eIF4B stimulates 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 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 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. 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 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 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 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 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 . 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. If eIF4B acts identity at a false-discovery rate (FDR) <0.01], whereas RNA primarily as a cofactor for eIF4A, then mRNAs whose relative sequencing (RNA-seq) data identified only about one-half that GENETICS TEs are reduced by impairing eIF4A should be similarly affected number with comparable changes in mRNA abundance (SI Ap- by eliminating eIF4B, and few mRNAs should be affected ex- pendix, Fig. S2F). By dividing the ribosome footprint reads by the clusively in cells lacking eIF4B. However, if eIF4B can function total RNA reads across the coding sequence (CDS), we identi- independently of eIF4A, e.g., to remodel the 40S mRNA entry fied 111 genes whose relative TE (henceforth “relative TE” is channel, then we should identify mRNAs exhibiting a heightened referred to simply as “TE”) is twofold or more higher in WT than requirement for eIF4B but not for eIF4A. Our results indicate in tif3Δ cells [ΔTE Δ = TE Δ /TE <0.5; FDR <0.01], that eIF4B cooperates with eIF4A to stimulate the translation of tif3 (37) tif3 (37) WT many mRNAs with a heightened propensity for 5′ UTR sec- indicating a heightened dependence on eIF4B; whereas 48 genes ondary structure, as is consistent with eIF4B functioning as an displayed less-than-average dependence on eIF4B and exhibited twofold or higher TE in the mutant [ΔTEtif3Δ(37) = TEtif3Δ(37)/ eIF4A cofactor. However, we also identified mRNAs with an > < enhanced requirement for eIF4B but not for eIF4A and ob- TEWT 2.0; FDR 0.01] (Fig. 1A, red dots). We refer to the genes with ΔTEtif3Δ(37) <0.5 as being “hyperdependent on served distinct effects of eliminating eIF4B and inactivating ” eIF4A on the expression of reporters with 5′ UTR SL insertions, eIF4B. suggesting that eIF4B also can stimulate translation indepen- dently of eIF4A. Interestingly, mRNAs that are hyperdependent Cooperation of eIF4B with eIF4A and Ded1 in Promoting Efficient on eIF4B, eIF4A, or Ded1 tend to be longer and less prone to Translation in Vivo. In our previous study (19), using the criteria forming the closed-loop intermediate mediated by eIF4G inter- defined above, only 36 genes were found to be hyperdependent on eIF4A. Despite the small number of eIF4A-hyperdependent actions with eIF4E bound to the cap and poly(A)-binding pro- Δ tein (Pab1) bound to the poly(A) tail of mRNAs. Closed-loop genes, the changes in TE conferred by tif1-ts and tif3 for all formation is thought to enhance eIF4F binding to mRNA and to genes exhibit a significant positive correlation (Fig. 1B), consis- facilitate its functions in PIC recruitment and eIF4A activation tent with functional cooperation between eIF4A and its cofactor and also might enable terminating to reinitiate eIF4B at a fraction of genes. However, only three genes show an translation efficiently on the same mRNAs (20). Surprisingly, unusually strong dependence on both eIF4A and eIF4B (Fig. mRNAs found to be hyperdependent on eIF4G have the op- 1C). That most eIF4A-hyperdependent genes are not equally posite characteristics, being relatively short, prone to closed-loop dependent on eIF4B could be explained by proposing that eIF4A formation, and devoid of 5′ UTR structure. We conclude that functions independently of eIF4B at these genes, e.g., by relying eIF4G has a critical function in vivo beyond stimulating eIF4A exclusively on eIF4G cofactor function. Alternatively, the eIF4A helicase activity; we believe this function involves enhancing activity in cells lacking eIF4B, although reduced from WT levels, closed-loop assembly and the advantages it provides for rapid could be sufficient to prevent a strong reduction in TE for most initiation. of the 33 eIF4A-hyperdependent genes in tif3Δ cells. On the other hand, our finding that only 3 of the 111 eIF4B-hyper- Results dependent genes are also hyperdependent on eIF4A (Fig. 1C) Ribosome Profiling of a tif3Δ Mutant Reveals a Cohort of eIF4B- suggests that eIF4B acts beyond its role as eIF4A cofactor, be- Hyperdependent Genes. We previously conducted ribosome foot- cause hyperdependence on eIF4B should dictate hyperdepen- print profiling of a yeast mutant lacking both chromosomal genes dence on eIF4A if eIF4B’s only function is enhancing eIF4A encoding eIF4A (TIF1/TIF2) and harboring the temperature- activity. An eIF4A-independent function for eIF4B is also con- − sensitive (Ts )alleletif1-A79V as the only source of eIF4A (tif1-ts) sistent with the slope of the trend-line of the ΔTEtif1-ts vs. − and an isogenic strain containing the Ts allele ded1-952 (ded1-ts). ΔTEtif3Δ(37) scatterplot being <1 (Fig. 1B), indicating that tif3Δ The mutants complemented by WT TIF1 or DED1 alleles were has a relatively greater effect than tif1-ts on TEs genome-wide examined in parallel as controls. The strains were cultured at despite similar reductions in bulk translation initiation in the two 30 °C followed by a shift to 37 °C for 1 h for the tif1-ts mutant and mutants (SI Appendix, Fig. S1 A and C).

