Structural analysis reveals the characteristicfeatures of Mtr4, a DExH helicase involvedin nuclear RNA processing and surveillanceJohn R. Weir, Fabien Bonneau, Jendrik Hentschel, and Elena Conti1

Max-Planck-Institute of Biochemistry, Department of Structural Cell Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany

Edited* by Juli Feigon, University of California, Los Angeles, CA, and approved June 1, 2010 (received for review April 12, 2010)

Mtr4 is a conserved RNA helicase that functions together with thenuclear exosome. It participates in the processing of structuredRNAs, including thematurationof 5.8S ribosomal RNA (rRNA). It alsointeracts with the polyadenylating Trf4-Air2 heterodimer to formthe so-called TRAMP (Trf4-Air2-Mtr4 Polyadenylation) complex.TRAMP is involved in exosome-mediated degradation of aberrantRNAs in nuclear surveillance pathways. We report the 2.9-Å resolu-tion crystal structure of Saccharomyces cerevisiae Mtr4 in complexwith ADP and RNA. The structure shows a central ATPase coresimilar to that of other DExH helicases. Inserted in the DExH coreis a region characteristic of Mtr4 orthologues that folds into anelongated stalk connected to a β-barrel domain. This domain showsunexpected similarity to the KOW domain of L24, a ribosomal pro-tein that binds 23S rRNA. We find that indeed the KOW domainof Mtr4 is able to bind in vitro transcribed tRNAiMet, suggesting itmight assist in presenting RNA substrates to the helicase core. Theinteraction ofMtr4with Trf4-Air2 ismediated not by the stalk/KOWinsertion but by the DExH core. We find that in the context of theTRAMP complex, the DExH core functions independently in vitroas an RNA helicase and a protein-binding platform. Mtr4 has thusevolved specific structural and surface features to perform itsmultiple functions.

The eukaryotic RNA exosome is an essential, ubiquitous, andconserved protein complex that acts in virtually all pathways

where ribonucleic acids undergo 3′–5′ processing or degradation(reviewed in ref. 1). In particular, the exosome participates in thediscrete trimming during the processing of structured RNA pre-cursors, in the turnover of both precursor and mature codingRNAs and in quality control pathways that detect and degradeaberrant RNA molecules (reviewed in ref. 2). The exosome isa complex of 10 different subunits (reviewed in ref. 3). Workon the yeast exosome proteins has shown that the RNase activ-ities of the core complex reside in a single subunit, Rrp44, whichcontains a 3′–5′ exoribonuclease activity in the C-terminal do-main and an endonucleolytic cleavage activity in the N-terminaldomain (4–8). The other nine core subunits are catalytically inert,but are nevertheless essential for the viability of yeast cells (9).The catalytically inactive core of the eukaryotic exosome has aring-like architecture (4) that functions both to recruit auxiliaryproteins and to bind RNA substrates by threading them to theRrp44 nuclease (reviewed in refs. 2 and 3). An important conse-quence of this arrangement of subunits is that the architecturerestricts and fine-tunes access to the exonuclease site (10). In-deed, the relatively poor ribonuclease activity of the exosomein vitro has been observed since its discovery (9), promptingthe quest for activating factors that would account for the rapidand processive activity that the complex shows in vivo.

Mtr4 (also known as Dob1p) is an auxiliary factor requiredfor most of the functions of the exosome in the yeast nucleus (re-viewed in refs. 11 and 12). It is an RNA-dependent ATPase that islocalized both in the nucleolus and nucleoplasm (13, 14). In vitro,Mtr4 is capable of unwinding RNA duplexes in the 3′–5′ direction(15, 16). In vivo, mutation or depletion of Mtr4 results in defects

in ribosome biogenesis similar to those observed in strainsdepleted of the exosome associated nuclease Rrp6 or of core exo-some subunits (14). In particular, the absence of functional Mtr4impairs the trimming of the 7S rRNA precursor to the mature5.8S rRNA and the degradation of an excised pre-rRNA spacerfragment (14). A similar accumulation of 3′-extended 5.8S rRNAprecursors is observed upon knockdown of the orthologue inhuman cells (17). Mtr4 is highly conserved across eukaryotes,with 50% sequence identity shared between the yeast and humanproteins.

