tfiiib subunit bdp1 participates in rna polymerase iii...

Research ArticleTFIIIB Subunit Bdp1 Participates in RNA Polymerase IIITranscription in the Protozoan Parasite Leishmania major

Fiordaliso C Romaacuten-Carraro1 Luis E Florencio-Mart-nez1 Gabriela Romero-Meza1

Tomaacutes Nepomuceno-Mej-a 1 Julio C Carrero 2 Rossana Arroyo 3

Jaime Ortega-Loacutepez 4 Rebeca G Manning-Cela 5 and Santiago Mart-nez-Calvillo 1

1Unidad de Biomedicina Facultad de Estudios Superiores Iztacala Universidad Nacional Autonoma de MexicoAv de Los Barrios 1 Col Los Reyes Iztacala Tlalnepantla Estado de Mexico CP 54090 Mexico2Departamento de Inmunologıa Instituto de Investigaciones Biomedicas Universidad Nacional Autonoma de MexicoCiudad de Mexico CP 04510 Mexico3Departamento de Infectomica y Patogenesis Molecular Centro de Investigacion y de Estudios Avanzados del IPNAv IPN 2508 Ciudad de Mexico CP 07360 Mexico4Departamento de Biotecnologıa y Bioingenierıa Centro de Investigacion y de Estudios Avanzados del IPN Av IPN 2508Ciudad de Mexico CP 07360 Mexico5Departamento de Biomedicina Molecular Centro de Investigacion y de Estudios Avanzados del IPN Av IPN 2508Ciudad de Mexico CP 07360 Mexico

Correspondence should be addressed to Santiago Martınez-Calvillo scalvunammx

Received 1 December 2018 Accepted 13 March 2019 Published 1 April 2019

Academic Editor Terry K Smith

Copyright copy 2019 Fiordaliso C Roman-Carraro et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Leishmania major a protozoan parasite that diverged early from the main eukaryotic lineage exhibits unusual mechanisms of geneexpression Little is known in this organismabout the transcription factors involved in the synthesis of tRNA 5S rRNA and snRNAstranscribed by RNA Polymerase III (Pol III) Here we identify and characterize the TFIIIB subunit Bdp1 in L major (LmBdp1)Bdp1 plays key roles in Pol III transcription initiation in other organisms as it participates in Pol III recruitment and promoteropening In silico analysis showed that LmBdp1 contains the typical extended SANTdomain aswell as other Bdp1 conserved regionsNevertheless LmBdp1 also displays distinctive features including the presence of only one aromatic residue in the N-linker regionWe were not able to produce null mutants of LmBdp1 by homologous recombination as the obtained double replacement cell linecontained an extra copy of LmBdp1 indicating that LmBdp1 is essential for the viability of L major promastigotes Notably themutant cell line showed reduced levels of the LmBdp1 protein and its growth was significantly decreased in relation to wild-typecells Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line and ChIP experimentsshowed that LmBdp1 binds to 5S rRNA tRNA and snRNA genes Thus our results indicate that LmBdp1 is an essential proteinrequired for Pol III transcription in L major

1 Introduction

In eukaryotic cells transcription of nuclear DNA is carriedout by three different classes of RNA polymerases (Pol) Pol III and III Each of these enzymes transcribes a specific groupof genes and each depends on transcription factors to bindits promoter regions Pol I produces 18S 58S and 28S rRNAs[1] while Pol II generates mRNAs and most microRNAs and

snRNAs [2] Pol III transcribes genes encoding 5S rRNAtRNAs U6 snRNA 7SL RNA and other small essential RNAmolecules [3 4] Pol III promoters have been divided intothree main types 5S rRNA genes possess a type 1 promotercomposed of three gene-internal elements (boxes A and Cand intermediate element) type 2 promoters are present ontRNA genes and they consist of two gene-internal sequences(boxes A and B) and U6 snRNA genes contain type 3

HindawiBioMed Research InternationalVolume 2019 Article ID 1425281 14 pageshttpsdoiorg10115520191425281

2 BioMed Research International

promoters which are constituted by three small sequenceelements located upstream of the genes (TATA box proximalsequence element and distal sequence element) [5]

General transcription factors TFIIIA TFIIIB and TFIIICare required for accurate initiation of Pol III transcription[6] TFIIIB is needed to form a transcriptionally activepreinitiation complex (PIC) at all three types of promoterregions as it participates in Pol III recruitment and promoteropening [7] TFIIIB consists of three subunits the TATA-binding protein (TBP) the TFIIB-related factor 1 (Brf1)and B double prime 1 (Bdp1) [8] Notably Bdp1 is specificto Pol III transcription since it does not show homologywith any of the general transcription factors of other RNApolymerases Early studies showed that Bdp1 contains aSANT (Swi3 Ada2 N-Cor and TFIIIB) domain which is ahighly conserved sim50-amino acid motif present in severalproteins involved in transcriptional regulation [9] Howeverrecent analysis established that the SANT domain present inBdp1 from human and yeast is actually larger and possessesother distinguished features and accordingly it was renamedextended SANT domain [10ndash12] In yeast the Bdp1 extendedSANT domain is essential for cell viability as it is neededfor Pol III PIC assembly and stability [13] The extendedSANT domain interacts directly with DNA andwith subunitsBrf1 and TBP [14 15] Other regions of Bdp1 associate withsubunits C128 C37 and C34 of Pol III [11 13 16] and subunitTfc4 of TFIIIC [17 18] Thus Bdp1 establishes an intricatenetwork of interactions within the Pol III PIC

The trypanosomatid parasite Leishmania major is theetiological agent of cutaneous leishmaniasis in Asia andAfrica [19] The parasite is transmitted to humans throughthe bite of infected sandflies of the genus Phlebotomus Inaddition to their medical importance L major and othertrypanosomatid protozoa such as Trypanosoma brucei andTrypanosoma cruzi are relevant because they express theirgenes by uncommon processes including Pol II polycistronictranscription and trans-splicing [20ndash22]

Little is known in trypanosomatids about either the DNAsequences or the proteins that participate in Pol III tran-scription initiation Unlike other organisms all snRNA genesare transcribed by Pol III in these parasites [23] NotablysnRNA expression in T brucei and L major is controlledby extragenic boxes A and B located within tRNA or tRNA-like sequences [24 25] Although 5S rRNA genes contain thecharacteristic gene-internal elements their promoter activityhas not been proven [26] Neither TFIIIA nor TFIIIC hasbeen identified in trypanosomatids [27] However ortho-logues of TFIIIB subunits TBP and Brf1 have been studied inT brucei TBP which participates in transcription by all threeRNA polymerases has been primarily investigated in thecontext of Pol II transcription of the spliced-leader (SL) RNAgenes [28 29] On the other hand knockdown analysis byRNA interference demonstrated that Brf1 is indeed involvedin Pol III transcription and that it is indispensable for cellsurvival of procyclic forms of T brucei [30] Another proteinthat participates in the regulation of Pol III transcription in Tbrucei isMaf1 which inhibits tRNA and snRNA transcriptionby associating with their promoter regions [31]

Here we identified and characterized the TFIIIB subunitBdp1 in L major (LmBdp1) Bioinformatic analysis showsthat LmBdp1 possesses the characteristic extended SANTdomain and other conserved regions that flank this domainAttempts to generate null mutants produced a cell line withan additional copy of LmBdp1 Nevertheless Western blotanalysis showed that the levels of LmBdp1 were considerablydiminished in the mutant cell line Notably the growth ofthis cell line was significantly reduced in relation to wild-type cells Nuclear run-on assays demonstrated that Pol IIItranscription was affected in the mutant cell line and ChIPexperiments showed that LmBdp1 binds to 5S rRNA tRNAand snRNA genes

2 Materials and Methods

21 In Silico Analyses Sequences were obtained fromTriTrypDB database (release 40) (httptritrypdborgtritrypdb) from the National Center for BiotechnologyInformation (NCBI) database (httpwwwncbinlmnihgov)and from Universal Protein Resource (UniProt) Multiplesequence alignments were made with the Clustal Omegaprogram (httpwwwebiacukToolsmsaclustalo) andshaded manually To predict the protein secondary structurethe PSIPRED Protein Sequence Analysis Workbench (httpbioinfcsuclacukpsipred) was used Homology modelingwas performedwith SWISS-MODEL v 37 (httpswissmodelexpasyorginteractive) Swiss-PDB Viewer program (httpwwwexpasyorgspdbv) and PyMOL v 211 (httpspymolorg2) using the crystallographic structure of humanextended SANT domain (PDB id 5n9g) and yeast Bdp1(PDB id 6F41) as templates Phosphorylated residues werepredicted with PhosTryp (httpphostrypbiouniroma2it)which was developed for the specific identification ofphosphorylated residues in Trypanosomatid proteins as themethod has been trained using phosphoproteomic data fromLeishmania T brucei and T cruzi [32]

22 Cell Culture and Transfection Promastigotes from Lmajor MHOMIL81Friedlin (LSB-1321) were grown in BMmedium (1times M199 medium pH 72 containing 10 heat-inactivated fetal bovine serum 95 gl brain heart infu-sion 40mM HEPES 001mgml hemin 00002 biotin100 IUml penicillin 100 120583gml streptomycin and 1times L-glutamine) at 26∘C and harvested in the mid-log phase Togenerate the L major cell line that expresses LmBdp1 fusedto a PTP tag wild-type promastigotes were transfected byelectroporation with the LmBdp1-PTP vector Briefly 4 times107 promastigotes in 04ml electroporation buffer (25mMHEPES 120mM KCl 015mM CaCl

2 5mM MgCl

2 10mM

KH2PO4 10mM K

2HPO4 2mM EDTA pH 76) were trans-

fected with 10 120583g of LmBdp1-PTP by electroporation at 1600V 50 120583F and 25 Ω (BTX Electro Square Porator ECM 830)Cells were grown for 24 h before spreading on plates contain-ing 07 Seaplaque GTG agarose (FMC Bioproducts) in BMmedium with 50 120583gml G418 Some of the isolated colonieswere selected for further analysis The same electroporationconditions were used for transfections with the pac and hygtargeting cassettes for the generation of knockout parasites