Sen et al. PNAS | September 20, 2016 | vol. 113 | no. 38 | 10465 Downloaded by guest on September 26, 2021 in ded1-ts cells are similarly altered in tif1-ts or tif3Δ cells but also A B -16 r = 0.39, N=4822, P<10 reveals many genes with a heightened requirement for Ded1 but 100 4 not for eIF4A or eIF4B (Fig. 2B, compare the right column with the left or middle column). Thus, although functional cooperation 10 2 is widespread, many genes exhibit a heightened requirement for tif1-ts

∆(37) only one or two of the three factors. 1 0 ∆TE tif3 2 TE, log The 5′ UTRs of eIF4B-Dependent mRNAs Are Atypically Long with 0.1 -2 Heightened Propensity for Structure. We analyzed the 5′ UTR features of eIF4B-hyperdependent genes using a compilation of 0.01 -4 0.01 0.1 110100 5′ UTR lengths and propensities for secondary structure (21), TE, WT -4 -2 0 2 4 log ∆TE 2 tif3∆(37) where each nucleotide in 3,000 different yeast transcripts was assigned a parallel analysis of RNA structure (PARS) score, C D E based on susceptibility to digestion with single- or double-strand– r = 0.36, N=4822, P<10-16 specific nucleases in mRNA reannealed in vitro; higher scores ∆TE <0.5 (N=111) ∆TE <0.5 (N=111) 4 tif3∆(37) tif3∆(37) denote greater residency in the double-stranded conformation. 174 108 78 2 For each transcript we calculated several cumulative PARS scores (Fig. 3A), including the sum of scores for all 5′ UTR 33 ded1-ts nucleotides (Total PARS); for the first 30 nucleotides (First30 P<10-20 0

3 ∆TE P<0.03 2 PARS); for 30 nucleotides surrounding the start codon (Start30 log 33 195 -2 ∆TEtif1-ts <0.5 (N=36) -4 Pearson’s correlation coefficient (r) ∆TEded1-ts <0.5 (N=228) -4 -2 0 24 A log ∆TE 2 tif3∆(37) ∆TEtif3∆(37) ∆TEded1-ts Fig. 1. Correlation between genome-wide changes in TE conferred by the ∆TEtif1-ts 0.39 (0.46) 0.39 (0.45) elimination of eIF4B or inactivation of eIF4A or Ded1 at 37 °C. (A) Scatterplot Δ Δ of TEs in tif3 vs. WT. Genes exhibiting twofold or greater changes in tif3 ∆TEtif3∆(37) 0.36 (0.40) cells at FDR <0.01 or >1.4-fold changes at FDR <0.05 are highlighted in red

and blue, respectively. (B) Density plot of log2ΔTE values for 4,822 expressed genes for tif1-ts vs. tif3Δ strains at 37 °C, using tif1-ts data from ref. 19, − > Δ > excluding genes with 6 log2 TE 6. The solid line is the determined re- B log ∆TE 2 gression line; the dotted line is the theoretical regression line for identical 0050020