Mtr4 participates not only in the processing of various struc-tured RNAs including snRNAs and snoRNAs (18), but also innuclear RNA surveillance pathways that involve the so-calledTRAMP (Trf4-Air2-Mtr4 Polyadenylation) complex (reviewedin ref. 11). TRAMP is formed by the direct interaction ofMtr4 with the noncanonical poly(A)polymerase Trf4 and itscofactor, the putative RNA-binding protein Air2 (19–21). Thecomplex acts by adding a short single-stranded poly(A) overhangto the 3′ end of aberrant or unstable transcripts such as a hypo-methylated initiator tRNA (tRNAiMet) (19, 21, 22). The helicaseis thought to unwind the structured regions of tRNAiMet andpresent it to the exosome as a substrate for degradation, althoughit is unclear how the association betweenMtr4 and the exosome isachieved.

Mtr4 shares more than 30% sequence identity with Ski2, aputative RNA helicase required for all known functions of theexosome in the cytoplasm (23). Both are DExH-box ATPases witha RecA-fold core related to that of archaeal Hel308, a DNAhelicase whose structure has been determined with a partiallyunwound DNA duplex (24). To address what are features char-acteristic of Mtr4 orthologues and their regulation, we have de-termined the crystal structure of Mtr4 in complex with RNA andcharacterized its mode of interaction with Trf4-Air2.

Results and DiscussionCrystal Structure Determination of Yeast Mtr4-Δ80. We identifiedby limited proteolysis the stable and conserved RNA-bindingregion of Saccharomyces cerevisiae Mtr4 encompassing residues81–1073 (Mtr4-Δ80) (Figs. S1 and S2). To crystallize Mtr4-Δ80, we incubated the purified protein with an A10 RNA oligoand ADP∶AlFx. We obtained crystals that diffracted to 2.9-Åresolution and solved the structure with a multiwavelength anom-alous diffraction (MAD) experiment on a crystal grown from

Author contributions: J.R.W. and E.C. designed research; J.R.W., F.B., and J.H. performedresearch; J.R.W., F.B., and J.H. contributed new reagents/analytic tools; J.R.W., F.B., and E.C.analyzed data; and J.R.W. and E.C. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 2xgj).1To whom correspondence should be addressed. E-mail: conti@biochem.mpg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1004953107/-/DCSupplemental.

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selenomethionine-substituted protein. The structure was refinedto 2.9-Å resolution to an Rfree of 24.8%, Rfactor of 19.9%, andgood stereochemistry (see Table S1 for data and refinementstatistics). Mtr4 folds into a globular DExH ATPase core and fea-tures an insertion that forms a protruding structural unit (Fig. 1).The crystal asymmetric unit contains two independent molecules.Molecule A includes most of theMtr4 polypeptide chain, with theexception of residues 363–391. Molecule B has well-defined elec-tron density for the DExH core, which is essentially identical tothat of molecule A. However, residues 672 to 809 in the protrud-ing region and three loops are disordered in molecule B andcould not be modeled. Five nucleotides of the A10 RNA oligoand ADP are also present in both molecules of the asymmetricunit. Here, we describe molecule A unless otherwise specified.

Architecture of the Central DExH Core. The DExH core of Mtr4 isformed by four domains (RecA-1, RecA-2, winged-helix, andhelical bundle) that interact to form a globular structure ofdimensions 55 × 55 × 35 Å. The structural analysis reveals thatthe architecture of the core has remarkable similarity to the cor-responding domains of the DExH helicases Hel308 (24) andPrp43 (25). Hence, we refer to it as the DExH core. The similarityof Mtr4 and Hel308 beyond the RecA region was not previouslydetected by sequence analysis probably due to the presence of theinsertion in the middle of the winged-helix domain (Fig. 1B).