BioMed Research International 3

23 Plasmid Constructs To generate the LmBdp1-PTPvector for immunofluorescence and ChIP assays the RPB6gene from the pB6-PTP plasmid [26] was substitutedwith theLmBdp1 gene To that end pB6-PTP was digested with XmaIand XbaI and the RBP6 gene was eliminated by agarose gelelectrophoresis The LmBdp1 gene (LmjF366530) (withoutthe terminal codon) was amplified by PCR using primersBdp1PTP-for-XmaI (51015840-ACCCGGGATGGACGACAACGA-GTTCGA) and Bdp1PTP-rev-XbaI (51015840-ATCTAGACTCAA-ACGAGAAGTCCGAGT) The PCR product was clonedinto pGEM-T Easy vector digested with XmaI and XbaIand ligated into the pB6-PTP backbone To produce plasmidpΔBdp1-pac for knockout experiments the pac-gene insert(17 kb) was obtained by digesting pΔ75-pac [33] with EcoRIand SacI The 51015840 targeting region from LmBdp1 (566 bp)was amplified by PCR with oligonucleotides Bdp1-for-XbaI(51015840-ATCTAGAGTCGGCCGTTGCGCTTCTC) and Bdp1-rev-EcoRI (51015840-AGAATTCGGCTCCACCGGTGCGTATC)The 31015840 targeting region (550 bp) was amplified withprimers Bdp1-for-SacI (51015840-AGAGCTCCGGTAGAACCA-AAAAGTTCG) and Bdp1-rev-XhoI (51015840-ACTCGAGTATAA-GTCCGACAGCGAAGA) To produce vector pΔBdp1-hyg the hyg-gene fragment (18 kb) from pΔ75-hyg [33] wasobtained after SpeI and SacI digestionThe 51015840 targeting regionwas amplified with primers Bdp1-for-XbaI and Bdp1-rev-SpeI(51015840-AACTAGTGGCTCCACCGGTGCGTATC) and the 31015840targeting region was amplified with oligonucleotides Bdp1-for-SacI and Bdp1-rev-XhoIThePCRproducts from 51015840 and 31015840targeting regions were digested with the restriction enzymesindicated in the name of each oligonucleotide For bothknockout vectors the selectable-marker gene and the twoflanking regions were cloned into pBluescript II KS (digestedwith XbaI and XhoI) by a single four-fragment ligation stepTo obtain the targeting cassettes pΔBdp1-pac and pΔBdp1-hyg were digested with XbaI and XhoI For nuclear run-onexperiments fragments of L major genes transcribed by thethree different RNA polymerases were amplified by PCRand cloned into the pGEM-T Easy vector The 24S120573 rRNAgene (LmjF27rRNA26 113 bp) was amplified with primersLmrRNA24S120573-51015840 (51015840-TTCCGGAGTCTTGTTTCGAG) andLmrRNA24S120573-31015840 (51015840-GTGGATTCGGTTGGTGAGTTG)and the 18S rRNA gene (LmjF27rRNA01 370 bp) wasamplified with oligonucleotides Lm-rRNA18S-51015840 (51015840-CGGCCTCTAGGAATGAAGG) and LmrRNA18S-31015840 (51015840-CCCCTGAGACTGTAACCTC) The 120572-tubulin gene(LmjF130330 338 bp) was amplified with primers alfa-tub-51015840(51015840-AGAAGTCCAAGCTCGGCTACAC) and alfa-tub-31015840 (51015840-GTAGTTGATGCCGCACTTGAAG) and the spliced-leaderRNA gene (LmjF02SLRNA0010 303 bp) was amplified withprimers SL-51015840 (51015840 GAGCGCGGTGGGCATGACA) and SL-31015840(51015840-ACTGCAAGGGTGCGCCCG) The LmjF060370 gene(521 bp) was amplified with oligonucleotides Lm06-0370-51015840(51015840-GAAGCGATGGACTGTTCTGG) and Lm06-0370-31015840(51015840-CGGTCCTTGCTGCGAATATC) and the LmjF060210gene (503 bp) was amplified with primers Lm06-0210-51015840(5-GCCGGAGACATTTGCGTAC) and Lm06-0210-31015840 (51015840-CTATGGCGACGGGATCATC) The LmjF060200 gene

(547 bp) was amplified with primers Lm06-0200-51015840 (51015840-CCATCCCATGACAAGAGC) and Lm06-0200-31015840 (51015840-TGT-AGTCGCTGTACTCGC) and the tRNA-Phe gene (LmjF09TRNAPHE01 338 bp) was amplified with oligonucleotidesLm09-TRNAPHE-51015840 (51015840-TTCATCCGCGCAAAGAGG) andLm09-TRNAPHE-31015840 (51015840-GGCCTTCCACGTATTTCG)ThetRNA-Tyr gene (LmjF36TRNATYR01 316 bp) was amplifiedwith primers Lm36-TRNATYR-51015840 (51015840-AGTGCCGAGAAG-TTCGACG) and Lm36-TRNATYR-31015840 (51015840-TCGTCTCCG-TTCCTGTTGC) and the 5S rRNAgene (LmjF155SrRNA01344 bp) was amplified with oligonucleotides Lm15-rRNA5S-51015840 (51015840-GAAAGCATCTCTGTG GGTTCGA) and Lm15-rRNA5S-31015840 (51015840CCCGGGGTCCTGCAAATG) The U2snRNA gene (LmjF31snRNA01 127 bp) was amplifiedwith primers U2-51015840 (51015840-AAACGTGGAACTCCAAGGAA)and U2-31015840 (51015840-TATCTTCTCGGCTATTTAGC) and thetRNA-Sec gene (524 bp) was amplified with primers Lm-TRNASEC524-51015840 (51015840-CCGGCTGCCTTCATCAACTC) andLm-TRNASEC524-31015840 (51015840-GCGCATACGTTTCGGAGTCC)After transformation of JM109 competent cells plasmidDNA was purified with NucleoSpin plasmid columns(Macherey-Nagel) as specified by the supplier The identityof each insert was confirmed by sequencing using the T7 andSP6 primers

24 Indirect Immunofluorescence Assays The cellular local-ization of LmBdp1 labeled with a PTP tag was determinedby indirect immunofluorescence as previously described[26 34] For these assays 15 times 107 mid-log promastigoteswere incubated with a rabbit anti-Prot C polyclonal antibody(Delta Biolabs) and a secondary goat anti-rabbit antibodyconjugated with Alexa-Fluor 488 (Life Technologies) Imageswere obtained with a Zeiss Axio VertA1 epifluorescencemicroscope and analyzed with the ZEN 2012 software (Blueedition)

25 Southern Blot and PCR Analyses For Southern blotexperiments 5 120583g of genomic DNA was digested with XhoIand SacI (for analysis of single-knockout parasites) orPst1 (for examination of double-knockout cells) The DNAwas separated by electrophoresis on 08 agarose gels andtransferred to Hybond-NC membranes (GE Healthcare)by capillary action Blots were hybridized with the 51015840targeting region from LmBdp1 (566 bp) labeled with [120572-32P]dCTP using the High Prime labeling system (Roche)Hybridizations were performed in 50 formamide 5times SSC02 SDS and 4times Denhardtrsquos reagent at 42∘C Filters werewashed at 68∘C in 02times SSC and 01 SDS To verify thereplacement of the LmBdp1 gene with the pac gene PCRanalysis was carried out with oligonucleotides PAC-LOC31015840-REV (51015840-GTGGGCTTGTACTCGGTCATGG) and Bdp1-for-upstream (51015840-TGTTGGCAACTTGCCACCGT) whichrecognizes sequences located upstream of the 51015840 targetingregion and primers PAC-DHFR-31015840-REV (51015840-GGAGGG-AGGAATGAGGTGAGCT) and Bdp1-for-upstream Thecorrect integration of the hyg gene was confirmed by PCRwith primers HYG-rev (51015840-GTCGGAGACGCTGTCGAA-CT) and Bdp1-for-upstream


26 Generation of LmBdp1 Polyclonal Antibody Competentcells of Escherichia coli BL21 (DE3) were transformed withplasmid pCold-LmBdp1 Expression of LmBdp1 recombinantprotein (LmBdp1r) was induced with 1mM isopropyl 120573-D-1-thiogalactopyranoside (IPTG) at 37∘C for 18 h Affinitychromatography with Ni-Sepharose 6 Fast Flow matrix (GEHealthcare) was carried out to purify the LmBdp1r pro-tein according to the manufacturerrsquos specifications Around50 120583ganimal of purified LmBdp1r was employed to inoculatesubcutaneously six-week-oldmale BALB-Cmice in TiterMaxGold adjuvant (Sigma) at a 11 ratio Pre-immune normalmouse serum was collected before inoculation Serum wascollected six weeks after antigen immunization Westernblot analyses against LmBdp1r and protein extracts frompromastigotes were performed to confirm the specificity ofthe anti-LmBdp1 polyclonal antibody

27 Western Blot Analysis Whole-cell protein extracts wereobtained by standard protocols [35] Briefly 2 times108 parasiteswere lysed in RIPA buffer (150mM NaCl 05 sodiumdeoxycholate 01 SDS 50mM Tris pH 74 and 1times proteaseinhibitors) The extract was incubated for 30min on iceand mixed every ten minutes and then centrifuged at 1500times g for 10min The supernatant was recovered and storedat minus70∘C For Western blot analysis 50120583g of total proteinwas fractionated by 10 SDS-PAGE and blotted onto PVDFmembranes (Bio-Rad) Membranes were incubated withrabbit Peroxidase Anti-Peroxidase (PAP) (11600 dilutionSigma) polyclonal anti-LmBdp1 antiserum (1100 dilution)or polyclonal human 120573-tubulin antibody (1500 Invitro-gen) Proteins were detected with a horseradish peroxidase-conjugated secondary antibody and developed using an ECLkit (GE Healthcare)

28 Nuclear Run-On Experiments These assays were per-formed as described before [30 36] with isolated nucleifrom 2 times 108 mid-log promastigotes Labeled nascent RNAwas hybridized to Hybond-N+ membranes (GE Healthcare)containing dots of 2 120583g of plasmid DNA These plasmidspossess fragments of genes transcribed by Pol I (18S and 24S120573rRNA) Pol II (120572-tubulin spliced-leader RNA LmjF060200LmjF060210 and LmjF060300) Pol III (5S rRNA U2snRNA tRNA-Phe and tRNA-Tyr) and by both Pol II and III(tRNA-Sec) Hybridization was performed for 48 h at 50∘C in50 formamide 5times SSC 02 SDS 4times Denhardtrsquos reagentand 100 120583gml salmon sperm DNA Filters were washed toa final stringency of 01times SSC and 01 SDS at 65∘C RNAhybridization signals were quantified by densitometry usingthe MultiGauge software

