-like (MultiplePeptidesResistanceFactor)proteinswithaDUF2156domain.Inbacteria,MprFproteins are aminoacyl-tRNA transferases that catalyse the transfer of lysinefromLys-tRNALysontomembranelipids.Aminoacylationoflipidsmodifiestheoverallchargeofthemembrane,whichincreasestheresistanceofbacteriatocationicantimicrobialpeptides.CharacterizationofthetwoMprF-likeproteinsinA.fumigatusshowsthatbothproteinsareinvolvedinthesynthesisofnovelaminoacylated lipids. Our preliminary results suggest that fungal DUF2156proteinsinfluencecentralcellularprocessessuchasvirulenceorsporulationinA.fumigatus.

216

P5.9

TransferRNA-derived fragments targetandregulateribosome-associatedaminoacyl-transferRNAsynthetases�AnnaM.Mleczko,PiotrCelichowski,KamillaBąkowska-Żywicka�InstituteofBioorganicChemistryPolishAcademyofSciences,NoskowskiegoSt.12/14,61-704Poznan,PolandRecently,anumberofribosome-associatednon-codingRNAs(rancRNAs)havebeen discovered in all domains of life. Our recent studies in Saccharomycescerevisiaehave shown thatprocessingof tRNAmolecules iswidespread in anumberofgrowthconditions.Moreover,wediscoveredthattRNAfragmentsbindtotheribosomesreveal inhibitoryactivityon invitrotranslation. In thispresent study, we functionally characterized the molecular activity of S.cerevisiae transfer RNA (tRNA)-derived fragments (tRFs) during proteinbiosynthesis.Ourresultsindicateribosome-associatedtRFsderivedfromboth5'(ranc-5'-tRFs)and3'-partoftRNAs(ranc-3'-tRFs)haveregulatoryrolesduringtranslation.Wedemonstratedfive3'-tRFsandone5'-tRFassociatewithasmallribosomal subunit and aminoacyl-tRNA synthetases (aa-RSs) in yeast.Furthermore,wediscoveredthatfouryeastaa-RSsinteractdirectlywithyeastribosomes. tRFs interactionswith ribosome-associated aa-RSs correlatewithimpairedefficiencyoftRNAaminoacylation.Thisworkwassupportedbygrants:POMOST/2011-4/1,2014/13/D/NZ1/00061,DEC-2017/27/B/NZ1/01416,KNOW.

217

P5.10

Alanine tRNAs translate environment into behavior inCaenorhabditiselegansDiana Andrea Fernandes De Abreu1, Thalia Salinas-Giegé2, LaurenceMaréchal-Drouard2,Jean-JacquesRemy11Genes,Environment,Plasticity,UMRCNRS7254, INRA1355,UniversitéNiceCôted’Azur,06903Sophia-Antipolis,France.Tel:33492386418,email: jean-jacques.remy@inra.fr2Institutdebiologiemoléculairedesplantes-CNRS,UniversitédeStrasbourg,F-67084Strasbourg,France.TheCaenorhabditiseleganswormsproduceandkeepimprintsoftheattractivechemosensorycuestowhichtheyareexposedearlyinlife.Imprintingenhancestransientlyorpermanentlychemoattractiontoearlyolfactorycues.We identified tRNAAla (UGC) as the unique olfactory imprinting molecule.Feeding naive unexposed worms on tRNAAla (UGC) from odor-exposedtransiently or stably enhance odor-specific responses, suggesting wormsproduceodor-specificformsoftRNAAla(UGC).DifferentmutationsinthetRNAmodifyingElongatorcomplexsub-units1or3geneseitherabolishchemo-attraction,orstablysuppressattractiontotheodorworms have been exposed to. Feeding Elongatormutants on tRNAAla (UGC)produced by wild-type unexposed worms however rescues both behavioralphenotypes.WeconcludethattRNAAla (UGC), throughtheElongatorcomplexactivity,cantranslatetheearlyolfactoryenvironmentandstablyreprogramtheC.eleganschemoattractivebehavior.

218

P5.11CharacterizationoftRNAIle(UAU)-likesmallRNAsinLactobacillusplantarum�C.Tomikawa1,S.Auxilien2,V.Guérineau3,K.Sakakibara1,G.Uesugi1,H.Hori1,D.Fourmy2,K.Takai1,S.Yoshizawa2�1Graduate School of Science and Engineering, EhimeUniversity,Matsuyama,Japan�2Institute for Integrative Biology of the Cell, CNRS, Gif-sur-Yvette,France�3Institut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette,FranceInmosteubacteria,asingletRNAIle(CAU)(whereC34ismodifiedtolysidine)isfoundfordecodingtherareAUAisoleucinecodoninsteadofatRNAIle(UAU),exceptforafewcasessuchasMycoplasmamobile.TilSenzymethatcatalyzeslysidine34 formation is present in all eubacteria lacking the tRNAIle (UAU)withoutanyexception.However,Lactobacillusplantarumhasgenesnotonlyfor tRNAIle (CAU) and TilS, but also for tRNAIle (UAU)-like small RNAs. If thetRNAIle(UAU)-likeRNAsareinvolvedindecoding,itmaycausesomeambiguityinthegeneticcodesincethetRNAcandecodeAUGmethioninecodonaswellasAUAisoleucinecodon.InordertoelucidatethefunctionalroleofthetRNAIle(UAU)-likeRNA in L.plantarum,wecharacterized the smallRNA.The tRNAIle(UAU)-likeRNAwasnotchargedwithisoleucinebyisoleucyl-tRNAsynthetase.Also, enzymatic probing results obtained on native tRNAIle (UAU)-like RNAsuggestthattheRNAadoptsastructurelessstablethantheonefoundcanonicaltRNAs.Theresults suggest the tRNAIle (UAU)-likeRNAdoesnot functionasacanonicalisoleucinetRNA.

219

P5.12Global identification of tRNA cleavage fragments in buddingyeastL.K.White,P.D.Cherry,S.E.Peach,J.R.Hesselberth�UniversityofColoradoAnschutzMedicalCampus,Aurora,Colorado,USASinceS.cerevisiaelacksRNAimachinery,thepoolofsmallRNAsinthismodeleukaryoteislimitedinitscomplexity.Small(18-70nt)RNAsequencingrevealeda population of RNAs predominantly comprised of tRNA halves and rRNAfragments(Drinnenbergetal.2008;Thompsonetal.2008).Wetookadvantageof this limited repertoire of small RNAs to examine tRNA cleavage andprocessinginS.cerevisiaecellsthatlack5́→3́RNAdecay.ThetRNAligaseTrl1is required for tRNA splicing and essential for growth but can be geneticallybypassedbyexpressionof "pre-spliced tRNAs." The5-́kinasedomainof Trl1phosphorylatesthe5́endof5́-OHRNAsubstrates,makingthemcompetentforligationor5́→3́decaybyXrn1.Wereasonedthatbypassedtrl1∆cellswouldaccumulateRNAproductsofendonucleasecleavageandthe5́-endsofthesefragmentswouldberesistanttoXrn1-mediateddecay.WeisolatedsmallRNAsfrom trl1∆ and wild-type cells and end-repaired these molecules to enabledetectionofendonuclease-cleavedRNAswith5́-OHand2́,3-́cyclicphosphateends.Asexpected,cells lackingTRL1accumulatetRNAsplicing intermediatesfromSENcleavage,including5-́and3́-exonsofintron-containingtRNAs,aswellas liberated tRNA introns. In addition, we identify other signatures ofendonuclease cleavage of tRNAs, including 5́- and 3́- tRNA half moleculesconsistent with Rny1 cleavage and a handful of smaller tRNA-derived RNAfragments (tRFs). Together, these data provide a comprehensive picture oftRNAprocessinginS.cerevisiaeandrevealpreviouslyuncharacterizedsitesofendonucleasecleavage.