changes in ΔTE values. Individual genes are shown by blue filled circles; the TNUOC coloring indicates increased density of genes (red is maximum). (C) Overlap tif1-ts tif3∆(37) ded1-ts between eIF4B- and eIF4A-hyperdependent genes exhibiting twofold or Δ ∆TE ∆TE ∆TE greater reductions in TE at 37 °C in tif3 and tif1-ts strains. (D) Overlap -2 0 2 between eIF4B- and Ded1-hyperdependent genes exhibiting twofold or greater reductions in TE at 37 °C in tif3Δ and ded1-ts strains. (E) Density plot

of log2ΔTE values for 4,822 expressed genes for ded1-ts vs. tif3Δ strains at 37 °C, using ded1-ts data from ref. 19, generated as in B.

Comparing changes in TE conferred by ded1-ts and tif3Δ for all − genes also revealed a significant correlation (r = 0.36, P < 10 16) (Fig. 1E) comparable to that observed in the tif1-ts/tif3Δ com- parison (r = 0.39) (Fig. 1B). Interestingly, a more substantial overlap of genes hyperdependent on both Ded1 and eIF4B was observed, representing 30% of the eIF4B-hyperdependent genes (Fig. 1D). Including the results of our previous comparison of genome-wide TE changes in ded1-ts vs. tif1-ts cells (19) reveals essentially the same degree of correlation for all three pairwise comparisons (Fig. 2A), whether all genes are compared or only those for which a statistically significant change in TE relative to WT (at FDR <0.05) was observed in any of the three Δ mutants (Fig. 2A, values in parenthesis). These findings suggest Fig. 2. Common and distinct changes in TE conferred by tif3 , ded1-ts, and tif1-ts mutations at 37 °C. (A) Correlation between TE changes con- similar degrees of functional cooperation of eIF4B with eIF4A ferred by tif3Δ, ded1-ts,ortif1-ts for all expressed genes (n = 4,822) or for and Ded1. genes with statistically significant TE changes in any of the three mutants The conclusions reached above are reinforced by hierarchical (FDR <0.05; n = 1,487). The r values for the latter subset of genes are given − clustering of TE changes for the 1,487 genes exhibiting signifi- in parentheses. P values for all correlations are <10 16. Published ΔTE cant differences from WT in any of the three mutants (Fig. 2B). values for tif1-ts and ded1-ts mutants were used from ref. 19. (B) Hierar- Thus, a substantial proportion of genes showing reduced TE in chical clustering analysis of ΔTE values conferred by tif1-ts, tif3Δ,and tif1-ts cells also display diminished TE in the tif3Δ mutant (Fig. 2B, ded1-ts at 37 °C for 1,487 genes with statistically significant TE changes in < red bars in left and center columns), as is consistent with the role any of the three mutants (FDR 0.05), presented as a heat-map using the R of eIF4B as eIF4A cofactor. However, numerous genes, including heatmap.2 function from the R gplots library and the default hclust hier- archical clustering algorithm. Only one gene (YNL042W-B)with>64-fold some of the most highly eIF4B-dependent genes, exhibit reduced Δ increaseinTEintif1-ts cells was excluded because including it would di- TE in tif3 cells (red) but no change or even increased TE (white minish the color differences among the remaining genes analyzed in the or blue) in tif1-ts cells (Fig. 2B). The corresponding ded1-ts heat heat-map. Published ΔTE values for tif1-ts and ded1-ts mutants were used map supports the conclusion that many genes whose TE is altered from ref. 19.