29 Chromatin Immunoprecipitation Assays The ChIP pro-cedures were performed as previously described [31] Briefly2 times 108 promastigotes were cross-linked with formaldehyde(final concentration of 1) for 5min at 37∘C A Vibra-CellVCX130 ultrasonic processor (Sonics) was employed to lysethe cells (15 s onoff 40 amplitude for 5min) Nucleiwere pelleted and resuspended in sonication buffer (1 SDS10mM EDTA 50mM Tris-HCl pH 81) Chromatin was

sonicated to an average DNA size of about 200 to 500 bpwith a BioRuptor UCD-200 (Diagenode) (30 s on30 s offhigh intensity) for 30 cycles The sonicated material was pre-cleared by adding protein AG plus-agarose beads (SantaCruz Biotechnology) and mixing for 1 h at 4∘C Chromatinsamples were incubated overnight at 4∘C with rabbit anti-Prot A antibody (Sigma) or nonspecific rabbit immune serum(negative control) Protein-DNA complexes were incubatedfor 1 h with protein AG plus-agarose beads and 200 ng ofsonicated salmon sperm DNA and washed as previouslydescribed [37] Cross-links were reversed with 200mMNaClat 65∘C overnight Samples were treated with RNase A andproteinase K DNA was precipitated with sodium acetateand ethanol and quantified Each ChIP experiment wasperformed at least three times

210 Quantitative Real-Time PCRExperiments ThePlatinumSYBR Green qPCR Super Mix-UDG kit (Invitrogen) wasemployed to examine around 5 ng of immunoprecipitatedDNA by quantitative real-time PCR The results wereanalyzed by the 2minusΔΔCq method as reported previously[30 37] Reactions were carried out with optimizedconditions that produce a single amplicon of the correct sizein triplicate Results are presented as fold enrichment overnegative control precipitations The promoter region of the18S rRNAgene (LmjF27rRNA01) was amplifiedwith primers18Sp-for (51015840-TTGTTTGGGTGGAGGTGAGA) and 18Sp-rev(51015840-CAAAATCATCAAACCCCGTTC) The promoter ofthe spliced-leader RNA gene (LmjF02SLRNA0010) wasamplified with oligonucleotides LmjF-SL-PromF (51015840-GAG-CGCGGTGGGCATGACA) and LmjF-SL-PromR (51015840-AAGCCATCACCACCGCAGC) The 120572-tubulin gene(LmjF130330) was amplifiedwith primers TUB-for (51015840-AGA-AGTCCAAGCTCGGCTACAC) and TUB-rev (51015840-GTA-GTTGATGCCGCACTTGAAG) The 5S rRNA gene(LmjF115SRRNA03) was amplified with primers 5S-for(51015840-GAAAGCATCTCTGTGGGTTCGA) and 5S-rev (51015840-AACCCTGAGTGCCGTACTC) The U2 snRNA gene(LmjF31snRNA01) was amplified with oligonucleotidesU2-for (51015840-TATCTTCTCGGCTATTTAGC) and U2-rev (51015840-AAACGTGGAACTCCAAGGAA) The tRNA-Ala gene(LmjF31TRNAALA01) was amplified with primers Ala-for(51015840-ATTGGGACGTTACCGCGTCG) and Ala-rev (51015840-ATT-GCGGCCCAGGCCTTTCA) The tRNA-like associated tothe U2 snRNA gene was amplified with primers Like-for (51015840-CCGAGAAGATATGTTAGTACCACC) and Like-rev (51015840-AGGAAAAGATGCTTTCGACGAG) The tRNA-Met gene(LmjF11TRNAMET01) was amplified with oligonucleotidestRNAmet-F (51015840-AAAGTTTGCGACCGGTGAG) andtRNAmet-R (51015840-CACAACTTTCACTCGTAGCCG)The U4snRNA (LmjF36snRNA01) was amplified with primers U4-51015840 (51015840-AAGCCTTGCGCAGGGAGATGT) and 51015840U4Lmjend(51015840-GACAAAAAGTAGTCCCCACCC) The tRNA-likeassociated to the U4 snRNA was amplified witholigonucleotides U4tRNA-like 51015840 (51015840-GAAAAAAGGAGC-GCCGCCCCA) and U4tRNA-like 31015840 (51015840-CGCAAGGCT-TGCCTTGGGTGT)


Extended SANT domainN-linker

H1 H4 H5H3

H1 H3 H4 H5H2

H2

(a)

LmBdp1

H1

H3

H4

H2 H2

H5

N-LINKER

HsBdp1

N-LINKERH1

H3

H4

Merge

N-LINKERH1

H3

H4

H2

H5H5

(b)

Figure 1 Sequence and structure analyses of the extended SANT andN-linker domains fromLmBdp1 (a)Multiple sequence alignment of theN-linker and extended SANT domains from L major (Lm) T brucei (Tb) T cruzi (Tc) A thaliana (At) D melanogaster (Dm) S cerevisiae(Sc) M musculus (Mm) and H sapiens (Hs) Identical residues in all species are indicated by black shading Conserved substitutions aredenoted by dark-grey shading with white lettering and semiconserved substitutions are indicated by light-grey shading with black letteringaccording to the ClustalΩ program Trypanosomatid-specific conserved residues are shaded in red with white letters Conserved amino acidsin at least three species different from trypanosomatids (At Dm Sc Mm and Hs) are shaded in blue with white lettering In the N-linkeraromatic residues are denoted in bold Below the Hs sequence the hash characters () indicate conserved residues that interact with the DNAminor groove (Y291 S293 F294 and Y299 in human Bdp1) The percent sign () indicates highly conserved amino acids (W303 E307 andR332 in human Bdp1) involved in the interaction between the N-linker and the extended SANT domain The triangle symbol (998771) shows theinvariably conserved R334 that forms a salt bridge with residue E191 from TBP and also interacts with the template DNA strand Asterisks (lowast)indicate conserved amino acids K338 N339 K342 R343 and E345 that contact both faces of the major groove of the DNA Symbols abovethe Lm sequence indicate conserved residues in LmBdp1 Predicted 120572-helices are denoted by rectangles (H1 to H5) for LmBdp1 (above thealignment) and human Bdp1 (below the alignment) (b) Predicted three-dimensional structure of the N-linker and extended SANT domainof LmBdp1 by homology-modeling using the crystal structure of human Bdp1 (HsBdp1) as a template The colors of the five 120572-helices areconserved in panels (a) and (b)

3 Results

31 LmBdp1 Possesses the Extended SANT Domain and OtherConserved Regions Based on the presence of the SANTdomain gene LmjF366530 was identified as a potentialorthologue of Bdp1 in L major (LmBdp1) [20] However asSANT domains are also present in other proteins includingthe subunits of many chromatin-remodeling complexes [9]we further analyzed the sequence and structure of LmBdp1to verify its identity Sequence alignments with Bdp1 ortho-logues from several species showed that LmBdp1 indeedcontains the typical extended SANT domain characterizedby the presence of five 120572-helices (Figure 1(a)) Moreovertwo sequences that flank the extended SANT domain theN-linker and the long arm [11 12] are conserved or semi-conserved in LmBdp1 The N-linker involved in the bindingto the minor groove of the DNA is distinguished by theoccurrence of aromatic residues including the invariably

conserved tryptophan (W) residue that is present in LmBdp1(Figure 1(a)) Nevertheless LmBdp1 does not contain otherconserved aromatic residues (Y291 F294 and Y299) that areimportant for the interaction Bdp1-DNA (Figure 1(a)) [10]The long arm [11] also known as helix C [16] or Bdp1 stem[12] which participates in interactions with Brf1 and subunitC34 of Pol III is also present in LmBdp1 (SupplementaryFigure 1(a)) The tether region located N-terminally to theN-linker (Supplementary Figure 1) interacts with Pol IIIsubunits C128 C37 and C34 and in most Bdp1 orthologuescontains several 120573-sheet structures [11] However unlikemost species the tether region in LmBdp1 is not predictedto fold into 120573-sheet structures (Supplementary Figure 1)Other amino acids that are important for the function ofhuman Bdp1 are conserved in LmBdp1 These include R334(R211 in LmBdp1) K338 (K215 in LmBdp1) and R343 (R220in LmBdp1) (Figure 1(a)) R334 interacts with TBP whileK338 and R343 contact the major groove of the DNA [10]


LmBdp1-PTP

WT

75 kDa

(a)

DAPI LmBdp1 Merge Brightfield

(b)

Figure 2 Nuclear localization of LmBdp1 (a) Western blot analysis with proteins isolated from the L major cell line that expresses theLmBdp1-PTP recombinant protein using an anti-Prot C monoclonal antibody Wild-type parasites were also analyzed as control (b) Thelocation of LmBdp1 labeled with a PTP tag was determined by indirect immunofluorescence assays using anti-Prot C monoclonal antibodyand an Alexa-Fluor 488 conjugated secondary antibody Nucleus (N) and kinetoplast (K) are stained with DAPI Size bars represent 5 120583m

The extended SANT domain is also conserved in Bdp1orthologues in T brucei and T cruzi (Figure 1(a)) as well asin other species of Leishmania (Supplementary Figure 2)

To further study the structure of the extended SANTdomain present in LmBdp1 its predicted three-dimensionalstructure was generated by homology modeling using astemplates the crystal structures of Bdp1 from human (Fig-ure 1(b)) and yeast (Supplementary Figure 1(b)) The archi-tectures of the obtained three-dimensional structures forthe extended SANT domain of LmBdp1 are very similar tothe ones reported for human and yeast [10ndash12] showing aconserved distribution of the five 120572-helices and the long armThus these results and the functional data showed belowdemonstrate that LmjF366530 is indeed the orthologue ofBdp1 in L major

32 LmBdp1 Is a Nuclear Protein In order to determine thecellular distribution of LmBdp1 in L major we performedindirect immunofluorescence experiments To that end a cellline where LmBdp1 was labeled with a C-terminal PTP tagwas generated The PTP tag is constituted by Protein A (ProtA) and Protein C (Prot C) epitopes separated by a tobaccoetch virus (TEV) protease cleavage site [38] The expressionof the recombinant LmBdp1-PTP protein was confirmedby Western blot analysis with an anti-Prot C antibody(Figure 2(a)) Immunofluorescence experiments were carriedout on fixed and permeabilized promastigotes with the sameantibody The images obtained demonstrated that LmBdp1localizes to the nucleus as anticipated for a protein thatregulates transcription (Figure 2(b)) The observed dottedpattern is similar to that obtained with Brf1 in T brucei [30]