The two RecA-like domains (RecA-1, and RecA-2, light bluein Fig. 1) pack against each other to bind RNA and ADP. Anunusual feature of Mtr4 is a 60-residue segment N-terminal toRecA-1 that folds into a long β-hairpin (dark blue in Fig. 1).The N-terminal β-hairpin packs with the β-strands againstRecA-1 and latches onto RecA-2. A linker of 15 residues connectsRecA-2 to the winged-helix (WH) domain (yellow in Fig. 1A).The WH domain packs against an 8-helix-bundle domain (pinkin Fig. 1), which in turn contacts the cleft between the two RecAdomains. A related helical bundle region was first described as theratchet domain in the structure of Hel308 because of its proposedrole in translocating the nucleic acid (24) and has subsequentlybeen found in the RNA splicing helicases Prp43 (25) and Brr2(26). Finally, the conserved hydrophobic residues at the C termi-nus of Mtr4 interact at an apolar patch between RecA-1 andRecA-2. The relative conformation of the two RecA domainsof Mtr4 is stabilized on one side (near the ADP) by the N-term-inal β-hairpin and on the other (near the RNA) by the helix-bundle domain and the C-terminal tail (Fig. 1A Right). Mutants

known to have phenotypes in vivo (M540I in Mtr4-20 and C942Yin Mtr4-1) map to the DExH core and are likely to indirectlyperturb RNA binding (Fig. S3A).

Architecture of the Stalk/KOW Insertion Domain. Mtr4 features aninsertion of 270 amino-acid residues that protrude from theglobular DExH core as a separate structural unit (Fig. 1A Left).This region is inserted between the third and fourth helices ofthe WH domain (Fig. S1). The insert starts with two α-helices or-iented roughly perpendicularwith respect to eachother (helicesα1and α2, orange in Fig. 1A Left). It continues into a globulardomain (in red) and ends with two α-helices (α3 and α4) thatare coiled around the preceding helices α1 and α2. The helicalportion of the insert thus forms an L-shaped stalk extending forabout 40Å from theDExH core. The globular portion of the insert(red in Fig. 1A) has a β-barrel architecture characterized by twolong loops (Fig. S1). A DALI search (27) revealed unexpectedstructural similarity to Tudor, SH3, and KOW (Kyrpides–Ouzounis–Woese) (28) domains (Z scores of about 4.5). Whereasthe similarity to the Tudor and SH3 domains appears restrictedto the overall fold, the similarity to KOW proteins extends to re-sidues that have been implicated in macromolecular interactions(see below).We therefore refer to the globular domain of theMtr4insertion as a KOW domain.

In one of the Mtr4 molecules in the asymmetric unit (moleculeA), the KOW domain is in close proximity to the DExH core. Thetwo long loops of the KOW domain contact the helical bundleand the RecA-2 domains, but this intramolecular interactionis most likely due to crystal packing. In molecule B, the KOWdomain is disordered. Thus, the KOW domain appears to be aflexible unit that could sample the conformational space aroundthe end of the stalk. The stalk helices α1 and α4 that emerge fromthe DExH core are in a similar orientation in the two moleculesof the asymmetric unit (rmsd 2.5 Å), whereas helices α2 and α3that connect to the KOW domain deviate more in their confor-mation (rmsd 7.5 Å) (Fig. S3B). Although the stalk is not a rigidunit, its conformational flexibility appears to be restricted byintramolecular contacts at the hinge regions (Fig. S3B).

ADP and RNA Binding by the DExH Core of Mtr4. The two RecA do-mains of Mtr4 pack against each other forming a cleft that is linedby the conserved motifs typical of the DExH/DEAD family ofSF2 helicases (reviewed in ref. 29). A DALI search finds thatthe relative orientation of the two domains is most similar to

stalk

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AFig. 1. Crystal structure of yeastMtr4-Δ80. (A) View of the S. cerevisiaeMtr4-Δ80 structure in two orienta-tions. The DExH core is formed bythe two RecA domains (in light blue)together with the N-terminal β-hair-pin (in blue), the WH domain (in yel-low), and the helical bundle domain(in pink). An insert in the sequenceof the WH domain folds into a longstalk (in orange) and a globularKOW domain (in red). The helices inthe stalk domain are labeled at Left.ADP and five nucleotides of a single-stranded poly(A) RNA are shown (inball-and-stick representation in black)bound at the DExH core. The N termi-nus and C terminus are labeled (resi-dues 81 and 1073). This and all otherstructure figures were generated withPyMOL (Delano Scientific). (B) Sche-matic representation of the domainorganization of Mtr4. The domainsare colored as in A.