10466 | www.pnas.org/cgi/doi/10.1073/pnas.1612398113 Sen et al. Downloaded by guest on September 26, 2021 compared these 161 genes with a corresponding group of 255 genes

A Δ < INAUGURAL ARTICLE Plus30 with greater-than-average eIF4B dependence [ TE Δ 0.7]. First30 Start30 tif3 (37) 5’ m7G AUG UGA AAAAAAAAAAAA Similar to our findings for the eIF4B-hyperdependent genes Δ < Max30 Plus15 (Fig. 3C), the 255 genes with TEtif3Δ(37) 0.7 show greater- ′ ′ Total PARS than-average 5 UTR lengths and PARS scores for all 5 UTR B features except the First30 PARS score (Table 1). Remarkably, 1.0 the 161 genes least dependent on eIF4B display below-average 5′ X =79 nt (N=2607) ′ 0.8 UTR lengths and PARS scores for all 5 UTR features (Table 1), All mRNAs X =141nt (N=61, P<10-5) strengthening the association between eIF4B dependence and 0.6 propensity for 5′ UTR structure. However, neither group of 0.4 genes shows an atypical propensity for secondary structure in the ∆TEtif3∆(37) < 0.5 CDS (Table 1, Plus PARS features). 0.2 Cumulative Fraction Cumulative Supporting these last conclusions, analysis of all 2,607 genes in 0.0 our study with available PARS data revealed significant negative 1 5 10 50 100 500 1000 Δ ′ C 5’UTR length correlations between TEtif3Δ(37) and each 5 UTR feature, with 25 the strongest associations for length, Total PARS, and Max30 PARS scores of 5′ UTRs (Fig. 3D, Left, red bars), whereas no * All mRNAs 20 * ∆TE <0.5 significant correlations were observed for any CDS features (Fig. tif3∆(37) D Right ded1-ts 15 3 , , red bars). Interestingly, changes in TE in cells observed previously (19) also show negative correlations with all 10 ′ * 5 UTR features similar in magnitude to those identified here for tif3Δ(37) cells (Fig. 3D, Left, gray bars). Relatively weaker, albeit Mean PARS score Mean PARS 5 significant, negative correlations were found for these same 0 features and the ΔTEtif1-ts values measured for the tif1-ts mutant Total First30 Start30 Max30 Plus15 Plus30 Plus45 Plus60 Plus75 (Fig. 3D, Left, green bars). Thus, greater-than-average 5′ UTR D length and propensity for secondary structure are important * 5’ UTR * determinants of heightened dependence on eIF4B, eIF4A, GENETICS * length tif3∆(37) Plus15 * * Total tif1-ts and Ded1. * ded1-ts Plus30 * We extended our comparative analysis of Ded1 and eIF4B * Avg * dependence with additional ribosome-profiling experiments on * Plus45 * First30 the tif3Δ mutant shifted from 30 °C to 15 °C for 10 min, desig- * Plus60 * Start30 nated “tif3Δ (15),” the regime used previously to inactivate a * * Max30 Plus75 cold-sensitive ded1-cs mutant (19). Polysome depletion in the * Δ -0.4 -0.3 -0.2 -0.1 0 -0.4 -0.3 -0.2 -0.1 0 tif3 mutant (12% of WT) was somewhat greater than seen in Correlation with ΔT TE Correlation with Δ TE ded1-cs (23% of WT) under these conditions (SI Appendix, Fig. (Spearman’s coefficients) (Spearman’s coefficients) S1 D and E). Comparing highly reproducible replicate ribosome Fig. 3. The 5′ UTRs of eIF4B-dependent genes exhibit longer-than-average footprint and RNA-seq data for tif3Δ and identically cultured length and PARS scores. (A) Schematic showing 5′ UTR and CDS intervals WT cells (r values >0.97) revealed a cohort of 156 genes assigned for calculating cumulative PARS scores for each gene. (B) Cumula- hyperdependent on eIF4B under these conditions (SI Appendix, tive distributions of 5′ UTR lengths for all 2,607 genes (black line) or for Fig. S3A). Again, we observed significant genome-wide negative eIF4B-hyperdependent genes (red line), curated by ref. 21, with the P value correlations between ΔTEtif3Δ(15) and 5′ UTR length and PARS indicating a statistically significant difference in mean length (X) for the 61 features (SI Appendix, Fig. S3B) and between TE changes in tif3Δ eIF4B-hyperdependent genes vs. all genes, as determined by the Student’s t test. (C) Average PARS scores calculated for the indicated gene set for each vs. ded1-cs cells shifted to 15 °C (SI Appendix, Fig. S3C), and 5′ UTR or CDS interval described in A, with P values from Student’s t tests there is extensive overlap between genes hyperdependent on indicated (*P < 0.05). (D) Spearman coefficients from correlations between eIF4B or Ded1 under these conditions (SI Appendix, Fig. S3D). ΔTE values conferred by the indicated mutations and 5′ UTR lengths or the Thus, eIF4B and Ded1 cooperate at both reduced and elevated − indicated cumulative PARS scores for all 2,607 curated genes (*P < 10 16). growth temperatures to enhance translation of a cohort of genes Results for tif1-ts and ded1-ts mutants are reproduced from ref. 19 for with a tendency to contain long, structured 5′ UTRs. comparison. In an effort to identify genes for which sequences conferring eIF4B hyperdependence are confined to the 5′ UTR, we analyzed ′ ≥ the expression of reporter genes constructed previously (19) PARS; for genes with a 5 UTR 15 nt); and the highest cu- containing the promoters and 5′ UTRs of candidate genes fused to mulative score in any 30-nt window across the 5′ UTR (Max30 ′ luciferase coding sequences (LUC)(SI Appendix,Fig.S4A). Eight PARS). We also calculated the average score over the 5 UTR reporters were analyzed that displayed appreciable reductions in (Avg PARS) and sums of scores for nucleotides downstream of TE in the tif3Δ mutant at 37 °C, which were driven primarily by + + + + the AUG, including intervals 1to 30 (Plus15), 16 to 45 reductions in ribosome density with minimal changes in mRNA + + + + + (Plus30), 31 to 60 (Plus45), 46 to 75 (Plus60), and 61 expression (SI Appendix,Fig.S4B). LUC expression was reduced + to 90 (Plus75). in tif3Δ vs. WT cells cultured at 37 °C for all eight reporters, and ′ Interestingly, the 61 eIF4B-hyperdependent genes for which 5 the magnitude of these reductions correlated with reductions in ′ UTR PARS scores were available exhibit a mean 5 UTR length ribosome density measured by ribosome profiling in the tif3Δ(37) of 140 nt, substantially greater than the mean length of 79 nt for experiment (SI Appendix,Fig.S4B and C). Thus, the 5′ UTRs of all 2,607 compiled genes examined in our study (P < 0.0001) these genes contribute to the reductions in TE conferred by (Fig. 3B). These genes also exhibit significantly higher mean elimination of eIF4B in vivo. Total and Max30 PARS scores (P < 0.01) and higher Avg PARS scores (0.06 ± 0.01 vs. 0.18 ± 0.04, P < 0.03) for their 5′ UTRs but eIF4B Is Critically Required for PIC Attachment to Reporter mRNA have typical PARS scores for all CDS intervals (Fig. 3C). Because Burdened with a Cap-Proximal SL. To provide independent support there are too few eIF4B-hypodependent genes with available for our conclusion that the 5′ UTRs of eIF4B hyperdependent PARS data, we adjusted the threshold to ΔTEtif3Δ(37) >1.4 and genes tend to be atypically long and prone to secondary structure,