222

P6.1

Atissue-specificexonskippingeventseparatesthecatalyticandalternativefunctionsofhumanleucyl-tRNAsynthetaseMaxwellBaymiller,ConnorForsyth,BenjaminNordick,andSusanA.MartinisDepartment of Biochemistry, University of Illinois at Urbana-Champaign,Urbana,USAThe aminoacyl-tRNA synthetases (AARS) set the genetic code by attachingamino acids to their cognate tRNAs. Throughout their lengthy evolutionaryhistory,theAARShavealsodevelopedmanyfunctionsbeyondaminoacylation.For example, human cytoplasmic leucyl-tRNA synthetase (LARS) has beenadapted as an intracellular leucine sensor for the mammalian target of therapamycincomplex1(mTORC1)masterregulatorypathway.Herewedescribeanalternatively-splicedtranscriptvariantofhumanLARSwhichlacksanexoncodingforkeyenzymaticresiduesintheaminoacylationactivesite.ExpressionofthisLARSsplicevariant(LSV1)iselevatedspecificallyinleukocytes,whereitcomprises greater than20%of LARS gene transcripts. In human cell culture,LSV1overexpressionresultsinastableproteinproduct.ThisLSV1proteinalsoincreases leucine-dependentmTORC1activitywithapotencycomparabletothat of full length LARS. These data indicate that LSV1may have evolved todecouple the canonical aminoacylation and alternative leucine-sensingfunctions of human LARS, and that this distinction is important in leukocytebiology.

223

P6.2

Aminoacyl-tRNA Synthetases: Investigations of Specificity forApplicationinProxiMAX/SyntheticBiologyM.M.FerreiraAmaral1,L.Frigotto2,C.G.Ullman2,G.Hermans2andA.V.Hine11School of Life and Health Sciences, Aston University, Aston Triangle,Birmingham,B47ET,UnitedKingdom2IsogenicaLtd,TheMansion,ChesterfordResearchPark,LittleChesterford,Essex,CB101XL,UnitedKingdom‘ProxiMAX’ randomisation technology (ColibraTM) is a defined saturationmutagenesisprocessthatdeliversprecisioncontrolofidentityandrelativeratioofaminoacids (AAs)at specified locationswithinaprotein library.Thisnon-degenerateprocessallowsencodingofall20oranydesiredsubsetofAAswithease,withoutconstraintsimposedbythegeneticcode.Usingamaximumof20codonsinsaturatedpositionsmeansmanycodonsremainavailabletoencodeadditional,unnaturalaminoacids(UAAs).IncorporationofUAAsisparticularlyrelevant in expanding engineered protein repertoires. Ultimately,we aim tocombineProxiMAXwithaninvitrotranscription/translationsystemtodesignand express synthetic protein libraries containing multiple UAAssimultaneously. We have employed ProxiMAX randomisation to encode 18naturalAAsinvarious,putativeAA-bindinglocationsofanE.colialanyl-tRNAsynthetase.Our poster describes the synthesis of two variant libraries, eachencoding >107 novel proteins, their composition, quality and our progresstowardsscreeninganddeconvolutionofthelibrariestodiscovernovelenzymes,withaninitialfocusonspecificityforD-AA.

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P6.3Contributiontothecharacterizationofthehumanmitochondrialarginyl-tRNAsynthetase:WhatweknowandwhatisnewLigiaElenaGonzalez-Serrano1,LoukmaneKarim1,FlorianPierre1,MarieSissler11UniversitédeStrasbourg,CentreNationaldelaRechercheScientifique(CNRS),Architecture et Réactivité de l’ARN, Strasbourg, France�E-mail:e.gonzalez@ibmc-cnrs.unistra.frHumanmitochondrialaminoacyl-tRNAsynthetases(mt-aaRSs)arekeyenzymesof the mitochondrial translation. Mutations in their nuclear genes arecorrelated with pathologies with a broad spectrum of clinical phenotypes.Despite being ubiquitously expressed, mutations on mt-aaRSs show aconvergenceofinjuriesonthecentralnervoussystem.Asremarkableexamples,mutations in mt-AspRS are correlated with a “mild” neuro-degenerativeleukodystrophy,whilemutationsinthemt-ArgRSarecorrelatedwithasevereneuro-developmental pontocerebellar hypoplasia. Previous investigationsperformed in the laboratory contributed to the characterization of the mt-AspRS.Inthisstudy,wefocusedonthedeterminationofcellularpropertiesofthemt-ArgRSanditsfunctionalcharacterization.Wefurtherinvestigatedtheimpactofaseriesofdisease-associatedmutationsontwomainpropertiesofmtArgRS:the in cellulo sub-mitochondrial localization and the in vitro aminoacylationactivity of this enzyme. Combined with previous works, the present resultsexpandtheknowledgeofthemt-aaRSs,sheddingnewlightonthelinkbetweenmt-aaRSs-mutationsanddisease.

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P6.4IleRSeditingprimarilytargetsnorvalinewhosemisincorporationismoretoxicthanofvalineM.Bilusa,M.Semanjskib,M.Mociboba,A.Toth-Petroczyc,B.Macekb,I.Gruic-SovuljaaDepartment of Chemistry, Faculty of Science, University of Zagreb, Zagreb,Croatia bProteome Center Tuebingen, University of Tuebingen, Tuebingen,Germany�cMax Planck Institute of Molecular Cell Biology and Genetics,Dresden,GermanyTranslationalerrorsmayinducelossofproteinstabilityandfunction.Theyarepartlypreventedbytheeditingactivityofaminoacyl-tRNAsynthetases(aaRSs).Norvaline(Nva)andvalinearetwo isoleucinesurrogateswith identicalmass:Val is a proteinogenic beta-branched amino acid, while the linear Nvasporadicallyaccumulatesincells,butisnotnormallyincorporatedtoproteins.WeshowedthatNvaandValareequallygoodnon-cognatesubstratesofthedeacylation-defective IleRS, both in vitro and in vivo. Surprisingly, however,IleRS distinguishes between Nva and Val at the CP1 editing domain thathydrolysesNva-tRNAIlefasterthanVal-tRNAIle.Wefurtherdemonstratedthatincorporation of Nva into proteome is more deleterious, although Nva ismistranslatedwithsimilarfrequencytoValandlargelyatthesameIlepositions.ComparisonofNva-tRNAeditingbyIleRS,LeuRSandValRSsuggestsNvaeditingwaspresentintheircommonancestor,priortoitsduplicationanddivergenceto three specialized enzymes. This raises an intriguing hypothesis thateliminationofNva,whichmayhadbeenpresentinprimordialproteins,likelydrovetheacquisitionoftheCP1editingdomain.

226

P6.5

Intracellular leucine sensingmechanismcoordinatedby leucyl-tRNAsynthetaseMinjiLee1,JongHyunKim2,InaYoon2,HeeChanYoo3,JeongHwaLee3,SunghoonKim2,JungMinHan1,31Department of IntegratedOMICS for Biomedical Science, Yonsei University,SouthKorea,2MedicinalBioconvergenceReserchCenter,CollegeofPharmacy,SeoulNationalUniversity,SouthKorea,3CollegeofPharmacy,YonseiUniversity,SouthKoreaLeucyl-tRNAsynthetase(LRS),anenzymeinvolvedinproteinsynthesis,servesas an intracellular leucine sensor formTORC1 signaling pathway, which is acentraleffectorforproteinsynthesis,metabolism,autophagy,andcellgrowth.The Rag GTPase cycle mediates leucine signaling to mTORC1. In thispresentation, we would like to introduce our current understanding ofintracellularleucinesensingmechanismcontrolledbyleucyl-tRNAsynthetase.HereweshowthedynamicsofRagGTPasecycleduringleucinesignalingandthat LRS serves as an initiating “ON” switch viaGTP hydrolysis of RagD thatdrives the entire RagGTPase cycle in theRagGTPase-mTORC1 axis. The LRS-RagDaxisshowedapositivecorrelationwithmTORC1activityincancercellsandtumortissues.Inaddition,weclarifytheroleofanotherleucinebindingprotein,Sestrin2,intheRagGTPase-mTORC1axis.ThisstudysuggeststhattheGTPasecycleofRagD/RagBcoordinatedbyLRSandSestrin2 is critical for controllingmTORC1activation,andthuswillextendthecurrentviewofaminoacidsensingbymTORC1andwillbeinvaluableforthedevelopmentofnovelapproachestocombatmTORC1-relatedhumandiseases.