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the close conformation observed in the structures of the DExHproteins Hel308 (24) and Prp43 (25) (in complex with a partiallyunwound DNA substrate and with ADP, respectively) and to theDEAD-box protein Dbp5 in complex with AMPPNP and RNA(30). ADP binds at one side of the interdomain cleft, with theadenine ring sandwiched between Arg547 and Phe148 and withthe adenine amino group recognized by Gln154 (that is part ofthe Q motif). Although the crystallization experiments were car-ried out in the presence of ADP-AlFx, we did not observe orderedelectron density for the AlFx moiety.

RNA binds at the opposite side of the interdomain cleft, withthe 5′ end at RecA-2 and the 3′ end at RecA-1 (Fig. 2). Binding ofthe five ribonucleotides occurs at similar binding pockets used byother DEAD/DExH helicases. In particular, nucleotides 2 to 5 inthe Mtr4 structure superpose with the first four unpaired bases ofthe DNA substrate in the Hel308 structure (position þ1 to þ4)(Fig. 2). The most 5′ nucleotide (nucleotide 1) is instead in a dif-ferent conformation as compared to DNA-bound Hel308, wherethe corresponding position in the nucleic acid is the last base pairbefore the duplex is unwound [position −1 (24)]. Nucleotide 1packs against Trp524 and Gly526. These residues, together withLys523 and Arg530, form a β-hairpin that is conserved at thesame structural position in Hel308, where it promotes strandseparation (24) (Fig. 2 and Fig. S4). An equivalent unwindingβ-hairpin is present in the structure of the DExH helicasePrp43 (25). Thus, the overall mode of substrate binding andunwinding is likely similar in DExH helicases.

The DExH Core Recruits Trf4-Air2. The presence of the stalk/KOWinsertion (hereon defined as SK) is a specific feature of Mtr4orthologues. We therefore tested whether the SK region

(residues 618–873) is required for binding Trf4-Air2, a specificinteracting partner of Mtr4. We expressed and purified a hetero-dimer containing the conserved poly(A)-polymerase domain ofTrf4 (residues 111–490) and the conserved zinc-knuckle regionof Air2 (residues 1–180). Both Mtr4 f.l. and Mtr4-Δ80 formeda ternary complex with Trf4111–490-Air21–180 that could be isolatedby size-exclusion chromatography (Fig. 3A and Fig. S2C). Next,we engineered a mutant of Mtr4-Δ80 lacking the SK region(Mtr4-Δ80-ΔSK). This mutant mimics the structure of Hel308,where a loop is present at the corresponding position of theSK insertion in the DExH core. Despite the significant removalof amino-acid residues, this mutant appears to be properly folded(melting temperature of 48 ºC for Mtr4-Δ80-ΔSK and of 46 ºCfor Mtr4-Δ80, Fig. S2A). The Mtr4-Δ80-ΔSK mutant formed aternary complex with Trf4111–490-Air21–180 by size-exclusion chro-matography (Fig. 3A and Fig. S2C), indicating that the DExHcore of Mtr4 rather than the stalk/KOW insertion mediatesthe formation of the TRAMP complex.

We next tested whether binding of Trf4-Air2 regulates the AT-Pase activity of Mtr4. For this experiment, we used a catalyticallyinactive mutant of Trf4 (DADA mutant) that retains Mtr4binding but is unable to contribute directly to ATP hydrolysis(20) (Fig. 3B, gray diamonds). Mtr4-Δ80 showed ATPase activityin the presence of an A15 RNA substrate (Fig. 3B, blue triangles).Adding Trf4DADA-Air2 did not affect the RNA-dependent AT-Pase activity of Mtr4 (Fig. 3B, blue squares). Inspection of themolecular surface reveals the presence of patches of residues con-served in Mtr4 orthologues and not in other helicases (Fig. S5).These conserved patches are far from the RNA/ADP bindingsites and are possible targets for Trf4-Air2 binding.

Mtr4-ADP-ssRNA

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Fig. 2. RNA binding by the DExH core of Mtr4. The structure of Mtr4 in complex with ssRNA is shown at Left (A). As a comparison, the structure of Hel308bound to a partially unwound duplex DNA (PDB ID code 2P6R) is shown after optimal superposition at Right (B). The domains in the Hel308 structure arecolored as the corresponding domains in the DExH core of Mtr4. The close-up views show the region around the unwinding β-hairpin. The numbering of thedeoxyribonucleotides of the product strand in the Hel308 structure is from ref. 24. The product strand being translocated through the ATPase is in black,whereas in gray is the complementary strand in the Hel308 structure.