33 Targeted Gene Replacement of LmBdp1 To analyze theeffects of the elimination of LmBdp1 on cell growth andtranscription in promastigotes of L major we intended togenerate LmBdp1 null mutant parasites by targeted gene

replacement Although L major Friedlin is a diploid organ-ism some chromosomes are aneuploid [39] However ithas been shown that this strain possesses two copies ofchromosome 36 [40] where the LmBdp1 gene is locatedConsequently two sequential rounds of targeted gene disrup-tion were planned to obtain null mutants of LmBdp1 usingplasmids that contain puromycin (pac) and hygromycin (hyg)resistance genes In these plasmids the selectable markergenes are bounded by 51015840 and 31015840 flanking regions of LmBdp1

The single-knockout cell line for LmBdp1 was generatedby transfecting wild-type promastigotes with the targetingcassette from pΔLmBdp1-pac and clones were selected withpuromycin Southern blot analysis using the 51015840 flankingregion as a probe showed a 62 kb band expected fromtargeted gene replacement with genomicDNA from a single-knockout clone digested with XhoI in addition to the 26 kbband that corresponds to the undisrupted gene (Figure 3)The expected bands of 25 kb (gene replacement) and 17 kb(undisrupted gene) were also observed with SacI-digestedgenomic DNAMoreover the pac gene was amplified by PCRfrom the single-knockout clone (Supplementary Figure 3)Thus these results demonstrate that one copy of LmBdp1 wasreplaced with the pac gene

To obtain the double-knockout cell line for LmBdp1 thesingle-knockout clone was transfected with the targeting cas-sette fromvector pΔLmBdp1-hyg and cells were selectedwithpuromycin and hygromycinThe obtained transfected clonesshowed growth defects (see below) Southern blots of PstI-digested genomic DNA from the double-knockout cell lineshowed that the second allelic copy of LmBdp1 was replacedwith the hyg gene (28 kb band) (Figure 3) The expectedband of 51 kb (pac gene) was also observed However a bandof 46 kb that corresponds to the undisrupted LmBdp1 genewas also detected (Figure 3) PCR analysis also showed thepresence of coding sequences for hyg and pac in the cellline (Supplementary Figure 3)These results indicated that anadditional copy of LmBdp1was present in themutant cell line


X

65256540 3acute5acute

26 Kb

Bdp1

X XP PS S

17 Kb46 Kb


5acute

62 KbPAC

X S XP PS

25 Kb51 Kb

6525654028 Kb

HYG

P PP

3acute

(a)

XhoI SacI PstI

62

26 25

17

5146

28

Kb Kb Kb

WT

SKO

DKO+1

SKO

WT

WT

(b)

Figure 3 Southern blot analysis of LmBdp1 knockout parasites (a) Restriction maps of the wild-type LmBdp1 locus (top map) and mutantloci with a copy of LmBdp1 substituted with the pac gene (middle map) or replaced with the hyg gene (bottom map) Restriction sites forXhoI SacI and PstI are indicated Sizes of predicted restriction fragments are shown The location of the fragment employed as a probe (51015840targeting region) in the Southern blot experiments is denoted with a black bar (b) Southern blot analysis with genomicDNA isolated from theLmBdp1 single-knockout (SKO) clone digested with XhoI and SacI (left figures) and with genomic DNA isolated from the double-knockout+1 (DKO+1) clone digested with PstI (right figure) The 51015840 flanking region was used as a probe

which was named double-knockout +1 (DKO+1)Thus thesedata strongly suggest that LmBdp1 is an essential gene

34 The Amount of the LmBdp1 Protein Was Reduced inthe Mutant Parasites To further analyze the growth defectin the mutant cells growth curves of the DKO+1 cell linewere obtained and compared to growth curves of LmBdp1single-knockout and wild-type parasites (Figure 4(a)) Thedata showed that the DKO+1 cell line is strongly impairedin growth as it multiplies more slowly and to a lower

stationary-phase cell density than single-knockout and wild-type promastigotes (Figure 4(a)) Growth curves of thesingle-knockout clone and wild-type parasites did not revealsignificant differences (Figure 4(a))

To determine the level of the LmBdp1 protein in theDKO+1 cell line recombinant LmBdp1 (LmBdp1r) proteinwas obtained to produce antibodies against it To that endthe complete LmBdp1 gene was amplified by PCR and clonedinto the pColdI vector where it was fused to a 6timesHis tagTheLmBdp1r protein was expressed in E coli purified by nickelaffinity chromatography and used as antigen for polyclonal


0 1 2 3 5 6Days

120

100

80

60

40

20

0

WTSKODKO+1

4 7 8

Cel

lsm

l (x1

06)

(a)

1008060

4020

0

WT DKO+1

60 kDa LmBdp1

TUB50 kDa

WT DKO+1

P

rote

in le

vel

(b)

Figure 4 Growth curves and Western blot analysis of LmBdp1 knockout parasites (a) Growth of promastigotes from wild-type (WT)single-knockout (SKO) and double-knockout +1 (DKO+1) cell lines Values represent means of three experiments Standard deviation barsare shown (b) Western blot analysis of LmBdp1 in wild-type (WT) and double-knockout +1 (DKO+1) parasites using the LmBdp1 antibodyThe bands shown here and from two more independent assays were quantified and plotted (lower graph) Protein levels of LmBdp1 werenormalized to the amount of 120572-tubulin which was used as loading control Standard deviation bars are shown

antibody production in mice (data not shown) Western blotanalysis with the LmBdp1 polyclonal antiserum showed thatcomparing to wild-type cells the amount of LmBdp1 proteinwas decreased by sim70 in the DKO+1 cell line (Figure 4(b))Thus in spite of the presence of an additional copy of theLmBdp1 gene in the DKO+1 parasites the expression of theLmBdp1 protein is considerably diminished

35 Pol III Transcription Is Affected in the Mutant Cell LineTo determine if the reduced levels of LmBdp1 have an effecton Pol III transcription run-on experiments were performedwith isolated nuclei from the DKO+1 cell line and wild-typepromastigotes (Figure 5) The genes analyzed were tRNA-Phe tRNA-Tyr 5S rRNA and U2 snRNA (transcribed byPol III) tRNA-Sec (transcribed by Pol II and Pol III) 120572-tubulinLmjF060200LmjF060210 andLmjF060370 (tran-scribed by Pol II) and the 18S rRNA (transcribed by Pol I)The hybridization signals observed in Figure 5(a) and twomore independent experiments were quantitated setting to100 the transcription signal obtained with wild-type cells(Figure 5(b)) Normalization was performed with 120572-tubulinAs anticipated Pol III transcription was decreased in theDKO+1 cell line since signal from U2 snRNA 5S rRNAtRNA-Phe and tRNA-Tyr was reduced to 33 47 49 and58 of the control value respectively (Figures 5(a) and 5(b))Thus the nuclear run-on data corroborate the involvementof LmBdp1 in Pol III transcription Signal of the tRNA-Sectranscribed by both Pol II and Pol III was diminished to78 of the control value Pol II transcription of 120572-tubulinLmjF060200 LmjF060210 and LmjF060370 was slightlyaffected Interestingly 18S rRNA signal was reproducibly

reduced to sim55 of the control (Figures 5(a) and 5(b))To further analyze this unexpected result the 24S120573 rRNAgene was included in new nuclear run-on experiments Asshown in Figures 5(c) and 5(d) similarly to the 18S rRNAtranscription of the 24S120573 rRNA gene was reduced to 39in the DKO+1 cell line Transcription of the spliced-leader(SL) RNAwas not affected (Figures 5(c) and 5(d)) supportingthe previous results that indicate that LmBdp1 does notparticipate in Pol II transcription

36 LmBdp1 Binds to Pol III Promoters To determinewhether LmBdp1 associates to Pol III promoter regions invivo chromatin immunoprecipitation (ChIP) experimentswere carried out using the L major cell line that expressesLmBdp1 fused to the PTP tag Immunoprecipitations wereconducted with a ChIP-grade anti-Prot A antibody whichrecognizes the two Prot A domains present in the PTP tag[41] andwith a nonspecificmouse immune serumas negativecontrol To assess the binding of LmBdp1-PTP to the L majorgenome qPCR assays were performed with the purifiedDNA Data obtained from three independent experimentsshowed that LmBdp1 is enriched in the 5S rRNA tRNA-AlatRNA-Met and the promoter regions from the U2 and U4snRNAs (tRNA-like sequences) (Figure 6) Enrichment wasalso detected within the U2 snRNA gene which contains aninternal promoter element [25] Thus these results confirmthe binding of LmBdp1 to Pol III promoters As anticipatedenrichment was not observed with the 120572-tubulin gene andthe SL RNA promoter region (Figure 6) Notably we did notfind association of LmBdp1 with the promoter region of therRNA transcription unit which suggests that the reduction


WT DKO+1

18S

200

210

370

TUB

PG

PHE

TYR

SEC

5S

U2

U2

(a)

20018S 210 370 TUB PHE TYR SEC 5S

120

U2

100

80

60

40

20

0

T

rans

crip

tion

WT DKO+1(b)

DKO+1

18S

SL

PG

WT

TUB

24S

(c)

18S TUB SL

T

rans

crip

tion

0

20

40

80

60

100

120

WT DKO+1

24S

(d)

Figure 5 Nuclear run-on analyses with the LmBdp1 double-knockout +1 cell line (a) Labeled nascent RNA from isolated nuclei from theLmBdp1 double-knockout +1 cell line (DKO+1) and wild-type (WT) parasites was hybridized to dot blots of double-stranded DNAs (2 120583g)cloned into pGEM-T Easy The 18S rRNA gene (18S) was tested as a representative Pol I gene The Pol II genes analyzed were LmjF060200(200) LmjF060210 (210) LmjF060370 (370) and 120572-tubulin (TUB) The Pol III genes examined were tRNA-Phe (PHE) tRNA-Tyr (TYR)5S rRNA (5S) and the U2 snRNA (U2) which was examined in duplicate dots The tRNA-Sec (SEC) transcribed by both Pol II and Pol IIIwas also analyzed As control an empty vector was assessed (PG) (b) The signals obtained for each gene in panel (a) and from two moreindependent experiments were quantified and plotted considering as 100 the signal obtainedwithwild-type promastigotes Values representmeans of the three experiments Standard deviation bars are shown RNA levels were normalized to the level of 120572-tubulin (c) Nuclear run-onanalysis performed as indicated in panel (a) The Pol I genes analyzed were 24S120573 rRNA (24S120573) and the 18S rRNA (18S) The Pol II genesexamined were SL RNA (SL) and 120572-tubulin (TUB) As control an empty vector was also analyzed (PG) (d) The signals obtained for eachgene in panel (c) and from two more independent experiments were quantified and plotted as indicated in panel (b)

in 18S and 24S120573 rRNA transcription that was observed in theLmBdp1 DKO+1 cell line (Figure 5) is an indirect effect