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P6.6Membraneaminoacyl-tRNAsynthetasesandsignalingpathwaysMarineHemmerle,HubertD.Becker�InstitutdeBotanique,Strasbourg,FranceAminoacyl-tRNA synthetases (aaRS) are ubiquitous enzymes, whichconventionalroleistoformaminoacyl-tRNA.Despitetheirwell-knownroleinprotein synthesis, studies have highlighted their implication in many othercellular processes including apoptosis and autophagy.�In the yeastSaccharomyces cerevesiae the methionyl-tRNA synthetase (MRS) and theglutamyl-tRNA synthetase (ERS) are found in a complex together with theproteinArc1.InsidethiscomplextheproteinArc1actsasacytosolicanchorandanaminoacylationcofactorforthetwoaaRS.EvenifArc1wasshowntobeessentiallycytosolic,studieshaverevealedthatitalso interactswith lipids such as phosphoinositides (PIPs).UsingmembranescoatedwithPIPs,subcellularfractionationexperimentsandanewfluorescenttool developed by our lab, we confirmed the interaction of Arc1 with PIPspresent in the vacuolar membrane. Moreover, these preliminary resultsindicate that the vacuolar Arc1 may trigger anchoring of two aaRS at thevacuolarmembranetoo.ThephysiologicalconditionsthatprovokethisvacuolarrelocationofArc1 and the role of vacuolarArc1 and theof twomembrane-boundaaRSswillbediscussed.

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P6.7Functional analysis of plant double-length tyrosyl-tRNAsynthetasefromArabidopsisthaliana.TetsuoKushiro1,YoheiToyoda1,YusukeSaga1,ShunsukeTatematsu1,MikiWada2,KeiEndo2,KoichiIto21Meiji University, School of Agriculture, Kawasaki, Japan�2The University ofTokyo,DepartmentofComputationalBiologyandMedicalSciences,Kashiwa,Japan�kushiro@meiji.ac.jpDespite large numbers of non-canonical functions of aminoacyl-tRNA synthetases(aaRSs)beingdiscoveredinmammals,thoseinplantsandfungiarelargelyunexplored.DuringourattempttoidentifythefunctionofplantaaRSs,wenoticedapresenceofadouble-length tyrosyl-tRNA synthetase (TyrRS) gene in the model plant Arabidopsisthaliana.SuchaTyrRSwasfoundonly in limitedplantsandweremorebroadlyfoundamong trypanosomatids. However, unlike those from trypanosomatids, both the N-terminalhalffulllenth(designatedTyrRS-1N)andtheC-terminalhalffulllength(TyrRS-2C)containedtheclassIconservedmotifsandwereseemedactive.FunctionalanalysisusingyeastcomplementaryassayshowedthatTyrRS-1NitselfwasinactivewhileTyrRS-2C itself was active. Trypanosomatids double-length TyrRS was known to form anintramolecularpseudo-dimeraccomodatingonemoleculeoftRNAacrossthepseudo-dimerinterface.InordertoanalyzethesimilarityanddiscrepancyofA.thalianaTyrRSfunction compared to trypanosomatids, mutations at active site residues wereintroducedintoTyrRS-1NandTyrRS-2Cportionsofthefulllength.Intriguingly,mutationsin already inactive TyrRS-1N portion resulted in inactivation of the full length TyrRS.Therefore,thesemutatedresiduesweresuspectedtosomehowparticipateinapseudo-dimer formation. By comparing the sequence of TyrRS-1N with TyrRS-2C and otherTyrRSs, we noticed several residues that were highly conserved among TyrRSs weremissing in TyrRS-1N. Although an attempt to restore TyrRS activity in TyrRS-1N bymutating back these residues have failed, the catalytic domain of TyrRS-1N seemedoriginally inactive while the anticodon binding domain was speculated to be active.Therefore,thetRNAsubstratemostlikelybindstothecatalyticdomainofTyrRS-2CandtheanticodonbindingdomainofTyrRS-1Nacrossthepseudo-dimerinterface.

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P6.8AsparginyltRNAsynthetase(NRS) isanovelregulator inhipposignaling�Kyu-SunLee,EunbyulYeonandKweonYu�Metabolism&Neurophysiologylab,KoreaResearchInstituteofBiotechnology&Bioscience,Daejeon,34141,SouthKoreaAnaminoacyltRNAsynthetase(ARS)isakeyenzymethatplaysanimportantrole in protein translation. Recent studies suggest that the non-canonicalfunction of ARSs is involved in various pathological mechanisms includingcancer, autoimmune disorders, and neurological disorders. However, themolecular mechanisms of ARSs in cancer are largely unknown. Here, weestablished Drosophila carcinomamodel by overexpressing of yorkie (yki), awell-knownoncogenicproteininHipposignalingpathway.Undergenome-wideRNAiscreeningofallDrosophilaARSs,weidentifiedasparginyltRNAsynthetase(NRS)as theoneofcancermodifiergene.Yki-inducedtumorphenotypewassignificantly rescued by knockdown of NRS in the adult eye. Furthermore,knockdownofNRSstronglysuppressedthemitoticindex(phosphor-H3)ofyki-induced tumor in the larval eye disc. We also confirmed that increasedphosphorylationactivityofAktandS6kcausedbyykioverexpressionwerealsoreducedbyNRSknockdown.Interestingly,NRSinhibitoralsosuppressedtumorphenotype. Thus, we hypothesize that NRS is a novel regulator in hipposignaling. Based on genetic and biochemical analysis, we showed that NRSmodulates hippo singlaing through interacting with Salvador, a bindingsubstrateofhippokinase.Takentogether,theseresultsprovideapossibilityfordevelopingananti-cancerdrugusingNRSasanoveltarget.

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Abiogenicevolutionofaminoacyl-tRNAsynthetaseKozoNaganoNaganoResearchInstituteofMolecularBiology,4-8-24Higiriyama,Kohnan-ku,Yokohama233-0015,JapanTheoriginof lifeonearthdemandsaprerequisiteforabiogenicevolutionofRNAwhich isbelievedtobehighlydestructiveontheearthcoveredbyboilingocean.Ihaveproposedamechanism of evolution of RNA as a left-handed single-stranded lattice with a trend ofcondensationanddehydrationoforganiccompoundsunderaconditionofhighpressureandhigh temperature in the mantle of the earth. All organic compounds and energy-richpolyphosphates were synthesized from a large amount of methane hydrate, inorganicphosphateandoxygenradicalsthatwereproducedfromferrousoxidesbydisintegrationofuranium(1).Thesixfoldsymmetricalstructureofthelatticeallowedtostackalotofenergy-richcompoundssuchasadenosinetriphosphateandphospho-lipidswhichwererequiredforformationof cellmembranereat the later stage (1).More importantly, the cavity formedinside the lattice allowed polymerization of small L-amino acids such as glycine, alanine,proline and serine that were required to protect RNAs from hydrolysis (1). The 6.5 foldsymmetricallatticeallowedalittlemorelargerL-aminoacidstoincorporatetheprotectiveproteins and formation of α–helices, but insertion of larger amino acids such as arginine,methionine, phenylalanine and tryptophanwere rather difficult (1). However, in the nextstage of evolution of the lattice formation of left-handed single strandedRNA, the 8 foldsymmetriclatticeseemstoincorporateargininesandalsothedimericrelationshipbetweenthe two neighbor strands suggests a possibility of aminoacyl-tRNA synthetase to evolvebecause some of the aminoacyl–tRNA synthetases were found to be in dimeric form.ExtensionofprotectingL-aminoacidpolymeratthedimericshapestronglysuggestsevolutionofaminoacyl-tRNAsynthetase.Moreover,formationofsmalltRNAseemspossibleunderthefunctionofaminoacyl-adenylateintheprocessofevolutionofthesynthetasesinitsdimericform.TheaminoacylgroupofthetRNAatthedimericform,whichbehavesasAsitetRNA,can be transferred to the neighbouring tRNA,which can be supposed to be P site tRNA.However,existenceofEsitetRNAcannotbeconfirmedinthisstageofevolution.References

1.Nagano,K.(2017)Integr.Mol.Med.4(3):2-22.