Sen et al. PNAS | September 20, 2016 | vol. 113 | no. 38 | 10467 Downloaded by guest on September 26, 2021 Table 1. Comparison of 5′ UTR lengths and PARS features for genes exhibiting significant changes in TE in tif3Δ mutant vs. WT cells at FDR <0.05 at 37 °C

5′ UTR feature All mRNAs, n = 2,607 ΔTEtif3Δ(37) <0.7, n = 255 P value ΔTEtif3Δ(37) >1.4, n = 161 P value

Total length 79.0 ± 1.6 119.3 ± 6.61 <0.0001 60.81 ± 6.57 <0.006 Total PARS 7.75 ± 0.49 18.55 ± 2.03 <0.0001 −2.79 ± 1.68 <0.0001 Average PARS 0.058 ± 0.008 0.147 ± 0.019 <0.002 −0.135 ± 0.049 <0.0001 First30 PARS 3.12 ± 0.26 4.16 ± 0.744 0.24 −1.37 ± 1.48 <0.0001 Start30 PARS 4.59 ± 0.31 7.08 ± 0.923 <0.02 −0.037 ± 1.58 <0.0005 Max30 PARS 14.8 ± 0.31 19.31 ± 0.917 <0.0001 8.5 ± 1.50 <0.0001 Plus15 PARS 9.15 ± 0.31 9.61 ± 0.944 0.66 7.44 ± 1.45 0.19 Plus30 PARS 8.84 ± 0.33 10.15 ± 0.936 0.23 8.57 ± 1.53 0.84 Plus45 PARS 9.01 ± 0.33 10.62 ± 1.002 0.14 8.99 ± 1.48 0.99 Plus60 PARS 9.38 ± 0.32 11.29 ± 1.14 0.08 8.86 ± 1.4 0.70 Plus75 PARS 9.65 ± 0.33 12.06 ± 1.03 <0.03 8.00 ± 1.25 0.22

The mean (± SEM) 5′ UTR length or indicated PARS feature was calculated for all 2,607 genes or for 255 of the 443 genes having

ΔTEtif3Δ(37) <0.7 that were compiled in the PARS database and for 161 of the 270 genes with ΔTEtif3Δ(37) >1.4 at FDR <0.05 that were compiled in the PARS database.

we examined the effects of tif3Δ on LUC reporters containing SL cells, the SL reduces but does not abolish the association of re- insertions at sites 5 nt (cap-proximal) or 44 nt (cap-distal) from porter mRNA with 48S PICs (compare TIF3, +SL in Fig. 4C vs. ′ ′ the 5 end of a 70-nt 5 UTR comprised primarily of (CAA)n TIF3, −SL in Fig. 4B), as is consistent with the reduced luciferase repeats and thus considered to be relatively unstructured. Be- expression of the corresponding +SL LUC reporter in WT (Fig. ∼ cause 43S PICs protect 45 nt of mRNA (22), we reasoned that 4A, rows 1 and 2, TIF3). In tif3Δ cells, the +SL mRNA was the cap-proximal SLs should inhibit PIC attachment, whereas the present almost exclusively in the free mRNP fractions (Fig. 4C, cap-distal SL insertions should inhibit scanning without imped- tif3Δ, +SL), whereas an appreciable pool of 48S-associated ing 43S attachment. Expression of the reporter lacking an SL insertion (−SL) was reduced by ∼60% in tif3Δ cells (Fig. 4A, Left, row 1, TIF3 vs. tif3Δ), as is consistent with the reduction in bulk translation Luciferase (million units) Ratio tif3∆/TIF3 A 0 0.4 0.8 1.2 0 0.2 0.4 0.6 SI Appendix A initiation in this mutant ( , Fig. S1 ). The presence of 70nt SL insertions of moderate predicted stability (−5.7 kcal/mol) at 1 cap TIF3 tif3∆ -10.5kcal/mol either cap-proximal or cap-distal locations had little or no impact 2 cap Δ on LUC expression both in WT and tif3 cells (Fig. 4A, Left, cap -5.7 kcal/mol rows 3 and 5 vs. row 1) and thus did not substantially alter the 3 -10.5 kcal/mol tif3Δ/TIF3 expression ratio from that observed for the −SL 4 cap construct (Fig. 4A, Right, rows 3 and 5 vs. row 1). The more cap -5.7 kcal/mol stable SL insertions (−10.5 kcal/mol) at cap-proximal or cap- 5 distal locations reduced reporter expression in WT cells relative to that of the −SL reporter, with the cap-distal SL exerting a BCcap 70nt cap -10.5 kcal/mol stronger effect (Fig. 4A, Left, rows 2 and 4 vs. row 1, TIF3). 60 Interestingly, the cap-proximal insertion was substantially more 60 TIF3, -SL TIF3, +SL tif3∆, -SL tif3∆, +SL inhibitory in tif3Δ cells, reducing the tif3Δ/TIF3 expression ratio 40 40 by a factor of approximately threefold compared with the −SL reporter (Fig. 4A, Right, row 2 vs. rows 1 and 3). That eliminating % total RNA 20 % total RNA 20 eIF4B exacerbates the inhibitory effect of the stable cap-proxi-

