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Eif4b Stimulates Translation of Long Mrnas with Structured 5′ Utrs and Low Closed-Loop Potential but Weak Dependence on Eif4g

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Eif4b Stimulates Translation of Long Mrnas with Structured 5′ Utrs and Low Closed-Loop Potential but Weak Dependence on Eif4g eIF4B stimulates translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G Neelam Dabas Sena, Fujun Zhoua, Michael S. Harrisb,c, Nicholas T. Ingoliab,c, and Alan G. Hinnebuscha,1 aLaboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; bDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and cDepartment of Biology, Johns Hopkins University, Baltimore, MD 21218 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2015. Contributed by Alan G. Hinnebusch, August 2, 2016 (sent for review May 26, 2016; reviewed by Mark P. Ashe and Nahum Sonenberg) DEAD-box RNA helicases eukaryotic translation initiation factor 4A eIF4B (7–9), recent results suggests that yeast eIF4B and (eIF4A) and Ded1 promote translation by resolving mRNA second- eIF4G equally stimulate ATP hydrolysis and RNA unwinding ary structures that impede preinitiation complex (PIC) attachment by eIF4A in the manner expected for increased coupling of to mRNA or scanning. Eukaryotic translation initiation factor 4B ATP hydrolysis to unwinding (10), and they appear to accel- (eIF4B) is a cofactor for eIF4A but also might function indepen- erate the transition between the open and closed states of dently of eIF4A. Ribosome profiling of mutants lacking eIF4B or eIF4A (11). In a reconstituted yeast translation system, eIF4B with impaired eIF4A or Ded1 activity revealed that eliminating cooperates with eIF4E·eIF4G, eIF4A, and eIF3 to promote the eIF4B reduces the relative translational efficiencies of many more rapid assembly of 48S PICs positioned at the start codons of genes than does inactivation of eIF4A, despite comparable reduc- native capped mRNAs (12), enhancing the functional affinity of tions in bulk translation, and few genes display unusually strong eIF4A for the PIC (13). Yeast eIF4B binding to the 40S subunit requirements for both factors. However, either eliminating eIF4B or is crucial for its functions in vivo and in vitro, and eIF4B binds inactivating eIF4A preferentially impacts mRNAs with longer, more to the head domain of the 40S subunit and alters the mRNA structured 5′ untranslated regions (UTRs). These findings reveal an entry channel (13), suggesting that eIF4B promotes an open eIF4A-independent role for eIF4B in addition to its function as conformation of the 40S subunit conducive to mRNA re- eIF4A cofactor in promoting PIC attachment or scanning on struc- cruitment or scanning. It is unknown whether eIF4B remodel- tured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially ing of the 40S mRNA entry channel occurs independently of its impair translation of longer mRNAs in a fashion mitigated by the role as an eIF4A cofactor. ability to form closed-loop messenger ribonucleoprotein particles Results from a mammalian reconstituted system indicated that (mRNPs) via eIF4F–poly(A)-binding protein 1 (Pab1) association, eIF4A can facilitate PIC attachment and scanning through a suggesting cooperation between closed-loop assembly and eIF4B/ stem–loop (SL) structure of moderate stability distal from the helicase functions. Remarkably, depleting eukaryotic translation cap (14), whereas helicases DHX29 and yeast DEAD-box pro- initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic tein Ded1 (an ortholog of mammalian DDX3X) were required translation initiation factor 4F (eIF4F), preferentially impacts short to resolve SLs of higher stability (15, 16). Consistently, in yeast mRNAs with strong closed-loop potential and unstructured 5′ cells, a ded1 mutation had a stronger effect than an eIF4A mutation UTRs, exactly the opposite features associated with hyperdepend- ence on the eIF4B/helicases. We propose that short, highly effi- Significance cient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly. Protein synthesis initiates in eukaryotes when the 40S ribosomal subunit, loaded with initiator tRNA, attaches to the 5′ end of the eIF4B | eIF4A | eIF4G | Ded1 | translation mRNA, scans the 5′ UTR, and selects the AUG start codon. Ri- bosome attachment and scanning are impeded by structures in he translation initiation codon in most eukaryotic mRNAs is the 5′ UTR that can be resolved by RNA helicases Ded1 and Tidentified by the scanning mechanism, which commences with eukaryotic translation initiation factor 4A (eIF4A), with cofactors binding of initiator Met-tRNAi to the small (40S) ribosomal sub- eIF4B and eIF4G. We show that eIF4B can stimulate translation unit in a ternary complex (TC) with eukaryotic translation initiation independently of eIF4A and that eIF4B, eIF4A, and Ded1 are factor (eIF) 2 and GTP. The resulting 43S preinitiation complex preferentially required for translating long mRNAs, burdened (PIC) attaches to the mRNA 5′ end in a manner facilitated by with 