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P6.10RegulationoftranslationalfidelityinSalmonellaTyphimuriumRebeccaSteinerandMichaelIbba�OhioStateUniversityTranslationisanessentialcellularprocessthatinvolvesintricatequalitycontrolmechanisms to ensure fidelity. Aminoacyl tRNA synthetases function duringtranslation to ligate tRNA to the cognate amino acid. Phenylalanyl tRNAsynthetase (PheRS) has a proofreading mechanism to preventmisaminoacylationofnon-cognateaminoacids to tRNA.Oxidative stresshasbeenshowntoalterqualitycontrolofthreonyltRNAsynthetase. Inaddition,treating E. coliwith hydrogenperoxide (H2O2) alters the amino acid pool byoxidizing phenylalanine into tyrosine isomers, increasing availability of non-cognate amino acids. Due to increased non-cognate availability, PheRSmustmaintain accuracy during oxidative stress; however, how PheRS changesenzymatically and structurally has yet to be elucidated. Upon treatment ofPheRS with H2O2, we observed differential oxidation patterns betweensubunits,pairedwithsecondarystructuralchanges.Interestingly,H2O2didnotimpactthecognateaminoacidaminoacylationactivity,but leadto increasedproofreadingactivity.Theseresultssuggestapossiblemechanismforthecellto

protectitselffromaminoacidpoolchangesduringoxidativestress.

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Lysyl-tRNA synthetase producing second messenger Ap4A totrigger polymerization of tumor suppressor HINT1 for allergyresponseJingYu1, JieZhang1,YuanyuanLiang1,ZaizhouLiu1,AlexMotzik2,FengWu3,MengmengZheng1,JingwuKang1,PengfeiFang1,CongLiu3,EhudRazin2,MinGuo1,JingWang11StateKeyLaboratoryofBioorganicandNaturalProductsChemistry,ShanghaiInstituteofOrganicChemistry,ChineseAcademyofSciences,345LinglingRoad,Shanghai200032,China2DepartmentofBiochemistryandMolecularBiology,Institute for Medical Research Israel–Canada, Hebrew University MedicalSchool, Jerusalem91120, Israel�3InterdisciplinaryResearchCenteronBiologyandChemistry,Shanghai InstituteofOrganicChemistry,ChineseAcademicofScience,Shanghai.China�E-mail:Jwang@sioc.ac.cnLysyl-tRNAsynthetase(LysRS)istheessentialenzymethatacylatestRNALyswithamino acid lysine, and also the major generator of second messengerdiadenosine tetraphosphates (Ap4A). Ap4A is a universal pleiotropic signalingmoleculeneededforvariouscellregulationpathways.Duringallergicmastcellactivation,LysRSisphosphorylatedonSer207andboostitsactivitytocondenseATPwithLys-AMPtoformAp4A.Accordingly,thecellularconcentrationofAp4Aincreasesupto3-10foldin15minanddropsbacktobasallevelin60min.Ap4Abinds the tumor suppressor HINT1 directly, and disrupts the interactionbetween HINT1 and transcription factor MITF, thus releasing MITF for thetargeted genes transcription essential for immune response. Using crystalstructures,emissionmicroscopyanalyses,andmultiplebiochemicalassays,wefoundthat,insteadofinducingallostericconformationalchangetopropagatesignalslikepreviousreportedsecondmessengers,Ap4AtriggersHINT1toformpolymerbothinvitroandinmastcellinconcentration-dependentmanner.Inaddition,thepolymerizationofHINT1blocktheinteractingsurfaceforMITFandrelease MITF transcriptional accordingly. These findings reveal a novelregulating mechanism of LysRS on gene transcription in allergy response,throughthetetheringmannerperformedbyitsnoncanonicalproduct-secondmessengerAp4A.

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P7.1FunctionalandstructuralstudyofPlasmodiumtRNAimportmachineryMarta Cela1, Renaud Geslain2, José Jaramillo1, Delphine Kapps1, BernardLorber1, Joëlle Rudinger-Thirion1, Claude Sauter1, Anne Théobald-Dietrich1,andMagaliFrugier11ArchitectureandReactivityofRNA,UniversityofStrasbourg,CNRS, InstituteforMolecularandCellularBiology,67084StrasbourgCedex,France2LaboratoryoftRNABiology,DepartmentofBiology,CollegeofCharleston,USAMalariacontinuestobeaglobalburdenforhealth,with445,000deathsand216millioncasesin2016(1).Ourresultsbringanunprecedentedobservation:mosquitoessalivaryglandsporozoitesimportexogenoushosttRNAs.ThistRNAtraffickingismadepossiblebyauniquesurfaceprotein,namedtRip(transferRNAimportprotein).TheproteintRipmediatestRNAentranceintheparasite,andthismechanismiscrucialforinfectionbyapresently unknownmechanism (2). So far, we established that (i) in vitro tRip bindstRNAswithhighaffinityandrecognizestheelbowofthetRNAmoleculethroughitsC-terminaldomain.(ii)InvivoimmunolocalizationexperimentsfoundtRipinboththeliverandthebloodstagesofPlasmodiuminthemammalianhost,aswellasinthemosquitovector.(iii)tRipislocatedatthesurfaceoftheparasite,itstRNAbindingdomainbeingexposed to the outside. (iv) In vitro, exogenous tRNAs enter rapidly live sporozoites.Proteinbiosynthesisisreducedinaknock-outparasite,deletedfortheTRIPgene(tRip-KO) and its infectivity is diminishedat theblood stage as compared to thewild-typeparasite.Moreover,thegene-encodingtRipisspecificforApicomplexaparasites,suchasPlasmodium,Toxoplasma,andCryptosporidium.HowaretRNAsimportedintoPlasmodium?Whatistheirroleintheinfectiousprocessof the parasite?We provide a functional and structural description of this parasite-specific import process by investigating recognition between tRip and host tRNAs,comparativeproteomicsbetweenwildtypeandtRip-KOparasites,identificationoftRippartnersandstructureoftRip,tRip/tRNAcomplexandthecomplexformedbetweentRipanditsPlasmodiumproteinpartners.1.WorldMalariaReport20172.BourT.,etal.(2016)Apicomplexa-specifictRipfacilitatesimportofexogenoustRNAsintomalariaparasites.ProcNatlAcadSciU.S.A.113:4717-4722.