mal SL implicates eIF4B in promoting PIC attachment at 0 0 structured 5′ ends. By contrast, tif3Δ did not further reduce the Fraction IN 1 2 3 4 56 7 8 9 10Fraction IN 1 2 3 4 56 7 8 9 10 Free mRNPs 40S 60S expression of the reporter harboring the more inhibitory cap- Free mRNPs 40S 60S distal SL (Fig. 4A, row 4). Because we showed previously that a Fig. 4. A cap-proximal SL increases eIF4B dependence for the expression of highly stable cap-distal SL introduces a strong block to ribosomal reporter mRNA at the step of 43S PIC attachment. (A, Left) Schema of LUC scanning that cannot be overcome efficiently in WT cells (19), we reporters indicating position and predicted stabilities of SLs inserted into a suggest that the defect in PIC attachment conferred by the ab- 70-nt unstructured 5′ UTR, either 5 nt or 44 nt from the cap. LUC expression sence of eIF4B in tif3Δ cells is no longer rate-limiting for initi- was determined as in SI Appendix, Fig. S4 for six independent transformants of WT (FJZ046) and tif3Δ (FJZ052) strains for each reporter following ap- ation in the presence of the cap-distal SL. proximately three cell doublings in SC-Ura at 23 °C. Mean (± SEM) luciferase To demonstrate that the reduced LUC expression of the expression in WT and tif3Δ cells (Center) and ratios of expression in tif3Δ vs. strong cap-proximal SL in tif3Δ cells (Fig. 4A, rows 1 and 2) WT cells (Right) are plotted. (B) WT and tif3Δ transformants harboring re- involves diminished PIC attachment, we measured the associa- porter plasmids derived from those described in A but with a truncated LUC tion of reporter mRNAs containing (+SL) or lacking (−SL) this CDS (78 nt), containing (+SL) or lacking (−SL) the cap-proximal −10.5 kcal/mol SL with native 48S PICs and free mRNPs resolved by sedimen- SL, were cultured as in A, and cells were cross-linked with 2% HCHO before tation through sucrose density gradients. These reporters were harvesting. WCEs were resolved by sedimentation through sucrose gradi- derived from the LUC constructs by truncation of the LUC CDS ents, and fractions were collected with continuous scanning at 254 nm to locate free 40S and 60S subunits. RNA was extracted, and the abundance of to 78 nt, which should accommodate only one translating ribo- reporter mRNA was quantified by quantitative RT-PCR, conducting three some (23), reducing the mRNA length to allow separation of 48S technical replicates for each fraction, and is reported as the percentage of PICs from free mRNPs. Cells were treated with formaldehyde to total RNA in fractions 1–10. Highly similar results were obtained in an in- prevent dissociation of PICs during sedimentation (24). In WT dependent experiment.

10468 | www.pnas.org/cgi/doi/10.1073/pnas.1612398113 Sen et al. Downloaded by guest on September 26, 2021 reporter lacking the SL is retained in tif3Δ cells (Fig. 4B, tif3Δ, observed for the tif1-ts, ded1-ts,andded1-cs mutants (Fig. 5A).