4 Discussion

In thisworkwe characterized the orthologue of the Bdp1 sub-unit of transcription factor TFIIIB in L major The presenceof Bdp1 in this ancient protozoan parasite indicates that thebasic mechanisms of regulation of Pol III transcription wereestablished early in the evolution of the eukaryotic lineagesNotably the molecular mass of Bdp1 varies considerablyacross evolution Whereas the predicted weights of isoforms1 of Bdp1 in Homo sapiens and Mus musculus are 2938 and2701 kDa respectively Bdp1 is predicted to be a sim341 kDa

protein in T brucei and a sim44 kDa protein in L majorMolecular masses of 677 and 782 are predicted for Bdp1 inSaccharomyces cerevisiae and Drosophila melanogaster (iso-form A) respectively Sequence identity of LmBdp1 rangesfrom 198 to 228 for the yeast and human orthologuesrespectively higher identities were observed with Bdp1 fromT brucei (285) and T cruzi (353) (Figure 1(a)) Asexpected LmBdp1 is very similar to its orthologues inother species of Leishmania showing identities that rangefrom 797 (with Leishmania panamensis) to 959 (withLeishmania gerbilli) (Supplementary Figure 2)

In spite of molecular mass and sequence variations Bdp1orthologues share the extended SANT domain and otherconserved sequences including the N-terminal region the


Pol I 18S

111 bp 18Sp

Pol II

584 bp

TUB

93 bp SLpSL

Pol III

5S rRNA

149 bp

101 bp

tRNA-Met

130 bp 192 bp

127 bp

U2tRNA-liketRNA-Ala

176 bp

130 bp

U4tRNA-like

tRNA-Pro

(a)

14

12

10

8

6

4Fold

enric

hmen

t

2

0

18Sp SL

p

-tu

b

5S rR

NA

U2

snRN

A

U4

tRN

A li

ke

tRN

A-A

la

tRN

A-M

et

U4

snRN

A

U2

tRN

A li

ke

(b)

Figure 6 Chromatin immunoprecipitation analysis of LmBdp1 (a) Schematic representation of the genes and promoter regions examinedby ChIP experiments (b) Chromatin from a cell line that expresses the recombinant protein LmBdp1-PTP was precipitated with a ChIP-grade anti-Prot A antibody Precipitated DNA was analyzed by qPCR The results from three independent ChIP experiments each analyzedby three qPCR reactions are shown Error bars indicate standard deviations Results are presented as fold enrichment over negative controlprecipitations


long arm and the tether region LmBdp1 possesses thecharacteristic extended SANT domain predicted to foldinto five 120572-helices (Figure 1) Similarly to Bdp1 from yeasta sixth 120572-helix that comprises the long arm is present inLmBdp1 (Supplementary Figure 1) Interestingly the longarm is predicted to be present in all the Bdp1 orthologuesthat we analyzed with the exception of the human protein(Supplementary Figure 1) [10] In yeast the long arm forms acoiled-coil structure with Brf1 homology domain II and endsnext to winged helix domains 2 and 3 from Pol III subunitC34 [11]TheN-linker region is characterized by the presenceof 3-6 aromatic amino acids (W F and Y) (Figure 1(a))However only the conserved W that is located at the endof the N-linker is present in LmBdp1 (Figure 1(a)) Since themissing aromatic residues anchor the N-linker to the majorgroove of the DNA through aromatic-sugar interactions withthe DNA backbone [11] a different type of contacts shouldoccur between the LmBdp1 N-linker and the promoter DNAThe Bdp1 tether involved in interactions with some PolIII subunits is folded into several 120573-sheet structures in Scerevisiae H sapiens Schizosaccharomyces pombe and Dmelanogaster (Supplementary Figure 1) [11 12] Nonethelessin L major and other trypanosomatid parasites this region isnot predicted to fold into 120573-sheets (Supplementary Figure 1)Thus LmBdp1 shares several characteristics with other Bdp1orthologues but it also shows some distinctive features

Our data demonstrate that LmBdp1 participates in PolIII transcription as the DKO+1 cell line showed reducedlevels of tRNAs 5S rRNA and U2 snRNA in nuclear run-on experiments (Figure 5) The association of LmBdp1 withgenes transcribed by Pol III was demonstrated by ChIPanalysis (Figure 6) While nuclear run-on assays exhibited areduction in the transcription levels of 18S and 24S120573 rRNAsChIP analysis did not reveal the binding of LmBdp1 to thepromoter region of the ribosomal transcription unit Thusthe observed decrease in 18S and 24S120573 rRNA transcriptionis most likely a secondary effect caused by the reductionof 5S rRNA as cells coordinate the expression levels ofall rRNA species to regulate ribosome biogenesis [42] Thetarget of rapamycin (TOR) signal-transduction pathway is akey regulator of this process [43] Interestingly while mosteukaryotes possess one or two TOR kinases L major andother trypanosomatids have three different TOR kinases[44] that might coordinate transcription by all nuclear RNApolymerases

We were unable to generate null mutants of LmBdp1as the DKO+1 cell line contained an extra copy of LmBdp1(Figure 3) It is possible that this additional copy wasgenerated while trying to delete the second endogenousgene of LmBdp1 as attempts to knockout essential genesin Leishmania commonly produce alteration in gene copynumber either by gene amplification or changes in ploidy[33 45ndash47]Thus this result strongly suggests that LmBdp1 isessential for the growth of Lmajor promastigotes as has beenreported in yeast [17] Regardless of the moment when theextra LmBdp1 copy was produced the growth of the DKO+1cell line is strongly impaired and the expression of LmBdp1 isreduced by 70 (Figure 4) We tried to restore the expressionlevels of LmBdp1 in theDKO+1 cell line by transfecting it with

the pLmBdp1-PTP vector (data not shown) However severalattempts to obtain transfected parasites were unsuccessfuldue to the inability of theDKO+1 cell line to reach the optimalcell densities for electroporation and to tolerate the drugselection process As an alternative approach we transfectedthe LmBdp1 single-knockout parasites with the pLmBdp1-PTP vector and then tried to knockout the second LmBdp1allele (data not shown) Nevertheless we were not capableof deleting the second endogenous copy of LmBdp1 whichsuggests that the expression of the recombinant LmBdp1-PTPprotein in the resultant cell line was not high enough to allowthe elimination of the second allelic copy of LmBdp1

The function of Bdp1 is regulated by phosphorylationof specific amino acids In logarithmically growing yeastcells four Bdp1 residues (S49 S164 S178 and S586) arephosphorylated by PKA Sch9 and CK2 kinases and Bdp1is dephosphorylated under conditions that reduce Pol IIItranscription [48] Human Bdp1 by contrast is phospho-rylated by CK2 during mitosis to inhibit U6 snRNA tran-scription [49] An in silico search allowed us to identifyseveral potential phosphorylation sites in LmBdp1 includingT308 (PhosTryp score of 0933) S129 (score of 0905) andS327 (score of 0841) (Supplementary Figure 4) NotablyPKA [50] and CK2 [51] are present in Leishmania Thussimilarly to yeast and human the activity of LmBdp1 couldbe controlled by phosphorylation This is supported by thefact that Western blot analysis of LmBdp1 often showed twoormore bands (Figure 4(b) and Supplementary Figure 5) thatmight correspond to different phosphorylation states of theprotein

Besides its role in Pol III transcription initiation Bdp1is also involved in maturation of tRNAs by interacting withRNAse P the enzyme required for the site-specific cleavageof the 51015840 leader sequence of precursor tRNAs [17] Also Bdp1participates in the integration of Ty1 a long terminal repeatretrotransposon upstream of tRNA genes in S cerevisiae[52] Moreover Bdp1 also interacts with Hmt1 a proteinarginine methyltransferase from yeast that inhibits tRNAtranscription by methylating an unknown factor of the PolIII complex [53] Bdp1 seems to also participate in Pol IItranscription termination of noncoding RNAs in the vicinityof tRNA genes [54] It remains to be determined whetherBdp1 also performs multiple functions in L major and othertrypanosomatids

5 Conclusions

In the present study we show that LmBdp1 shares severalsequence and structural features with Bdp1 orthologues fromother species such as the presence of the extended SANTdomain and the long arm But some differences were alsoobserved including the occurrence of only one aromaticresidue in the N-linker and the lack of predicted 120573-sheetstructures in the tether region of LmBdp1 Our data alsodemonstrate that LmBdp1 localizes to the nucleus where itregulates the Pol III transcription of tRNAs 5S rRNA andU2snRNA by associating with their promoter regions Targetedgene replacement analysis strongly suggests that LmBdp1 isessential for the growth of promastigotes of L major Thus


LmBdp1 could be considered a suitable candidate for drugdevelopment against Leishmania

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors have no conflicts of interest to declare

Acknowledgments

This work was supported by grant 251831 from CONACyTby grants IN214715 and IN207118 from UNAM-PAPIIT to SMartınez-Calvillo and by grant 240086 from CONACYT toRebecaGManning-CelaThe authors thankAnaMCevallosand Yolanda I Chirino-Lopez for fruitful discussions andLeticia Avila-Gonzalez and Claudia I Flores-Pucheta fortechnical assistance This work is one of the requirements toobtain the PhD degree in Posgrado en Ciencias Biomedicas(UNAM) for Fiordaliso C Roman-Carraro who was therecipient of a doctoral fellowship from CONACyT (Fellow-ship 294812 CVU 549461)

Supplementary Materials

Supplementary Figure 1 sequence and structure analyses ofconserved regions of Bdp1 Supplementary Figure 2 sequencealignment of the N-linker and extended SANT domain indifferent species of Leishmania Supplementary Figure 3PCR analysis of LmBdp1 single- and double-knockout +1parasites Supplementary Figure 4 predicted phosphory-lated residues in LmBdp1 using the PhosTryp web toolSupplementary Figure 5 Western blot analysis of LmBdp1(Supplementary Materials)