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P7.2

ContributionofthetRNAIle4317A>Gmutationtothephenotypicmanifestation of the deafness-associated mitochondrial 12SrRNA1555A>GmutationFeilongMeng1,2,ZheyunHe3,XiaowenTang3,JingZheng1,2,XiaofenJin2,XiaoyanRen3,MiZhou1,2,MengWang1,2,Min-XinGuan1,2,3*1DivsionofMedicalGeneticsandGenomics,TheChildren'sHospital,ZhejiangUniversity School of Medicine, Hangzhou, China�2Institute of Genetics,Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 3AttardiInstitute of Mitochondrial Biomedicine, School of Life Sciences, WenzhouMedicalUniversity,Wenzhou,Zhejiang,ChinaThe 1555A>G mutation in mitochondrial 12S rRNA has been associated withaminoglycoside-induced and nonsyndromic deafness in many individuals worldwide.Mitochondrialgeneticmodifiersareproposedtoinfluencethephenotypicexpressionofm.1555A>Gmutation.Here,wereportthatadeafness-susceptibilityallele(m.4317A>G)in the tRNAIle gene modulates the phenotype expression of m.1555A>G mutation.Strikingly, a large Han Chinese pedigree carrying both m.4317A>G and m.1555A>Gmutations exhibited much higher penetrance of deafness than those carrying onlym.1555A>Gmutation.Them.4317A>Gmutationaffectedahighlyconservedadenineatposition59 in theT-loopof tRNAIle.Wethereforehypothesized that them.4317A>GmutationaltersbothstructureandfunctionoftRNAIle.Usinglymphoblastoidcelllinesderived from members of Chinese families (three carrying both m.1555A>G andm.4317A>Gmutations,threeharboringonlym.1555A>Gmutation,andthreecontrolslacking thesemutations), we found that the cell lines bearing bothm.4317A>G andm.1555A>Gmutations exhibitedmore severemitochondrial dysfunctions than thosecarrying only m.1555A>G mutation. We also found that the m.4317A>G mutationperturbedtheconformation,stability,andaminoacylationefficiencyoftRNAIle.Thesem.4317A>Gmutation-inducedalterationsintRNAIlestructureandfunctionaggravatedthe defective mitochondrial translation and respiratory phenotypes associated withm.1555A>G mutation. Furthermore, mutant cell lines bearing both m.4317A>G andm.1555A>GmutationsexhibitedgreaterreductionsinthemitochondrialATPlevelsandmembranepotentialsandincreasingproductionofreactiveoxygenspeciesthanthosecarrying only m.1555A>G mutation. Our findings provide new insights into thepathophysiology of maternally inherited deafness arising from the synergy betweenmitochondrial12SrRNAandtRNAmutations.

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P7.3

Mutations in H. sapiens tRNase ZL(ELAC2) associated withhypertrophiccardiomyopathyimpairmitochondrialtRNA3’-endprocessing�LouisLevinger,MakenzieSaoura,Kyla-GayePinnock,MariaPujantell-Graell,BenjaminLucasYorkCollege/CUNY,Jamaica,NY11451,USAChristopherPowell,PedroRebelo-Guiomar,MichalMinczukMRCMitochondrialBiologyUnit,UniversityofCambridge,CambridgeCB20XY,UKDysfunctionofmitochondrialgeneexpression,causedbymutations ineitherthemitochondrial or nuclear genomes, is associatedwith adiverse groupofhuman disorders characterized by impaired mitochondrial respiration. AnincreasingnumberofmutationshavebeenidentifiedinnucleargenesinvolvedinmitochondrialRNAmetabolism.Withinthisgrouparepathogenicmutationswhich have been identified in the genes encoding enzymes involved in theprecursortranscriptprocessing, includingELAC2codingforthemitochondrialtRNaseZL.Here,wereporttheidentificationofsixteennovelELAC2variantsinindividuals presenting with mitochondrial respiratory chain deficiency,hypertrophic cardiomyopathy and lactic acidosis. First, we provide furtherevidence for the pathogenicity of the three previously reported variants(p.Phe154Leu,p.Leu423Pheandp.Thr520Ile)bystudyingthetRNaseZactivityinaninvitrosystem.Next,weappliedthisrecombinantsystemtoinvestigateallnovelmissensevariants,confirmingthepathogenicroleofthesenewELAC2mutations. The residues in which HCM-associated substitutions were foundwereeffectivelymodelledinsolvedtRNaseZstructures,providinginsightintoenzymestructureandfunction.Finally,weshowthatprimaryfibroblastsfromtheindividualswithnovelELAC2variantshaveelevatedlevelsofunprocessedmitochondrialRNAprecursors.Ourstudythusbroadlyconfirmsacorrelationofgenotype/phenotypeforvariantsinELAC2andsuggeststhevalueofscreeningfor mutations in the ELAC2 gene in severe infantile-onset of hypertrophiccardiomyopathyandmitochondrialrespiratorychaindysfunction.

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P7.4

Fine-tuning of protein synthesis by tRNA 2’-O-methylation isrequiredforlearningandmemory�YuNagayoshi1, Fan-YanWei1, Takeshi1 Chujo1, Suzuki Takeo2, SuzukiTsutomu2andKazuhitoTomizawa11Department of Molecular Physiology, Faculty of Life Science, KumamotoUniversity�2DepartmentofChemistry&BiotechnologyFacultyofEngineering/SchoolofEngineering,TheUniversityofTokyo�FTSJ1isatRNAmodificationenzymefor2’-O-methylationofcytosolictRNAsatpositions32and34.PathologicalmutationsinFTSJ1genehavebeenlinkedtothenonsyndromicX-linkedmentalretardation,buttheprecisemechanismhasbeenunclear.WehavegeneratedFtsj1knockout(KO)miceandinvestigatedthepathophysiologicalroles.DeficiencyofFtsj1resultedinhypomodificationofasubset of tRNAs, leading to the translational stalling in the ribosome.Consequently,theoverallproteinsynthesiswassignificantlyreducedinFtsj1KObrain. The aberrant protein synthesis led to the electrophysiological andmorphological abnormalities in both cortical and hippocampal neurons.Importantly,thespatialmemoryandfearmemorywere impaired inFtsj1KOmice,whichmirrorsthesymptomsofhumanpatients.Takentogether, theseresultsdemonstratethatFtsj1-medicatedtRNAmodificationisessentialforthefine-tuningofproteinsynthesis,anddysregulationofthisprocesscancausethedevelopmentofneurologicaldisease.

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P7.5

RNA modification plays a role in resistance tochemotherapeutics�Priyanka Nair1, JohnanesWagner1, DaphnaMokady2, Priyanka Vaz2,RobRottapel2&ZoyaIgnatova11Institute of Biochemistry and Molecular Biology, University of Hamburg,Hamburg,Germany.2PrincessMargaretCancerCentre,UniversityofToronto,CanadaOvariancancerinwomenisamongcancerswithlowfive-yearsurvivalrate.Sofar,thetreatmentisbasedonoxidativealterationsofthegrowthanddivisionofcancercellswithchemotherapeuticdrugs,likecisplatinandcisplatin-relatedcompounds.However,developmentofcisplatinresistanceiscurrentlyamajorchallengeleadingtorecurrenceofthedisease.Theunderlyingmechanismsofcisplatinresistanceareelusiveandelucidatingthemwillfosterthedevelopmentof new approaches to overcome resistance.We compared ovarian cell lineswhichspanthespectrumfromcisplatin-susceptibletocisplatin-resistant.UsingtRNA-microarray-based technology, we observed alterations in charging ofsome tRNA isoacceptor families following cisplatin treatment. WecomplementedthisdatawithRibo-Seq(orribosomeprofiling)andRNA-Seqandelucidatedderegulationsinamino-acidmetabolism.GuidedbyourobservationfromthedeepsequencinganalysisondisbalanceintheSAMsynthesis inthecisplatin-resistantcells,wedeterminedtheRNAmethylationpatternusingm6A-specificantibodies. Interestingly,followingcisplatintreatmentweobservedastrong persistence on them6A-modification pattern in the cisplatin-resistantcells contrasting the lossofm6Amodifications in the cisplatin-sensitive cells.Ourresultssuggestthatmetabolicactivitieslinkedtom6AmodificationofRNAsplayaroleincisplatin-resistance.

