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Roles of Individual Domains in the Function of DHX29, an Essential Factor Required for Translation of Structured Mammalian Mrnas

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Roles of Individual Domains in the Function of DHX29, an Essential Factor Required for Translation of Structured Mammalian Mrnas Roles of individual domains in the function of DHX29, PNAS PLUS an essential factor required for translation of structured mammalian mRNAs Vidya Dhote, Trevor R. Sweeney, Natalia Kim, Christopher U. T. Hellen, and Tatyana V. Pestova1 Department of Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203 AUTHOR SUMMARY Ribosomes initiate translation on the majority of eukaryotic N noigerlanimret- Central region noigerlanimret-C RecA1 RecA2 mRNAs by attaching to the dsRBD Insert WHD Ratchet domain OB domain 5′ end followed by base-by-base 1 534 758 1007 1078 1194 1369 scanning of the 5′ UTR until 377 448 766 842 they reach the initiation codon. W386 The heterotrimeric eukaryotic initiation factor (eIF) 4F pro- D385 OB domain motes ribosomal attachment F382 WH by binding to the cap via its K409 domain Ratchet eIF4E subunit and locally K401 K380 domain unwinding mRNA through the W408 R406 Y407 concerted action of its eIF4G RecA2 Insert and eIF4A (RNA helicase) BIOCHEMISTRY subunits together with eIF4B to generate a ribosomal- RecA1 binding site. The eIFs 4A, 4G, RecA2 and 4B also assist ribosomal complexes during scanning, but ribosomal movement through Fig. P1. Domain organization of DHX29. (Upper) Sequence alignment revealed that DHX29 consists of a DHX29- highly stable secondary struc- specific 534-aa–long NTR containing a putative dsRBD, a central catalytic core comprising tandem RecA-like ture elements requires an domains that contain a large RecA2 insert, and the C-terminal part, which includes winged-helix (WHD), ratchet, additional factor, DHX29, which and OB domains that are characteristic of DEAH/RHA helicases. (Lower Left) Ribbon diagram of the predicted belongs to the poorly charac- structure of DHX29’s dsRBD modeled on the basis of the solution structure of the related dsRBD from the mouse terized DEAH/RNA helicase A tRNA dihydrouridine synthase 2-like protein [Protein Data Bank (PDB) ID: 1WHN]. (Lower Right) Ribbon diagram of (RHA) family of RNA helicases the predicted structure of DHX29’s RecA1/2, WH, ratchet, and OB domains were generated by the SwissModel server, using the Prp43p crystal structure (PDB ID: 3KX2) as a template. Corresponding domains in DHX29 are that use energy derived from α α ATP hydrolysis to unwind shown in the same colors as in the upper diagram. The 1 and 2 helices of the WH domain are indicated. duplex RNA or to remodel RNA–protein complexes. DHX29 binds directly to ribosomal 40S subunits near the mRNA A unique feature of these helicases is the presence of a con- entrance and possesses nucleoside triphosphatase (NTPase) served C-terminal region after the helicase core. The structure activity that is strongly stimulated by 40S subunits (1). Although of the yeast DEAH/RHA helicase Prp43p (2, 3), which is the ribosomal position of DHX29 would allow it to unwind involved in ribosome biogenesis and mRNA splicing, contains mRNA directly before it enters the mRNA-binding cleft of the three distinct domains in this region: a winged helix (WH) 40S subunit, DHX29 is not a processive helicase, and evidence domain, a “ratchet” domain, and a β-barrel oligonucleotide/ suggests that it might work instead by remodeling ribosomal oligosaccharide-binding (OB) domain. The WH and ratchet complexes, inducing opening and closing of the mRNA entry domains of Prp43p are homologous to the corresponding channel and thus indirectly helping 43S complexes unwind en- tering mRNA stems. Here, to gain insights into DHX29’s domains in the Ski2-like DNA helicase Hel308 (4), whereas the fi mechanism of action, we generated a structural model of DHX29 C-terminal OB domain is speci c for the DEAH/RHA family. to identify its individual domains and characterized their roles in The structural homology between Hel308 and Prp43p led to the ribosome binding, nucleoside triphosphate hydrolysis, and pro- proposal that DEAH/RHA RNA helicases also may function moting the formation of a ribosomal initiation complex by by a mechanism of processive translocation, in which the DHX29. Taking into account the homology between DHX29 and other DEAH/RHA helicases, our findings also provide generally applicable insights into the function of the various structural Author contributions: V.D., T.R.S., C.U.T.H., and T.V.P. designed research; V.D., T.R.S., and domains that are specific to this class of protein. N.K. performed research; V.D., T.R.S., C.U.T.H., and T.V.P. analyzed data; and C.U.T.H. and DEAH/RHA helicases possess a catalytic core comprising T.V.P. wrote the paper. twin catalytic domains (designated “RecA1” and “RecA2”) The authors declare no conflict of interest. containing characteristic motifs (I–VI) that are involved in ATP This article is a PNAS Direct Submission. binding and hydrolysis, RNA binding, and coupling of ATP 1To whom correspondence should be addressed. E-mail: [email protected]. hydrolysis with nucleic acid unwinding as well as a prominent See full research article on page E3150 of www.pnas.org. β-hairpin protruding from between motifs V and VI of RecA2. Cite this Author Summary as: PNAS 10.1073/pnas.1208014109. www.pnas.org/cgi/doi/10.1073/pnas.1208014109 PNAS | November 13, 2012 | vol. 109 | no. 46 | 18641–18642 Downloaded by guest on September 30, 2021 β-hairpin acts as a wedge for separating the strands of a duplex a single structural unit. Its deletion did not influence the asso- nucleic acid, and the long ratchet helix of the ratchet domain ciation of DHX29 with 40S subunits but abrogated the ability interacts with the unwound single strand to facilitate processive of DHX29’s NTPase activity to respond to stimulation by 40S translocation of the enzyme along this strand during unwinding (4). subunits, thus resulting in the loss of DHX29’s activity in initi- Using sequence analysis and structural modeling, we found ation. This deletion also increased DHX29’s intrinsic NTPase – that DHX29 comprises a unique 534-aa long N-terminal region activity, similar to the effects of mutations in the RecA2 insert. (NTR), central catalytic RecA1/RecA2 domains containing a Both the OB domain and the insert are likely positioned near the large insert in the RecA2 domain that is not present in Prp43p, mRNA entrance and therefore might cooperate in suppressing and a C-terminal segment, which includes WH, ratchet, and OB the basal level of DHX29’s NTPase activity. Taken together, domains that are characteristic of DEAH/RHA helicases and fi are homologous to those of Prp43p (Fig. P1). The C-terminal these data suggest that the OB domain, which is speci c for region after the OB domain is more homologous to that of RHA DEAH/RHA helicases, plays an important role in their NTPase ’ than that of Prp43p and is predicted to form a series of β-strands cycle. The observation that mutations in DHX29 s predicted fl and two α-helices. The only recognizable element in the DHX29- ratchet helix did not in uence its activity in initiation on struc- specific NTR was a putative double-stranded RNA-binding tured mRNAs would be consistent with DHX29 functioning by domain (dsRBD) (Fig. P1) that adopts a compact fold and fre- remodeling of 43S complexes at the mRNA entrance rather than quently occurs in RNA-binding proteins. by direct unwinding of mRNAs before they enter the mRNA- Functional characterization of DHX29 mutants revealed that binding cleft of the 40S subunit. specific binding of DHX29 to 40S subunits is essential for its The importance of DHX29 for translation is underlined by function in initiation on structured mRNAs and is determined by observations that silencing its expression in human cells led to the dsRBD in the NTR and elements in the N-terminal half of a general impairment of translation, particularly of mRNAs with the WH domain, which include the α1/α2 helices and the α2-α3 long, structured 5′ UTRs (which typically encode regulatory loop (Fig. P1). These interactions are likely to be established proteins) and to impairment of cancer cell growth (5). Significant sequentially, indicating that DHX29 undergoes induced confor- overexpression of DHX29 in various types of cancers therefore mational changes following initial ribosomal association. suggests that it might be a potential target for therapeutic in- Mutational analyses of conserved motifs in the RecA domains fi fi ’ hibition (5), and our identi cation of functional domains that con rmed the requirement for NTP hydrolysis in DHX29 s fi fi β have roles that are speci c to DHX29 (e.g., its interaction with function in initiation and validated the signi cance of a -hairpin fi fi protruding from RecA2. Although the large insert in the RecA2 40S subunits) might aid in the identi cation of speci c sites domain is not required for the ability of DHX29’s NTPase ac- for inhibitors. tivity to respond to stimulation by 40S subunits or for DHX29’s function in initiation, we found that it has an autoinhibitory role 1. Pisareva VP, Pisarev AV, Komar AA, Hellen CU, Pestova TV (2008) Translation initiation ’ on mammalian mRNAs with structured 5’UTRs requires DExH-box protein DHX29. Cell in suppressing DHX29 s intrinsic NTPase activity. The insert 135(7):1237–1250. likely forms a minidomain near the putative entrance to the 2. He Y, Andersen GR, Nielsen KH (2010) Structural basis for the function of DEAH mRNA-binding channel. Interestingly, RHA and other RHA- helicases. EMBO Rep 11(3):180–186. like helicases also contain inserts at the analogous location, even 3. Walbott H, et al. (2010) Prp43p contains a processive helicase structural architecture though they are of variable size and possess little sequence with a specific regulatory domain.
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