References

[1] I Grummt ldquoLife on a planet of its own Regulation of RNApoly-merase I transcription in the nucleolusrdquo Genes amp Developmentvol 17 no 14 pp 1691ndash1702 2003

[2] X Liu D A Bushnell and R D Kornberg ldquoRNA polymeraseII transcription structure and mechanismrdquo Biochimica et Bio-physica Acta (BBA) - Gene RegulatoryMechanisms vol 1829 pp2ndash8 2013

[3] G Dieci G Fiorino M Castelnuovo M Teichmann and APagano ldquoThe expanding RNA polymerase III transcriptomerdquoTrends in Genetics vol 23 no 12 pp 614ndash622 2007

[4] E Lesniewska andM Boguta ldquoNovel layers of RNApolymeraseIII control affecting tRNA gene transcription in eukaryotesrdquoOpen Biology vol 7 2017

[5] G Dieci M C Bosio B Fermi and R Ferrari ldquoTranscriptionreinitiation by RNA polymerase IIIrdquo Biochimica et BiophysicaActa vol 1829 pp 331ndash341 2013

[6] E P Geiduschek andGA Kassavetis ldquoTheRNApolymerase IIItranscription apparatusrdquo Journal of Molecular Biology vol 310pp 1ndash26 2001

[7] G A Kassavetis S Han S Naji and E P Geiduschek ldquoTheRoleof Transcription Initiation Factor IIIB Subunits in PromoterOpening Probed by Photochemical Cross-linkingrdquoThe Journalof Biological Chemistry vol 278 no 20 pp 17912ndash17917 2003

[8] JHuet C ConesaNManaudNChaussivert andA SentenacldquoInteractions between yeast TFIIIB componentsrdquo Nucleic AcidsResearch vol 22 no 16 pp 3433ndash3439 1994

[9] L A Boyer R R Latek andC L Peterson ldquoThe SANTdomaina unique histone-tail-binding modulerdquo Nature Reviews Molec-ular Cell Biology vol 5 no 2 pp 158ndash163 2004

[10] J Gouge N Guthertz K Krammet al ldquoMolecularmechanismsof Bdp1 in TFIIIB assembly and RNA polymerase III transcrip-tion initiationrdquo Nature Communications vol 8 p 130 2017

[11] M K Vorlander H Khatter R Wetzel W J Hagen and C WMuller ldquoMolecular mechanism of promoter opening by RNApolymerase IIIrdquo Nature vol 553 no 7688 pp 295ndash300 2018

[12] G Abascal-Palacios E P Ramsay F Beuron E Morris and AVannini ldquoStructural basis of RNA polymerase III transcriptioninitiationrdquo Nature vol 553 no 7688 pp 301ndash306 2018

[13] H Hu CWu J Lee andH Chen ldquoA Region of Bdp1 Necessaryfor Transcription Initiation That Is Located within the RNAPolymerase III Active Site CleftrdquoMolecular andCellular Biologyvol 35 no 16 pp 2831ndash2840 2015

[14] G A Kassavetis R Driscoll and E P Geiduschek ldquo Mappingthe Principal Interaction Site of the Brf1 and Bdp1 Subunitsof Saccharomyces cerevisiae TFIIIB rdquo The Journal of BiologicalChemistry vol 281 no 20 pp 14321ndash14329 2006

[15] S K Khoo C C Wu Y C Lin J C Lee and H T ChenldquoMapping the protein interaction network for TFIIB-relatedfactor Brf1 in the RNA polymerase III preinitiation complexrdquoMolecular and Cellular Biology vol 34 pp 551ndash559 2014

[16] Y Han C Yan S Fishbain I Ivanov and Y He ldquoStructuralvisualization of RNApolymerase III transcriptionmachineriesrdquoCell Discovery vol 4 2018

[17] A Ishiguro G A Kassavetis and E P Geiduschek ldquoEssentialRoles of Bdp1 a Subunit of RNAPolymerase III Initiation FactorTFIIIB in Transcription and tRNA ProcessingrdquoMolecular andCellular Biology vol 22 no 10 pp 3264ndash3275 2002

[18] Y Liao IMWillis andRDMoir ldquoTheBrf1 andBdp1 Subunitsof Transcription Factor TFIIIB Bind to Overlapping Sites inthe Tetratricopeptide Repeats of Tfc4rdquoThe Journal of BiologicalChemistry vol 278 no 45 pp 44467ndash44474 2003

[19] S E von and S Tenzer ldquoCutaneous leishmaniasis Distinctfunctions of dendritic cells and macrophages in the interactionof the host immune system with Leishmania majorrdquo Interna-tional Journal of Medical Microbiology 2017

[20] A Gunzl L Vanhamme and P J Myler ldquoTranscription intrypanosomes a different means to the endrdquo in Trypanosomesafter the Genome J D Barry R McCulloch J C Mottramand A Acosta-Serrano Eds pp 177ndash208 Horizon BioscienceWymondham UK 2007

[21] S Martinez-Calvillo J C Vizuet-de-Rueda L E Florencio-Martinez R G Manning-Cela and E E Figueroa-AnguloldquoGene expression in trypanosomatid parasitesrdquo Journal ofBiomedicine and Biotechnology Article ID 525241 2010

[22] C Clayton ldquoThe regulation of trypanosome gene expression byRNA-binding proteinsrdquoPLoS Pathogens vol 9 article e10036802013

[23] V Nakaar A O Dare D Hong E Ullu and C TschudildquoUpstream tRNA genes are essential for expression of small


nuclear and cytoplasmic RNA genes in trypanosomesrdquoMolec-ular and Cellular Biology vol 14 no 10 pp 6736ndash67421994

[24] V Nakaar A Gunzl E Ullu and C Tschudi ldquoStructure of theTrypanosoma brucei U6 snRNA gene promoterrdquoMolecular andBiochemical Parasitology vol 88 no 1-2 pp 13ndash23 1997

[25] S Rojas-Sanchez E Figueroa-Angulo R Moreno-Campos LE Florencio-Martinez R G Manning-Cela and S Martinez-Calvillo ldquoTranscription of Leishmania major U2 small nuclearRNA gene is directed by extragenic sequences located within atRNA-like and a tRNA-Ala generdquo Parasites amp Vectors vol 9 p401 2016

[26] RMoreno-Campos L E Florencio-Martınez TNepomuceno-Mejıa et al ldquoMolecular characterization of 5S ribosomal RNAgenes and transcripts in the protozoan parasite Leishmaniamajorrdquo Parasitology vol 143 no 14 pp 1917ndash1929 2016

[27] A C Ivens C S Peacock E A Worthey et al ldquoThe genome ofthe kinetoplastid parasite Leishmania majorrdquo Science vol 309no 5733 pp 436ndash442 2005

[28] J Ruan G K Arhin E Ullu and C Tschudi ldquoFunctional Char-acterization of a Trypanosoma brucei TATA-Binding Protein-Related Factor Points to a Universal Regulator of Transcriptionin Trypanosomesrdquo Molecular and Cellular Biology vol 24 no21 pp 9610ndash9618 2004

[29] A Das Q Zhang J B Palenchar B Chatterjee G A Crossand V Bellofatto ldquoTrypanosomal TBP Functions with theMultisubunit Transcription Factor tSNAP To Direct Spliced-Leader RNA Gene ExpressionrdquoMolecular and Cellular Biologyvol 25 no 16 pp 7314ndash7322 2005

[30] D E Velez-Ramirez L E Florencio-Martinez G Romero-Meza et al ldquoBRF1 a subunit of RNA polymerase III transcrip-tion factor TFIIIB is essential for cell growth of Trypanosomabruceirdquo Parasitology vol 142 Article ID S0031182015001122 pp1563ndash1573 2015

[31] G Romero-Meza D E Velez-Ramırez L E Florencio-Martınez et al ldquo Maf1 is a negative regulator of transcriptionin Trypanosoma bruceirdquoMolecular Microbiology vol 103 no 3pp 452ndash468 2017

[32] A Palmeri P F Gherardini P Tsigankov et al ldquoPhosTryp Aphosphorylation site predictor specific for parasitic protozoa ofthe family trypanosomatidaerdquo BMC Genomics vol 12 p 6142011

[33] S Martınez-Calvillo K Stuart and P J Myler ldquoPloidy changesassociated with disruption of two adjacent genes on Leishmaniamajor chromosome 1rdquo International Journal for Parasitologyvol 35 no 4 pp 419ndash429 2005

[34] T Nepomuceno-Mejıa L E Florencio-Martınez and SMartınez-Calvillo ldquoNucleolar division in the promastigotestage of leishmania major parasite A Nop56 point of viewrdquoBioMed Research International vol 2018 article 1641839 2018

[35] H Ji ldquoLysis of cultured cells for immunoprecipitationrdquo ColdSpring Harbor Protocols vol 2010 2010

[36] N E Padilla-Mejıa L E Florencio-Martınez R Moreno-Campos et al ldquoThe Selenocysteine tRNA Gene in Leishmaniamajor Is Transcribed by both RNA Polymerase II and RNAPolymerase IIIrdquo Eukaryotic Cell vol 14 no 3 pp 216ndash227 2015

[37] J C Vizuet-de-Rueda L E Florencio-Martınez N E Padilla-Mejıa R Manning-Cela R Hernandez-Rivas and S Martınez-Calvillo ldquoRibosomal RNA Genes in the Protozoan ParasiteLeishmania major Possess a Nucleosomal Structurerdquo Protistvol 167 no 2 pp 121ndash135 2016

[38] B Schimanski T N Nguyen and A Gunzl ldquoHighly efficienttandem affinity purification of trypanosome protein complexesbased on a novel epitope combinationrdquo Eukaryotic Cell vol 4no 11 pp 1942ndash1950 2005

[39] Y Sterkers L Lachaud L Crobu P Bastien and M PagesldquoFISH analysis reveals aneuploidy and continual generationof chromosomal mosaicism in Leishmania majorrdquo CellularMicrobiology vol 13 no 2 pp 274ndash283 2011

[40] M B Rogers J D Hilley N J Dickens et al ldquoChromosomeand gene copy number variation allow major structural changebetween species and strains of Leishmaniardquo Genome Researchvol 21 no 12 pp 2129ndash2142 2011

[41] J H Lee G Cai A K Panigrahi et al ldquoA TFIIH-AssociatedMediatorHead Is a Basal Factor of Small Nuclear Spliced LeaderRNA Gene Transcription in Early-Diverged TrypanosomesrdquoMolecular and Cellular Biology vol 30 no 23 pp 5502ndash55132010