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P7.6

Modulationof translationefficiency: anewplayer indendriticcellfunctionMarisaReverendo1,PhilippePierre1,21CNRS,INSERM,CIML,AixMarseilleUniversity,Marseille,France�2InstituteforResearch in Biomedicine - iBiMED and Aveiro Health Sciences Program,UniversityofAveiro,Aveiro,PortugalDendritic cells (DC) are immune cells with the capacity to initiate immuneresponsesthoughthepresentationofantigenstonaïveTcells.DCschangetheirgene expression pattern rapidly after activation by microbes and are keyimmuneregulators.ThecontrolofproteinsynthesisduringDCactivation isamajordeterminantofproperfunction;tRNAsarefundamentalforthisprocessastheytranslatemRNAtemplatesintocorrespondingproteins.However,stressconditionscanaltertheirpoolandmodificationsdynamics,whichon itsowncanskewproteinsynthesisaccuracyandefficiency.CurrentlywearestudyingthemechanismsthatallowDCstocoordinatetheirgeneexpressionprogramboth at the transcriptional and translational levels upon activation. UsingtranscriptomicanalysisandtRNAsexpression,uponmicrobesensing,wehaveobserved a connection between variations in the tRNA species and immunefunction.IndeedproteinsynthesisregulationandtRNAabundanceregulationarevitalforDCactivationandTcellpriming.ThereforewebelieveregulationofproteinsynthesisqualitycanaffectDCfitnessandimmunefunction.

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P7.7Overcoming a “molecular ruler” mechanism: the unusualheterotrimeric tRNA splicing endonuclease of TrypanosomabruceiGabriel Silveira d’Almeida1, Mary Anne Rubio1, Christopher Trotta2,ArthurGünzl3,JuanAlfonzo11DepartmentofMicrobiologyandOSUCenterforRNABiology,TheOhioStateUniversity2PTCTherapeuticsInc�3SchoolofMedicine,UniversityofConnecticutIntrons interrupt tRNAsequences inallmajor linesofdescent (Bacteria,ArchaeaandEukarya), rendering them nonfunctional for protein synthesis. Intron removal istherefore,essential.Inallknowneukaryotes,introncleavage,thefirststepinthetRNAsplicing pathway, is catalyzed by a conserved heterotetrameric tRNA splicingendonuclease (Sen) composed of four subunits: Sen54, 34, 15 and 2. BioinformaticsanalysisusingpreviouslypublishedeukaryoticSensequencesledustotheidentificationofonlyonehomologofthetRNAsplicingendonucleaseinTrypanosomabrucei(Sen34),suggestingthat,eithertheothersubunitsaremissingor,asawhole,theenzymeishighlydivergent in these organisms. In thiswork,we present evidence for a divergent anduniqueenzymecomposedofthreesubunits:homologsofSen34,15and2.Byperformingtandemaffinity chromatography followedbymass spectrometry analysiswe purifiedandidentifiedT.bruceiSen(TbSen)subunitsfromaT.bruceiS100fraction.Gelfiltrationchromatographyandinvitroactivityassaysrevealedthattheactiveenzymehadasizewithintherangeof58to72kDa,consistentwiththatofaheterotrimer.Furthermore,immunofluorescence localizationassays showed that theenzymewas cytoplasmic, instark contrast to the nuclear localization of Sen in most eukaryotes. The resultspresented here demonstrate that TbSen greatly diverges from previously describedeukaryoticenzymesinbothstructureandlocalization.Interestingly,inmosteukaryotes,Sen54servesasa“molecularruler”thatcarefullymeasuresthedistancebetweenthesplicesitesandthebackboneofthefoldedtRNA,aidinginsubstrateidentificationandcatalyticsitepositioning.OurfindingofaheterotrimericendonucleasethenobviatestheneedforaSen54subunitandmayremovethesubstraterecognitionrestrictionsetforthby the “molecular ruler”mechanism. Theseobservationshavedirect implications forboth the evolution of the enzyme in trypanosomes and its potential for targeting ofadditionalsubstrateswhilenotjustbeinglimitedtotRNAs.

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P7.8

IdentificationoftRNAmodifyingenzymesasnovelproteostasisplayersinhumancellsMarisa Pereira1, Cristiana Paulo1, Miguel Mano2, Sofia Varanda1,ManuelSantos1,AnaRaquelSoares11iBiMED–InstituteofBiomedicine,DepartmentofHealthSciences,Universityof Aveiro, Aveiro, Portugal 2CNC/UC-Biotech, Parque Tecnológico deCantanhede,Cantanhede,PortugalAccumulationofproteinaggregates,impairedproteinsynthesisandproteotoxicstress are hallmarks of protein conformational diseases. Previously,wehaveshownthattRNAmistranslationinducesproteotoxicstressandtranscriptomederegulation in zebrafish. Since tRNA modifying enzymes catalyze tRNAmodifications,whichareessentialforbothtRNAstabilityandefficientcodon-anticodon recognition,we hypothesize that deregulation of thesemoleculesalso affect translation fidelity, resulting in protein aggregation and UPRactivationinhumancells.High content screenings using HeLa cells expressing a protein aggregationfluorescent sensor were performed and particular tRNAmodifying enzymesthat catalyse modifications at position 34 were identified. Besides affectingproteome stability, absence of these enzymes decreased both proteasomeactivityandproteinsynthesisrateandinducedstressresponsepathways.Wearenowperformingcodonusageanalysisand identifyingwhichproteinsaremorepronetoaggregate.Together this data will contribute to elucidate the role of tRNA modifyingenzymesinproteostasisandindiseaseonset.Acknowledgements: This research was supported by the PortugueseFoundationforScience&Technology,POCI-COMPETE2020andFEDERthroughgrants SFRH/BPD/77528/2011, PTDC/BIM-MEC/1719/2014, POCI-01-0145-FEDER-007628andUID/BIM/04501/2013.

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AhomoplasmicmitochondrialtRNAPromutationcausingexerciseintolerancewithmuscleswellingK. Auré1,2,3, C. Jardel1,4, I. Chicherin5 , N. Entelis5, A.-M. Heckel5, F.Caillon6,Y.Péréon7,C.L’Hermitte-Stead1,I.Lemière4,A.Lombès1,7,8,I.Tarassov5.1InsermInstitutCochinU1016,Paris,France;2HôpitalAmbroiseParé,Boulogne-Billancourt,France;�3UniversitéVersailles-Saint-QuentinenYvelines,France;�4CHUPitié-Salpêtrière, Paris, France;� 5Universitéde Strasbourg - CNRSUMR7156 GMGM, Strasbourg, France 6CHU Nantes, Hôtel Dieu, Nantes,France�7CNRS UMR 8104, Paris, France;� 8Université Paris-Descartes-Paris5,Paris,France.MitochondrialDNAmutationsareattheoriginofincurablehumandiseases(1).If the vast majority of mtDNA mutations are hetroplasmic (simultaneouspresenceofwild-typeandmutantmtDNA),averyfewnumberofhomoplasmicpathogenicmutationsofmtDNAwerealsodescribed (2).WedescribeanewhomoplasmicmtDNAmutationfoundin5independentfamiliescharacterizedbyexerciseintoleranceandmuscleswelling,m.15992A>T.Itislocalizedinthemt-tRNAProgenecreatinganU34>Achange in theanticodonwobbleposition.A34hadneverbeendescribedinmt-tRNAsandispredictedtoinduceproblemsof decoding or aminoacylation. Patients' fibroblasts and cybrids werecharacterizedbylowactivityofmitochondrialcomplexesIandIII.Mitochondrialtranslation was only moderately decreased in fibroblasts. The level of mt-tRNAProwasnotchanged,meaningthatthemutationdoesnotaffectthetRNAstability. On the other hand, we found that the relative amount of othermitochondrialtRNAswerealteredinhumancellsbearingthemutation.References(1)MITOMAP:AHumanMitochondrialGenomeDatabase(2)McFarlandetal.,NatureGeneticsv.30145-146(2002)