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The KOW Domain of Mtr4 Shares Similarities with rRNA-Binding Pro-teins. The β-barrel structure in the insertion domain of Mtr4shows intriguing similarities with the KOW domains present inribosomal proteins. In addition to conserved residues importantfor the structural integrity of the domain, the KOW domains ofbacterial L24 and eukaryotic L26 and L27 proteins contain aninvariant glycine residue present in a loop region (28). Structuralstudies of the ribosome have shown that the invariant glycine ofL24 makes a base contact with ribosomal RNA (rRNA) (31, 32).In addition, rRNA is contacted by positively charged residuespresent in the long loops that characterize the L24 β-barrel.The β-barrels of Mtr4 and L24 superpose with a rmsd of2.6 Å. The superposition places Gly686 in yeast Mtr4 at the si-milar structural position of Gly15 in Escherichia coli L24 (yellow,Fig. 4A). Yeast Mtr4 also features conserved positively charged

residues at similar positions as rRNA-binding residues of E. coliL24 (Fig. 4A).

To test whether the KOW domain of Mtr4 contributes to theRNA-binding properties of Mtr4, we used an electrophoretic gel-mobility retardation assay with in vitro transcribed tRNAiMet,which lacks modifications and therefore mimics the physiologicalhypomodified tRNAiMet substrate of Mtr4. Mtr4-Δ80 efficientlybound tRNAiMet even in the absence of ATP (Fig. 4B, lanes 2–5),consistent with previous reports that nucleic acid bindingby DExH proteins does not depend on the presence of ATP

B

A

Fig. 3. The DExH core of Mtr4 is a binding platform for Trf4-Air2. (A) TRAMPcomplex formation by size-exclusion chromatography (Superdex 200 HR10∕30, GE Healthcare). The SDS-PAGE Coomassie-stained gel includes samplesof the peak fractions indicated. The overlay of the corresponding chromato-grams is shown in Fig. S2C. (B) The ATPase assay was performed in thepresence of an A35 RNA with Mtr4-Δ80 either alone (blue triangles),Trf4111–490 DADA-Air21–180 alone (gray diamonds), or both in complex together(blue squares). The activity of Mtr4-Δ80-ΔSK is also shown (green circles). Thedata represent mean values and standard deviations from three independentexperiments. The proteins used in the ATPase assay were analyzed bySDS-PAGE and are shown in Fig. S2D.

E.coli 23S rRNA

Mtr4-KOW

E.coli L24

K16

K687

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R678

R774

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Mtr4-∆80 Mtr4-∆80-∆SK0 0

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1 2 3 4 5 6 7 8 9 10 11 12 13 14

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Fig. 4. Mtr4 has a KOW domain that contributes to RNA binding. (A) Struc-tural similarity between the KOW domain of Mtr4 (in red) and the ribosomalprotein L24 (in dark gray, PDB ID code 3I1N). E. coli L24 is shown bound to 23SrRNA (light gray). In yellow is the conserved glycine residue characteristic ofKOW domains. Conserved positively charged residues of yeast Mtr4 andRNA-binding residues of L24 at similar structural positions are highlighted.Some extended loop regions outside of the KOW core have been omittedfor clarity. (B) The KOWdomain is involved in RNA binding. Gel-mobility assayconducted with a labeled tRNAiMet probe and the purified recombinantproteins indicated above the lanes (at increasing concentrations of 0.45,1.5, 45, and 150 μM protein). The position of the free RNA probe (lanes 1and 14) and of the RNA-bound complexes (asterisk) is shown at Right. Theproteins used in the gel-shift assay that were analyzed by SDS-PAGE areshown in Fig. S2B (Right).

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(16, 24, 29). Mtr4-Δ80-SK showed reduced RNA binding in thegel-shift assay (Fig. 4B, lanes 6–9) and a lower RNA-dependentATPase activity (Fig. 3B, green circles) as compared to Mtr4-Δ80. When expressed and purified separately, the KOW domain(residues 666–818) bound tRNAiMet (Fig. 4B, lanes 10–13).