[42] C Mayer and I Grummt ldquoRibosome biogenesis and cellgrowth mTOR coordinates transcription by all three classes ofnuclear RNA polymerasesrdquoOncogene vol 25 no 48 pp 6384ndash6391 2006

[43] C K Tsang H Liu and X S Zheng ldquomTOR binds to thepromoters of RNA polymerase I- and III-transcribed genesrdquoCell Cycle vol 9 no 5 pp 953ndash957 2014

[44] S L Madeira da and S M Beverley ldquoExpansion of the targetof rapamycin (TOR) kinase family and function in Leishmaniashows that TOR3 is required for acidocalcisome biogenesis andanimal infectivityrdquo in Proceedings of the National Academy ofSciences of the United States of America vol 107 pp 11965ndash119702010

[45] A K Cruz R Titus and S M Beverley ldquoPlasticity in chromo-some number and testing of essential genes in Leishmania bytargetingrdquo Proceedings of the National Acadamy of Sciences ofthe United States of America vol 90 no 4 pp 1599ndash1603 1993

[46] J C Mottram B P McCready K G Brown and K MGrant ldquoGene disruptions indicate an essential function forthe LmmCRK1 cdc2-related kinase of Leishmania mexicanardquoMolecular Microbiology vol 22 no 3 pp 573ndash582 1996

[47] R Balana-Fouce C Garcia-Estrada Y Perez-Pertejo and RMReguera ldquoGene disruption of the DNA topoisomerase IB smallsubunit induces a non-viable phenotype in the hemoflagellateLeishmania majorrdquo BMCMicrobiology vol 8 p 113 2008

[48] J Lee RDMoir and IMWillis ldquoDifferential Phosphorylationof RNA Polymerase III and the Initiation Factor TFIIIB inSaccharomyces cerevisiaerdquo PLoS ONE vol 10 article e01272252015

[49] P Hu K Samudre S Wu Y Sun and N Hernandez ldquoCK2Phosphorylation of Bdp1 Executes Cell Cycle-Specific RNAPolymerase III Transcription Repressionrdquo Molecular Cell vol16 no 1 pp 81ndash92 2004

[50] M Genestra L Cysne-Finkelstein and L Leon ldquoProtein kinaseA activity is associated with metacyclogenesis inLeishmaniaamazonensisrdquo Cell Biochemistry amp Function vol 22 no 5 pp315ndash320 2004

[51] P M L Dutra D P Vieira J R Meyer-Fernandes M AC Silva-Neto and A H Lopes ldquoStimulation of Leishmaniatropica protein kinase CK2 activities by platelet-activatingfactor (PAF)rdquo Acta Tropica vol 111 no 3 pp 247ndash254 2009

[52] N Bachman M E Gelbart T Tsukiyama and J D BoekeldquoTFIIIB subunit Bdp1p is required for periodic integration ofthe Ty1 retrotransposon and targeting of Isw2p to S cerevisiaetDNAsrdquoGenes ampDevelopment vol 19 no 8 pp 955ndash964 2005


[53] E J Milliman Z Hu and M C Yu ldquoGenomic insights ofprotein arginine methyltransferase Hmt1 binding reveals novelregulatory functionsrdquo BMCGenomics vol 13 no 1 p 728 2012

[54] K Roy J Gabunilas A Gillespie D Ngo and G F Chanf-reau ldquoCommon genomic elements promote transcriptional andDNA replication roadblocksrdquo Genome Research vol 26 no 10pp 1363ndash1375 2016

Hindawiwwwhindawicom

International Journal of

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Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



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GenomicsInternational Journal of


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The Scientific World Journal

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BioinformaticsAdvances in

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Cell BiologyInternational Journal of



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MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


promoters which are constituted by three small sequenceelements located upstream of the genes (TATA box proximalsequence element and distal sequence element) [5]

General transcription factors TFIIIA TFIIIB and TFIIICare required for accurate initiation of Pol III transcription[6] TFIIIB is needed to form a transcriptionally activepreinitiation complex (PIC) at all three types of promoterregions as it participates in Pol III recruitment and promoteropening [7] TFIIIB consists of three subunits the TATA-binding protein (TBP) the TFIIB-related factor 1 (Brf1)and B double prime 1 (Bdp1) [8] Notably Bdp1 is specificto Pol III transcription since it does not show homologywith any of the general transcription factors of other RNApolymerases Early studies showed that Bdp1 contains aSANT (Swi3 Ada2 N-Cor and TFIIIB) domain which is ahighly conserved sim50-amino acid motif present in severalproteins involved in transcriptional regulation [9] Howeverrecent analysis established that the SANT domain present inBdp1 from human and yeast is actually larger and possessesother distinguished features and accordingly it was renamedextended SANT domain [10ndash12] In yeast the Bdp1 extendedSANT domain is essential for cell viability as it is neededfor Pol III PIC assembly and stability [13] The extendedSANT domain interacts directly with DNA andwith subunitsBrf1 and TBP [14 15] Other regions of Bdp1 associate withsubunits C128 C37 and C34 of Pol III [11 13 16] and subunitTfc4 of TFIIIC [17 18] Thus Bdp1 establishes an intricatenetwork of interactions within the Pol III PIC

The trypanosomatid parasite Leishmania major is theetiological agent of cutaneous leishmaniasis in Asia andAfrica [19] The parasite is transmitted to humans throughthe bite of infected sandflies of the genus Phlebotomus Inaddition to their medical importance L major and othertrypanosomatid protozoa such as Trypanosoma brucei andTrypanosoma cruzi are relevant because they express theirgenes by uncommon processes including Pol II polycistronictranscription and trans-splicing [20ndash22]

Little is known in trypanosomatids about either the DNAsequences or the proteins that participate in Pol III tran-scription initiation Unlike other organisms all snRNA genesare transcribed by Pol III in these parasites [23] NotablysnRNA expression in T brucei and L major is controlledby extragenic boxes A and B located within tRNA or tRNA-like sequences [24 25] Although 5S rRNA genes contain thecharacteristic gene-internal elements their promoter activityhas not been proven [26] Neither TFIIIA nor TFIIIC hasbeen identified in trypanosomatids [27] However ortho-logues of TFIIIB subunits TBP and Brf1 have been studied inT brucei TBP which participates in transcription by all threeRNA polymerases has been primarily investigated in thecontext of Pol II transcription of the spliced-leader (SL) RNAgenes [28 29] On the other hand knockdown analysis byRNA interference demonstrated that Brf1 is indeed involvedin Pol III transcription and that it is indispensable for cellsurvival of procyclic forms of T brucei [30] Another proteinthat participates in the regulation of Pol III transcription in Tbrucei isMaf1 which inhibits tRNA and snRNA transcriptionby associating with their promoter regions [31]

Here we identified and characterized the TFIIIB subunitBdp1 in L major (LmBdp1) Bioinformatic analysis showsthat LmBdp1 possesses the characteristic extended SANTdomain and other conserved regions that flank this domainAttempts to generate null mutants produced a cell line withan additional copy of LmBdp1 Nevertheless Western blotanalysis showed that the levels of LmBdp1 were considerablydiminished in the mutant cell line Notably the growth ofthis cell line was significantly reduced in relation to wild-type cells Nuclear run-on assays demonstrated that Pol IIItranscription was affected in the mutant cell line and ChIPexperiments showed that LmBdp1 binds to 5S rRNA tRNAand snRNA genes

2 Materials and Methods

21 In Silico Analyses Sequences were obtained fromTriTrypDB database (release 40) (httptritrypdborgtritrypdb) from the National Center for BiotechnologyInformation (NCBI) database (httpwwwncbinlmnihgov)and from Universal Protein Resource (UniProt) Multiplesequence alignments were made with the Clustal Omegaprogram (httpwwwebiacukToolsmsaclustalo) andshaded manually To predict the protein secondary structurethe PSIPRED Protein Sequence Analysis Workbench (httpbioinfcsuclacukpsipred) was used Homology modelingwas performedwith SWISS-MODEL v 37 (httpswissmodelexpasyorginteractive) Swiss-PDB Viewer program (httpwwwexpasyorgspdbv) and PyMOL v 211 (httpspymolorg2) using the crystallographic structure of humanextended SANT domain (PDB id 5n9g) and yeast Bdp1(PDB id 6F41) as templates Phosphorylated residues werepredicted with PhosTryp (httpphostrypbiouniroma2it)which was developed for the specific identification ofphosphorylated residues in Trypanosomatid proteins as themethod has been trained using phosphoproteomic data fromLeishmania T brucei and T cruzi [32]

22 Cell Culture and Transfection Promastigotes from Lmajor MHOMIL81Friedlin (LSB-1321) were grown in BMmedium (1times M199 medium pH 72 containing 10 heat-inactivated fetal bovine serum 95 gl brain heart infu-sion 40mM HEPES 001mgml hemin 00002 biotin100 IUml penicillin 100 120583gml streptomycin and 1times L-glutamine) at 26∘C and harvested in the mid-log phase Togenerate the L major cell line that expresses LmBdp1 fusedto a PTP tag wild-type promastigotes were transfected byelectroporation with the LmBdp1-PTP vector Briefly 4 times107 promastigotes in 04ml electroporation buffer (25mMHEPES 120mM KCl 015mM CaCl

2 5mM MgCl

2 10mM

KH2PO4 10mM K

2HPO4 2mM EDTA pH 76) were trans-

fected with 10 120583g of LmBdp1-PTP by electroporation at 1600V 50 120583F and 25 Ω (BTX Electro Square Porator ECM 830)Cells were grown for 24 h before spreading on plates contain-ing 07 Seaplaque GTG agarose (FMC Bioproducts) in BMmedium with 50 120583gml G418 Some of the isolated colonieswere selected for further analysis The same electroporationconditions were used for transfections with the pac and hygtargeting cassettes for the generation of knockout parasites















H1 H4 H5H3

H1 H3 H4 H5H2

H2

(a)

LmBdp1

H1

H3

H4

H2 H2

H5

N-LINKER

HsBdp1

N-LINKERH1

H3

H4

Merge

N-LINKERH1

H3

H4

H2

H5H5

(b)


3 Results




LmBdp1-PTP

WT

75 kDa

(a)


(b)










X


26 Kb

Bdp1

X XP PS S

17 Kb46 Kb


5acute

62 KbPAC

X S XP PS

25 Kb51 Kb

6525654028 Kb

HYG

P PP

3acute

(a)