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P7.10

Biochemical and structural studies of the plasmodial proteintRip,freeorinthepresenceofvariousligands�José Jaramillo,DelphineKapps, Bernard Lorber, Claude SauterAnneThéobald-DietrichandMagaliFrugierAminoacylation of eukaryotic transfer RNA: Control and pathogenicity,ArchitectureetRéactivitédel’ARN,CNRS,UniversitédeStrasbourg,IBMC,15rueRenéDescartes,67000Strasbourg,FranceMalariaisadiseasecausedbyPlasmodiumparasitestransmittedtohumansbyinfected mosquitoes. These parasites develop into two hosts: the mosquitoAnopheles and a vertebrate organism.�In the genome of Plasmodium, weidentifiedasurfaceproteincontainingatRNAbindingdomain.Thisproteinwasnamed tRip (tRNA import protein) in reference to its capacity to importexogenoustransferRNA(tRNA)byanactiveprocess.In the absence of tRip, the parasite has a reduced protein biosynthesis anddevelopsslowlyinthebloodofthevertebratehost.Theseobservationssuggestthat host tRNAs affect the protein synthesis and/or the regulation of geneexpressionintheparasite.However,themechanismbywhichthishappensisstillunclear.�BesideitsabilitytoformacomplexinthepresenceoftRNA,tRipinteracts also specifically with plasmodial cytosolic aminoacyl-tRNAsynthetases.Weareaimingtoreconstituteafullcomplex invitro inordertostudyitsfunction.Inadditiontocrystallizationattempts,differentbiochemical(PAGE,HPLC)andbiophysical (MS, DLS, CD, AUC, SAXS, ...) techniques are used in order tocharacterizethesize,shapeandoligomerizationstateof tRip inthedifferentcomplexes.

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P7.11

Physiological alterations in HEK293 cells caused by tRNAmisexpression�Ana Sofia Varanda1,Mafalda Santos1,2, Ana Soares1, CarlaOliveira2,3

andManuelA.S.Santos11iBiMED&Health Sciences, University of Aveiro, 3810-193 Aveiro,Portugal�2IPATIMUP,InstitutodeInvestigaçãoeInovaçãoemSaúde,I3S,4200-465Porto,Portugal.3FacultyofMedicine,UniversityofPorto,4200-465Porto,PortugalAlterations in protein synthesis components, namely tRNAs, aminoacyl-tRNAsynthetasesortRNAmodifyingenzymes,increasethelevelofproteinsynthesiserrors(PSE)andarerelatedwithdiseases,fromcancertoneurodegeneration.Still, the cause-effect mechanisms remain to be elucidated. In order tounderstand the physiological effects of PSE in human cells,wemodified theanticodon of a human tRNASer, to incorporate serine at various non-cognatesites.StableHEK293celllinesexpressingthesetRNAswereanalyzedatdifferentcell passages. tRNAs misexpression led to the accumulation of misfoldedproteins and protein aggregates, but activation of the ubiquitin-proteasomesystem and the unfolded protein response (UPR) maintained cell viability.Deregulated genes encoding plasma membrane and extracellular proteinssuggestthatlevelsofmistranslationthathaveminorornovisibleeffectsoncellviabilityimpactcellphysiologythroughderegulationofionhomeostasisandcelladhesion.

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P7.12

A deafness-associated tRNA mutation alters the m1G37modificationintRNAIleandmitochondrialdysfunctionMengWang1,2,FeilongMeng1,2,MiZhou2,Min-XinGuan1,2,31.DivisionofMedicalGeneticsandGenomics,TheChildren'sHospital,ZhejiangUniversitySchoolofMedicine,Hangzhou,Zhejiang,310052,China.�2.Instituteof Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang,310058,China.�3. Joint InstituteofGeneticsandGenomeMedicinebetweenZhejiangUniversityandUniversityofToronto,Hangzhou,Zhejiang,China.Severalmitochondrial tRNAmutationshavebeenassociatedwithmaternallyinherited deafness. However, the pathophysiology of these tRNAmutationsremainspoorlyunderstood. Inpresentstudy,weinvestigatedthepathogenicmechanismunderlyingthedeafness-associatedmitochondrialtRNAIle4295A>Gmutation,which is localizedatahighlyconservednucleotide (A37) for thefidelity of codon recognition and stabilization. Primer extension andmethylationactivity assays indeed confirmed that them.4295A>GmutationcreatedatRNAmethyltransferase5(TRMT5)-catalyzedm1G37modificationoftRNAIle.Usingcybridcell lines,wedemonstratedthesignificantdecreases intheefficiencyofaminoacylationandsteady-statelevelofmt-tRNAIleinmutantcybrids,comparedwithcontrolcybrids.Afailureinmetabolismofmt-tRNAIlecaused the variable reductions inmitochondrial functions inmutant cybrids.OurfindingsmayprovidenewinsightsintothepathophysiologyofmaternallytransmitteddeafnessthatwasmanifestedbyalterednucleotidemodificationofmitochondrialtRNA.

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P7.13

A futile tRNA cycle drives energy expenditure and reprogramsmetabolisminMaf1-/-mice.I.M.Willis1,R.D.Moir1,N.Hernandez2�1AlbertEinsteinCollegeofMedicine,BronxNY,USA,2UniversityofLausanne,Lausanne,SwitzerlandAs a master regulator of transcription by RNA polymerase (Pol) III, Maf1respondstochangesinnutrientsandcellularstressconditionstobalancetheproductionof tRNAs,5SrRNAandothersmallnon-codingRNAswithproteinsyntheticneed,cellgrowthandmaintenance.SowhyareMaf1-/-miceleanandwhydotheyresistweightgainonnormalandhighfatdiets(1)?Oneproposalis that these phenotypes result from increased energy expenditure due to afutilecycleoftRNAsynthesisanddegradationthatisenhancedinMaf1-/-mice.Inagreementwiththisview,PolIIIChIP-seq,tRNAexpressionandmetabolomicdatasupportthefunctionofMaf1asachronicrepressorofPolIIItranscription.MetaboliteprofilingpointstoincreasedactivityintheTCAcycle,thepentosephosphatepathwayandtheureacycleandconfirmskeypredictionsofthefutiletRNA cycle hypothesis by identifying changes in pathways of nucleotidesynthesisandturnover.Thus,constitutivelyhighlevelsofPolIIItranscriptioninMaf1-/- mice reprogram central metabolic pathways and waste metabolicenergythroughafutiletRNAcycle.1.Bonhoure.etal.,(2015)GenesDev.29,934-947.

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ADDITIONALPOSTERS

250

P2.51

The critical role of queuosine modifications in the humanoxidativestressresponseS.M.Huber,U.Begley,T.J.Begley,P.C.Dedon�Massachusetts InstituteofTechnology,DepartmentofBiologicalEngineering,CambridgeMA,USACellsrespondtoenvironmentalchangesandxenobioticexposuresbyalteringtheir phenotypes through signal transduction, transcriptional regulation, andproteinsecondarymodifications.Usingauniquecomputationalandanalyticalplatform,werecentlydescribedanoveltRNA-basedmechanismoftranslationalcontrolofthecellstressresponseinbacteriaandyeast.Inthismodel,stressorsandtoxicantsinducethereprogrammingofdozensofmodifiedribonucleosidesintRNAthatmodulatelevelsofproteinproductionbypromotingtheselectivetranslation of codon- biasedmRNAs representing families of stress-responsegenes.Here,weuseLC-MS/MSanalysisandmultivariatestatisticstomovethisdiscovery into human cells and show that oxidative stress induces specificchangesinqueuosinemodificationsthatinturnregulateproteinlevelsbywayofselectivetranslationofcodon-biasedstressresponsetranscripts.Ourfindingshavethepotentialtotransformourunderstandingofhumancellandexposurebiology to reveal newmechanism of toxicant-induced pathology with directrelevance to many human diseases and to be exploited for diagnostic andtherapeuticpurposes.