Upon superposition of theMtr4 andHel308 structures, we findthat in this crystal form the KOW domain is positioned near theduplex region of the nucleic acid bound to Hel308 (Fig. 2). In theconformation observed in molecule A, the long β3–β4 loop ofthe KOW domain would clash against the two unpaired basesof the unwound 5′-3′ strand (Fig. S4). However, the flexibilityof the KOW domain would allow it to adopt a different confor-mation relative to the ATPase domain upon nucleic acid binding.

ConclusionsMtr4 is a conserved helicase of the DExH family that cooperateswith the eukaryotic nuclear exosome in RNA processing anddegradation. Three aspects are particularly important for itsbiological functions: the binding and unwinding of RNAs, the in-teraction with Trf4-Air2 to form the TRAMP complex, and theinteraction with the exosome. What are the features of Mtr4 thatsupport these activities?

The RNA-binding and -unwinding activities of Mtr4 requirethe DExH core domain. The conformation of the two RecA do-mains we observe in the DExH core is similar to that previouslyobserved with the DExH helicases Hel308 and Prp43 either in theunbound form or bound to nucleic acid or ADP (24, 25). Theactive conformation of the two RecA domains appears to bestabilized even in the absence of ligands by intramolecular inter-actions with the additional regions that form the DExH core (theN-terminal β-hairpin, the winged-helix, and the helical bundledomains). Although interdomain movements in Mtr4 are likelyto occur upon ATP hydrolysis and RNA translocation, we donot expect major conformational changes of the RecA domainsas those observed for example between the active and inactivestates of the DEAD-box protein eIF4AIII (33, 34). The differ-ence in the extent of conformational changes between the onand off states might rationalize why in DEAD-box proteinsbinding of ATP and nucleic acid is cooperative, whereas in DExHproteins it is uncoupled.

Formation of the TRAMP complex is also mediated by theDExH core. Although it might be expected that Trf4-Air2 discri-minates Mtr4 from other DExH helicases by binding to the mostdistinctive region of the molecule (i.e., the stalk/KOW insertion),it instead binds at the most common region. This is strikingbecause Mtr4 appears to be only a docking platform for Trf4-Air2. It has previously been reported that Mtr4 does not affectthe polyadenylation activity of Trf4-Air2 (19). Here, we find thatTrf4-Air2 does not affect the ATPase activity of Mtr4. Thus, theTrf4-Air2 binding and helicase properties of Mtr4 are apparentlyuncoupled despite being mediated by the same DExH core. Thecharacteristic insertion of Mtr4 contributes instead to RNA bind-ing. The insertion contains a fold that is structurally similar toKOW domains of ribosomal RNA-binding proteins. We find thatthe KOW domain of Mtr4 is indeed able to bind a structuredRNA such as in vitro transcribed tRNAiMet. Given the functionof Mtr4 in processing structured RNAs and given its conforma-tional flexibility around the end of the stalk domain, it is temptingto speculate that the KOW domain might assist the helicase inrecognizing structured RNAs and present them to the unwindingβ-hairpin of the DExH core. The Ski2 helicase is also predicted tohave an insertion in the DExH core, although little similarity canbe found at the sequence level with the KOW domain of Mtr4.Ski2 functions together with the exosome in the cytoplasm, and assuch it encounters different RNA substrates.

Wheras the 5′ end of the short single-stranded RNA bound intheMtr4 structure is positioned at the top of the DExH core, nearthe unwinding β-hairpin, the 3′ end exits at the base of the core. In

the Hel308 structure, the 3′ end of the unwound product strandwraps around a helix-loop-helix (HLH) domain that packsagainst the DExH core. Mtr4 does not have the equivalent ofthe Hel308 HLH domain. We speculate that an equivalent bind-ing to the RNA 3′ end might be exerted by exosome proteins. Wefind that the surface of Mtr4 from which the 3′ end protrudes iswell conserved in the sequences of Mtr4 and Ski2 orthologues(Fig. S5), suggesting it might function as a docking site for acommon binding partner of the two helicases. How such an inter-action might occur is a question for future studies.