5′ UTR structures, that inefficiently form the closed-loop eIF4F, comprised of cap-binding protein eIF4E, scaffolding protein intermediate with the mRNA ends joined by eIF4G. In contrast, eIF4G, and DEAD-box RNA helicase eIF4A. The helicase activity eIF4G appears to be most crucial for closed-loop assembly on of eIF4A is thought to facilitate PIC attachment by resolving sec- short, highly translated, and unstructured mRNAs. ondary structures in cap-proximal mRNA nucleotides. Interactions between eIF4G and eIF3 (in mammals) and eIF5 and eIF1 (in Author contributions: N.D.S., F.Z., N.T.I., and A.G.H. designed research; N.D.S., F.Z., and M.S.H. budding yeast) stabilize PIC association with the eIF4F–messenger performed research; N.D.S., F.Z., N.T.I., and A.G.H. analyzed data; and N.D.S., F.Z., N.T.I., and A.G.H. wrote the paper. ribonucleoprotein particles (mRNPs) (reviewed in refs. 1 and 2). Reviewers: M.P.A., The University of Manchester; and N.S., McGill University. Eukaryotic translation initiation factor 4B (eIF4B) also promotes The authors declare no conflict of interest. PIC attachment to mRNA, and mammalian eIF4B stimulates the Data deposition: Sequencing data from this study have been deposited in the Gene ATPase and RNA helicase activitiesofeIF4A(1,3,4),increases Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo/ (accession no. coupling of ATP hydrolysis to duplex unwinding by eIF4A (5), and GSE81966). increases the processivity of eIF4A helicase function (6). 1To whom correspondence should be addressed. Email: [email protected]. Although yeast eIF4B lacks the C-terminal RNA-binding do- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. main involved in stimulating eIF4A helicase activity by mammalian 1073/pnas.1612398113/-/DCSupplemental. 10464–10472 | PNAS | September 20, 2016 | vol. 113 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1612398113 Downloaded by guest on September 26, 2021 (tif1-A79V in a strain lacking TIF2) on translation of a reporter its WT control or for 2 h for the ded1-ts and WT strains, evoking harboring a long 5′ UTR (17), and a ded1 mutation impaired marked reductions in bulk polysome assembly in the mutant INAUGURAL ARTICLE scanning through a cap-distal SL (18). By ribosome footprint strains (19). To analyze cells lacking eIF4B under similar con- profiling of eIF4A and Ded1 mutants, we previously identified a ditions, a tif3Δ mutant and isogenic TIF3 strain were cultured at much larger cohort of mRNAs with a greater-than-average re- 30 °C and shifted to 37 °C for 1 h. Bulk polysomes in tif3Δ cells quirement for Ded1 vs. eIF4A in achieving WT translational were reduced to ∼23% of the WT level, a slightly smaller re- efficiencies (TEs) and found that Ded1-hyperdependent mRNAs duction than seen for the tif1-ts strain (17% of WT) and some- tend to have unusually long or structured 5′ UTRs. That only a what greater than observed in the ded1-ts mutant (36% of WT) small number of mRNAs were found to be hyperdependent on (SI Appendix, Fig. S1 A–C). [Henceforth, results obtained from eIF4A, despite a strong reduction in bulk translation in the this experiment are designated as “tif3Δ (37)”.] Because bulk eIF4A mutant, implied that the absolute TEs of most mRNAs polysomes are diminished in the mutant cells, absolute TEs of were reduced comparably by inactivation of eIF4A. Neverthe- most mRNAs will be reduced compared with WT, but these less, moderate reductions in relative TE conferred by the eIF4A reductions are dampened by normalization to total ribosome mutation were associated with increased propensity for 5′ UTR footprint reads, and TE changes are determined relative to all structure. These results, combined with the differential effects of other mRNAs. Those genes exhibiting reductions in relative TE Ded1 and eIF4A mutations on reporter mRNAs with SLs at in tif3Δ vs. WT cells display a greater-than-average dependence ′ different 5 UTR locations, suggested that Ded1 is crucial for on eIF4B, whereas genes exhibiting increased relative TE in the ′ scanning through structured 5 UTRs, whereas eIF4A performs mutant show a lower-than-average dependence on eIF4B and an essential function common to virtually all mRNAs, e.g., en- might even be repressed by eIF4B. hancing PIC attachment, and that in the presence of Ded1 Both ribosome footprinting and RNA-seq results were highly eIF4A is either ineffective or dispensable in resolving highly reproducible in biological replicates (Pearson’s r ∼0.99) (SI Ap- stable structures (19). pendix, Fig. S2 A–D). Comparing ribosome footprint densities in To shed light on the functional overlap between eIF4A and tif3Δ and WT cells revealed a set of ∼530 genes with substantially eIF4B in yeast cells, we have conducted ribosome profiling of a Δ altered translation in the mutant in both replicates [SI Appendix, tif3 mutant (lacking eIF4B) under the same growth conditions Fig. S2E; red dots indicate a greater than twofold deviation from used previously to profile tif1 and ded1 mutants.
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