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P2.52

Nano LC-MS using capillary columns enables accuratequantification of modified ribonucleosides at low femtomollevelsSandra D. Kienast1,2, L. Peter Sarin3, Johannes Leufken1,2, Hannes C. A.Drexler4,ChristianFufezan5andSebastianA.Leidel1,2,61Max Planck Research Group for RNA Biology, Max Planck Institute forMolecular Biomedicine, Münster, Germany 2Cells-in-Motion Cluster ofExcellence, University of Münster, Münster, Germany�3The RNAciousLaboratory, Department of Biosciences, University of Helsinki, Helsinki,Finland�4Bioanalytical Mass Spectrometry Unit, Max Planck Institute forMolecular Biomedicine, Münster, Germany 5Cellzome, Molecular DiscoveryResearch, GlaxoSmithKline, Heidelberg, Germany 6Department of ChemistryandBiochemistry,UniversityofBern,SwitzerlandtRNAmodifications are crucialmodulators of translation and gatekeepers ofproteome integrity. Despite their biological importance, technical challengeshave limited their detection and accurate quantification.�We established asensitive capillary nanoflow liquid chromatographymass spectrometry (nLC-MS)workflowforquantitativehigh-resolutionanalysesofmodifiednucleosides.Weevaluateddifferentstationaryphases forreversed-phaseseparation: twoporousgraphiticcarbon(PGC)andanend-cappedC18material.Whenusedina nLC-MS setup, PGC and C18 capillaries provided excellent signal-to-noiseratiosspanninguptosixordersofmagnitude,allowingtheanalysisofindividualnucleosidesdown to femtomol levels.Thematricesdiffer in their separationcapability and nucleoside sensitivity, nicely complementing one another.Absoluteorrelativequantificationwasattainedbydefiningthelineardetectionrange through synthetic nucleosides or by normalizing signal intensities to astableisotopelabelledspike-in.WeshowthatcapillarycolumnsinanLC-MSsetupareapowerfulandsensitivetool to quantitatively analyzemodified ribonucleosides in complex biologicalsamples.

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P2.53

ApipelinefortotaltRNAcompositionanalysisbrokendowntoisoacceptorsanddifferentialmodificationcontentMarkHelm1&YuriMotorin21 Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University ofMainz, Staudingerweg 5, D-55128 Mainz, Germany 2 Laboratoire IngénierieMoléculaire et Physiopathologie Articulaire (IMoPA) UMR7365 CNRS- UL,BioPôledel’UniversitédeLorraineCampusBiologie-Santé,9,avenuedelaForêtdeHaye,CS50184,54505Vandoeuvre-les-Nancy,FranceThe composition of a cell’s tRNA population crucially affects its various functions,including in particular protein biosynthesis but also immunostimulation andfragmentation into tRNA fragments (tFRs). The tRNA population of a cell is a highlydiversemixture,whosecompositionistraditionallyanalyzedinpreparationsofso-calledtotaltRNA.Thesearethoughttocontainsomeforty-oddmajorspecies inacanonicalsystem,i.e.inbacteria,archeae,orineukaryoticcytosol.However,acloserlookrevealshigher diversity: in higher eukaroytes, tRNA genes number in the hundreds, manycontainingminorsequencedeviations.Moreimportantly,extensivepost-transcriptionalmodificationoftRNAs,whichwaslongthoughttobestaticandofuniformstoichiometry,hasturnedouttobedynamicallyinvolvedincellularresponsestooutsidestimuli.Hence,adetailedunderstandingof functionalaspectsof total tRNA including translationbutalso non-canonical effects such as immunostimulation, requires quantification ofdifferenttRNAsequencesaswellasoftheirmodificationstatus.HerewepresentanintegratedapproachallowingfastquantificationofindividualtRNAsequencesbymicroscalethermophoresis(MST).UsingcomplementarycDNAcarryingafluorophore and a biotin residue, the content of a given tRNA in total tRNA can bequantified in lessthanonehour,allowinganear-comprehensiveanalysisoftheforty-oddconventionalcomponentsoftotaltRNAinsidethreedays.SuchquantificationalsoconstitutesanoptimizationstepforsubsequentaffinitybasedtRNAisolationusingthesamebiotinylatedcDNA.Thus“sequence-purified”tRNAscanthenbeassessedfortheirmodification content, allowing the definition of a so-called modivariants, i.e. tRNAsequencesthatdifferonlyintheirmodificationstatus.CombinationwithRNA-seqbasedmethodscanpinpointobservedchangestodefinedpositionsinsidethetRNAsequences.ThiscombinationofMST,LC-MSandRNA-seqbasedanalyticsallowstracingchangesinthetotal tRNA landscape in responsetovariousoutsidestimuli liable tochangegeneexpressionpatterns.

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P2.54HumanmitochondrialtRNAprocessingbyribonucleaseP�AgnesKarasik,MarkosKoutmos�University of Michigan, Department of Chemistry and Department ofBiophysics,AnnArbor,MI48105,USAProtein coding and rRNA genes are separated (or ‘punctuated’) by tRNAencoding sequences in the human mitochondrial DNA. Therefore,mitochondrial tRNA ((mt)tRNA)maturation is essential for the processing ofotherkeymitochondrialRNAs.While(mt)tRNAsequencescompriseonlyasmallportionofthemitochondrialDNAsequence(~10%),over50%ofthedisease-causingmutationsinDNAarelocatedwithin(mt)tRNAcodingsequences.Here,we investigate how patient mutations found in precursor (mt)tRNAs impair(mt)pre-tRNArecognitionbythethree-proteincomplexresponsiblefortRNA5’endmaturation,mitochondrialRNaseP(mtRNaseP).Wefindthatonlyasubsetofmutationsinfluence(mt)pre-tRNAbinding,andthatallofthemutationsweinvestigated impact the rate of (mt)pre-tRNA processing bymtRNase P. Ourfindingsmaybepartially explainedby thealteredUVmeltingprofilesof themutant(mt)pre-tRNAswhicharelikelytoadoptalternativelyfoldedstructures.Our results demonstrate that disease-linked (mt)DNA mutations can impairearlystepsofmitochondrialtRNAmaturation.

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P4.13ApplicationofimprovedsequencingmethodsrevealsadominantisoacceptorinangiospermtRNAprofilesJessicaWarren,DanSloanDepartmentofbiology,ColoradoStateUniversity,USA. The development of improved tRNA sequencing methods has nowopenedthedoortoexploringthetRNAprofilesofmultipleorganisms.Here,IpresenttRNA-seqdatafromfourangiospermspecies(floweringplants),usingacombinationofAlkBtreatmenttoremoveinhibitorybasemodifications(Cozenetal.,2015andZhengetal.,2015)andYAMAT-seq(Y-shapedAdapter-ligatedMAture TRNA sequencing; Shigematsu et al. 2017). Mature tRNAs weresequencedfromtotalcellularRNAfromfourdiverseangiosperms:Arabidopsisthaliana, Solanum tuberosum, Medicago truncatula, and Oryza sativa.Utilization of adapters complementary to CCA tails resulted in librariescomposed almost entirely ofmature tRNAs (up to 99%), demonstrating theutilityofthemethodtogeneratelibrarieshighlyenrichedintRNAsequences.TheresultingreadsweredominatedbyasingletRNAfamily,astRNAsfromthecytosolicprolineisoacceptorfamilymadeupthemajorityofsequencedreadsinallangiospermspecies(upto78%),followedbytheplastidtrnG(GCC).DropletdigitalPCR(ddPCR)analysisconfirmedtheexceptionallyhighexpressionleveloftrnP.WhyasingletRNAwouldbeinsuchhighrelativeabundanceisyettobedetermined, but the advancement of tRNA profiling methods will lead to abetter understanding of the multiple roles tRNAs play in diverse biologicalsystems.