Materials and MethodsProtein Expression and Purification. Mtr4-Δ80 was expressed as a 6x-His-tagged protein (cleavable with Tobacco Etch Virus TEV protease) in E. coliBL21(DE3) cells or in B834 cells for selenomethione substitution. It was pur-ified using a Ni-nitrilotriacetate (NTA) affinity step followed by TEV cleavageand heparin Sepharose chromatography (GE Healthcare). The final purifica-tion step by size-exclusion chromatography (Superdex 200, GE Healthcare)was carried out in 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM MgCl2, and2 mM DTT. The same protocol was used to purify Mtr4 f.l., Δ80-ΔSK, andKOW. The complex of S. cerevisiae Trf4-Air2 was obtained by coexpressionin E. coli of TEV-cleavable His-tagged Trf4111–490 and untagged Air21–180.The complex was affinity-purified on Ni-NTA beads (GE Healthcare) followedby TEV cleavage, heparin and size-exclusion chromatography. The Trf4DADA

mutation was engineered with the QuikChange kit (Stratagene), verified byDNA sequencing and purified like the wild type.

Crystallization and X-Ray Structure Solution. Mtr4-Δ80 was concentrated to40 mg∕mL after gel filtration and incubated with 1 mM ADP∶AlFx and a1.2 M excess of A10 RNA for 10 min at 22 ºC. Crystals of the native proteinwere grown by sitting-drop vapor diffusion at 18 ºC using 50 mM MES pH6.0, 200 mM ammonium acetate, and 20% (wt∕vol) PEG 3350 in the reservoir.The crystals were cryoprotected with the reservoir solution supplementedwith 20% ethylene glycol prior to data collection at 100 K. All diffraction datawere collected at the Swiss Light Source (SLS) PXII beamline (Villigen, Switzer-land) and processed using XDS (35). Phase information was obtained by athree-wavelength MAD experiment with a selenomethionine-substitutedcrystal. SHELXD (36) was used to locate the selenium sites and SHARP (37)to calculate the phases. The initial electron density map was improved usingsolvent flipping and noncrystallographic symmetry averaging with DM (38).The model was built using Coot (39) and refined using Refmac5 (40) againstthe native data to 2.9-Å resolution.

ATPase Assay. The ATPase reactions were carried out in 50 mM Hepes, pH 7.5,100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 10% (vol∕vol) glycerol and0.1 mg∕mL BSA. We added 1.5 pmol of Mtr4 with or without equimolaramounts of Trf4-Air2 proteins in a 20-μL reaction volume containing2 mM ATP, 2 pmol A15 RNA, and traces of [γ-32P]ATP. Reactions were incu-bated for 0, 30, 60, and 90 min at 30 ºC and stopped by adding 400 μL ofice-cold acid-washed charcoal (Sigma) in 10 mM EDTA. After 30 min of cen-trifugation at 16,000 g, the supernatants containing γ-32P were countedusing a Packard Tri-carb 2100TR Liquid Scintillation Analyzer.

Gel-Shift Assay. The coding sequence for S. cerevisiae tRNAiMet preceded by aT7 promoter sequence was cloned in a pUC19 vector by assembly of overlap-ping phosphorylated oligonucleotides. RNA was transcribed using the T7RNA polymerase MEGAshortscript kit (Ambion) in the presence of [α-32P]UTP and purified on a 10% (wt∕vol) polyacrylamide gel containing 7 M Urea.For the gel-shift assay, 0.5 pmol labeled tRNAiMet was mixed with 4.5, 15, 45,or 150 pmol protein in a 10-μL reaction containing 20 mM Hepes at pH 7.5,100 mM potassium acetate, 30 mM KCl, 5% (vol∕vol) glycerol, 5 mM magne-sium diacetate, 0.1% (vol∕vol) NP-40, 2 mM DTT, and 30 μg∕mL heparin as anonspecific competitor. The mixtures were incubated 1 h at 4 °C before add-ing 2 μL 50% (vol∕vol) glycerol containing 0.25% (wt∕vol) xylene cyanole.Samples were run on a 6% (wt∕vol) polyacrylamide gel at 4 °C and visualizedby phosphorimaging (GE Healthcare).

ACKNOWLEDGMENTS. We thank Clemens Schulze-Briese, Anuschka Pauluhn,and the staff at SLS for excellent assistance with data collection; JeromeBasquin and the staff of theMPI-Martinsried crystallization facility for crystal-lization screenings; and Claire Basquin for the biophysics measurements. Wealso thank members of our lab for critical reading of the manuscript. Thisstudy was supported by the Max Planck Gesellschaft, the Sonderforschungs-bereich SFB646, and the Leibniz Program of the Deutsche Forschungsge-meinschaft.

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