XhoI SacI PstI

62

26 25

17

5146

28

Kb Kb Kb

WT

SKO

DKO+1

SKO

WT

WT

(b)







0 1 2 3 5 6Days

120

100

80

60

40

20

0

WTSKODKO+1

4 7 8

Cel

lsm

l (x1

06)

(a)

1008060

4020

0

WT DKO+1

60 kDa LmBdp1

TUB50 kDa

WT DKO+1

P

rote

in le

vel

(b)







WT DKO+1

18S

200

210

370

TUB

PG

PHE

TYR

SEC

5S

U2

U2

(a)


120

U2

100

80

60

40

20

0

T

rans

crip

tion

WT DKO+1(b)

DKO+1

18S

SL

PG

WT

TUB

24S

(c)

18S TUB SL

T

rans

crip

tion

0

20

40

80

60

100

120

WT DKO+1

24S

(d)



4 Discussion





Pol I 18S

111 bp 18Sp

Pol II

584 bp

TUB

93 bp SLpSL

Pol III

5S rRNA

149 bp

101 bp

tRNA-Met

130 bp 192 bp

127 bp

U2tRNA-liketRNA-Ala

176 bp

130 bp

U4tRNA-like

tRNA-Pro

(a)

14

12

10

8

6

4Fold

enric

hmen

t

2

0

18Sp SL

p

-tu

b

5S rR

NA

U2

snRN

A

U4

tRN

A li

ke

tRN

A-A

la

tRN

A-M

et

U4

snRN

A

U2

tRN

A li

ke

(b)









5 Conclusions




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018
















H1 H4 H5H3

H1 H3 H4 H5H2

H2

(a)

LmBdp1

H1

H3

H4

H2 H2

H5

N-LINKER

HsBdp1

N-LINKERH1

H3

H4

Merge

N-LINKERH1

H3

H4

H2

H5H5

(b)


3 Results




LmBdp1-PTP

WT

75 kDa

(a)


(b)










X


26 Kb

Bdp1

X XP PS S

17 Kb46 Kb


5acute

62 KbPAC

X S XP PS

25 Kb51 Kb

6525654028 Kb

HYG

P PP

3acute

(a)

XhoI SacI PstI

62

26 25

17

5146

28

Kb Kb Kb

WT

SKO

DKO+1

SKO

WT

WT

(b)







0 1 2 3 5 6Days

120

100

80

60

40

20

0

WTSKODKO+1

4 7 8

Cel

lsm

l (x1

06)

(a)

1008060

4020

0

WT DKO+1

60 kDa LmBdp1

TUB50 kDa

WT DKO+1

P

rote

in le

vel

(b)







WT DKO+1

18S

200

210

370

TUB

PG

PHE

TYR

SEC

5S

U2

U2

(a)


120

U2

100

80

60

40

20

0

T

rans

crip

tion

WT DKO+1(b)

DKO+1

18S

SL

PG

WT

TUB

24S

(c)

18S TUB SL

T

rans

crip

tion

0

20

40

80

60

100

120

WT DKO+1

24S

(d)



4 Discussion





Pol I 18S

111 bp 18Sp

Pol II

584 bp

TUB

93 bp SLpSL

Pol III

5S rRNA

149 bp

101 bp

tRNA-Met

130 bp 192 bp

127 bp

U2tRNA-liketRNA-Ala

176 bp

130 bp

U4tRNA-like

tRNA-Pro

(a)

14

12

10

8

6

4Fold

enric

hmen

t

2

0

18Sp SL

p

-tu

b

5S rR

NA

U2

snRN

A

U4

tRN

A li

ke

tRN

A-A

la

tRN

A-M

et

U4

snRN

A

U2

tRN

A li

ke

(b)









5 Conclusions




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



LmBdp1-PTP

WT

75 kDa

(a)


(b)










X


26 Kb

Bdp1

X XP PS S

17 Kb46 Kb


5acute

62 KbPAC

X S XP PS

25 Kb51 Kb

6525654028 Kb

HYG

P PP

3acute

(a)

XhoI SacI PstI

62

26 25

17

5146

28

Kb Kb Kb

WT

SKO

DKO+1

SKO

WT

WT

(b)







0 1 2 3 5 6Days

120

100

80

60

40

20

0

WTSKODKO+1

4 7 8

Cel

lsm

l (x1

06)

(a)

1008060

4020

0

WT DKO+1

60 kDa LmBdp1

TUB50 kDa

WT DKO+1

P

rote

in le

vel

(b)







WT DKO+1

18S

200

210

370

TUB

PG

PHE

TYR

SEC

5S

U2

U2

(a)


120

U2

100

80

60

40

20

0

T

rans

crip

tion

WT DKO+1(b)

DKO+1

18S

SL

PG

WT

TUB

24S

(c)

18S TUB SL

T

rans

crip

tion

0

20

40

80

60

100

120

WT DKO+1

24S

(d)



4 Discussion





Pol I 18S

111 bp 18Sp

Pol II

584 bp

TUB

93 bp SLpSL

Pol III

5S rRNA

149 bp

101 bp

tRNA-Met

130 bp 192 bp

127 bp

U2tRNA-liketRNA-Ala

176 bp

130 bp

U4tRNA-like

tRNA-Pro

(a)

14

12

10

8

6

4Fold

enric

hmen

t

2

0

18Sp SL

p

-tu

b

5S rR

NA

U2

snRN

A

U4

tRN

A li

ke

tRN

A-A

la

tRN

A-M

et

U4

snRN

A

U2

tRN

A li

ke

(b)









5 Conclusions




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



X


26 Kb

Bdp1

X XP PS S

17 Kb46 Kb


5acute

62 KbPAC

X S XP PS

25 Kb51 Kb

6525654028 Kb

HYG

P PP

3acute

(a)

XhoI SacI PstI

62

26 25

17

5146

28

Kb Kb Kb

WT

SKO

DKO+1

SKO

WT

WT

(b)







0 1 2 3 5 6Days

120

100

80

60

40

20

0

WTSKODKO+1

4 7 8

Cel

lsm

l (x1

06)

(a)

1008060

4020

0

WT DKO+1

60 kDa LmBdp1

TUB50 kDa

WT DKO+1

P

rote

in le

vel

(b)







WT DKO+1

18S

200

210

370

TUB

PG

PHE

TYR

SEC

5S

U2

U2

(a)


120

U2

100

80

60

40

20

0

T

rans

crip

tion

WT DKO+1(b)

DKO+1

18S

SL

PG

WT

TUB

24S

(c)

18S TUB SL

T

rans

crip

tion

0

20

40

80

60

100

120

WT DKO+1

24S

(d)



4 Discussion





Pol I 18S

111 bp 18Sp

Pol II

584 bp

TUB

93 bp SLpSL

Pol III

5S rRNA

149 bp

101 bp

tRNA-Met

130 bp 192 bp

127 bp

U2tRNA-liketRNA-Ala

176 bp

130 bp

U4tRNA-like

tRNA-Pro

(a)

14

12

10

8

6

4Fold

enric

hmen

t

2

0

18Sp SL

p

-tu

b

5S rR

NA

U2

snRN

A

U4

tRN

A li

ke

tRN

A-A

la

tRN

A-M

et

U4

snRN

A

U2

tRN

A li

ke

(b)









5 Conclusions




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0 1 2 3 5 6Days

120

100

80

60

40

20

0

WTSKODKO+1

4 7 8

Cel

lsm

l (x1

06)

(a)

1008060

4020

0

WT DKO+1

60 kDa LmBdp1

TUB50 kDa

WT DKO+1

P

rote

in le

vel

(b)







WT DKO+1

18S

200

210

370

TUB

PG

PHE

TYR

SEC

5S

U2

U2

(a)


120

U2

100

80

60

40

20

0

T

rans

crip

tion

WT DKO+1(b)

DKO+1

18S

SL

PG

WT

TUB

24S

(c)

18S TUB SL

T

rans

crip

tion

0

20

40

80

60

100

120

WT DKO+1

24S

(d)



4 Discussion





Pol I 18S

111 bp 18Sp

Pol II

584 bp

TUB

93 bp SLpSL

Pol III

5S rRNA

149 bp

101 bp

tRNA-Met

130 bp 192 bp

127 bp

U2tRNA-liketRNA-Ala

176 bp

130 bp

U4tRNA-like

tRNA-Pro

(a)

14

12

10

8

6

4Fold

enric

hmen

t

2

0

18Sp SL

p

-tu

b

5S rR

NA

U2

snRN

A

U4

tRN

A li

ke

tRN

A-A

la

tRN

A-M

et

U4

snRN

A

U2

tRN

A li

ke

(b)









5 Conclusions




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



WT DKO+1

18S

200

210

370

TUB

PG

PHE

TYR

SEC

5S

U2

U2

(a)


120

U2

100

80

60

40

20

0

T

rans

crip

tion

WT DKO+1(b)

DKO+1

18S

SL

PG

WT

TUB

24S

(c)

18S TUB SL

T

rans

crip

tion

0

20

40

80

60

100

120

WT DKO+1

24S

(d)



4 Discussion





Pol I 18S

111 bp 18Sp

Pol II

584 bp

TUB

93 bp SLpSL

Pol III

5S rRNA

149 bp

101 bp

tRNA-Met

130 bp 192 bp

127 bp

U2tRNA-liketRNA-Ala

176 bp

130 bp

U4tRNA-like

tRNA-Pro

(a)

14

12

10

8

6

4Fold

enric

hmen

t

2

0

18Sp SL

p

-tu

b

5S rR

NA

U2

snRN

A

U4

tRN

A li

ke

tRN

A-A

la

tRN

A-M

et

U4

snRN

A

U2

tRN

A li

ke

(b)









5 Conclusions




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Pol I 18S

111 bp 18Sp

Pol II

584 bp

TUB

93 bp SLpSL

Pol III

5S rRNA

149 bp

101 bp

tRNA-Met

130 bp 192 bp

127 bp

U2tRNA-liketRNA-Ala

176 bp

130 bp

U4tRNA-like

tRNA-Pro

(a)

14

12

10

8

6

4Fold

enric

hmen

t

2

0

18Sp SL

p

-tu

b

5S rR

NA

U2

snRN

A

U4

tRN

A li

ke

tRN

A-A

la

tRN

A-M

et

U4

snRN

A

U2

tRN

A li

ke

(b)









5 Conclusions




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









5 Conclusions




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Data Availability




Acknowledgments




References




























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


tfiiib subunit bdp1 participates in rna polymerase iii...

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