−SL). These last results are consistent with the greater reduction [Note that transcript length is highly correlated with CDS INAUGURAL ARTICLE in luciferase expression observed for the +SL than for the −SL length in yeast (r = 0.98) (26).] Dividing all genes into 14 bins LUC reporter in the tif3Δ mutant (Fig. 4A, rows 1 and 2, tif3Δ). basedonCDSlength(Fig.5B) reveals that reductions in TE Δ Δ < The findings shown in Fig. 4 B and C indicate that a stable cap- occur in tif3 cells at 37 °C [ TEtif3Δ(37) 1] only for genes with ∼ proximal SL increases the requirement for eIF4B to achieve CDS lengths longer than 1,500 nt, whereas genes with CDS ∼ Δ robust PIC attachment and translation initiation. shorter than 1,100 nt exhibit increased TEs in tif3 cells (Fig. 5B). Similar findings were made for the ded1-ts and tif1-ts eIF4B Promotes Efficient Translation of Long mRNAs Underenriched mutants (SI Appendix, Fig. S5A), confirming that longer mRNAs for eIF4E and Pab1 Association. In budding yeast, translation ini- generally display heightened dependence on eIF4A, eIF4B, tiation is less efficient on longer mRNAs than on shorter ones and Ded1. (23, 25). Interestingly, we discovered that eIF4B hyperdependence A possible mechanism underlying the inverse relationship is also linked to mRNA length, because changes in TE conferred between mRNA length and initiation rate is that longer mRNAs can form more RNA structures involving 5′ UTR base-pairing by tif3Δ at 37 °C [ΔTE Δ ] exhibit a pronounced negative tif3 (37) with CDS sequences that in sum impede PIC attachment or ri- correlation with CDS lengths (Fig. 5A), and a significant but Δ bosomal scanning (25). Indeed, we discovered genome-wide smaller negative correlation also was observed for tif3 cells at positive correlations between CDS length and the length of 5′ Δ − 15 °C [Fig. 5A, TEtif3Δ(15)]. Negative correlations also were UTRs [correlation coefficient (ρ) = 0.26; P = e 16], Total PARS − scores (ρ = 0.18; P = e 16), and average PARS scores (ρ = 0.12; − P = e 16) but not with CDS PARS features. That shorter mRNAs A C tend to have shorter, less structurogenic 5′ UTRs could help ex- * ∆TEtif3∆(37) plain their reduced requirement for eIF4B, eIF4A, and Ded1. 1.0 All mRNAs (N=2670) To determine whether CDS length also contributes to eIF4B

o * i ∆TEded1-ts 0.8 car dependence beyond its participation in 5′ UTR structures, we

Fn 0.6 * ∆TE et P<10-15 Δ tif1-ts v conducted local polynomial regression (LOESS) of TE Δ

ita tif3 (37)

l ′ u vs. CDS length, 5 UTR length, or Total PARS scores and ex-

* ∆TEtif3∆(15) m GENETICS u SCL mRNAs 0.2 0.4 C amined correlations between the residuals of each regression and (N=373) * ∆TE the other two variables (SI Appendix, Fig. S5B). After controlling

ded1-cs 0.0 3210-1-2-3 -0.1-0.2-0.3-0.4-0.5-0.6 0 for CDS length, both 5′ UTR length and Total PARS scores still log ∆TE 2 tif3∆(37) Correlation with CDS length make significant contributions to the variance of ΔTEtif3Δ(37), B (Spearman’s coefficients) E although the correlation coefficients are reduced compared with 4 1.0 All mRNAs those shown in Fig. 5A for correlations of total ΔTEtif3Δ(37) vs. 5′ o (N=2757)

i

t 0.8

c

ar UTR or Total PARS scores (compare columns 1 and 2 in SI

2 Fn

0.6 ′ evital P<10-15 Appendix, Fig. S5B). Similarly, after controlling for 5 UTR tif3∆(37)

0 u length, both CDS length and Total PARS scores still contribute

m ∆TE

u

2 SCL mRNAs SI Appendix B C significantly ( , Fig. S5 , column 3), and after con- 0.2(N=390) 0.4 log -2 trolling for Total PARS scores, both CDS length and 5′ UTR 0.0 0-0.5-1-1.5 0.5 1.51 length make significant contributions to the variance of ΔTEtif3Δ(37) log ∆TE -4 2 tif4631-td (SI Appendix,Fig.S5B, column 4). This analysis indicates that contributions of 5′ UTR length and RNA structure to variations in 4 5 9 4 8 5 2 5 0 7 08 76 1 70 1 1 9 3 9 8 33 4 5 7 8 14 58 7 100 1 13 14 16 19 22 28 4 Δ Δ D 14 TEtif3 (37) are at least partly independent of CDS length and Maximum CDS size (nt) that 5′ UTR length per se is an important determinant independent 3 of its contribution to total 5′ UTR structure. Furthermore, CDS 2 length is the strongest determinant of ΔTEtif3Δ(37), independent ′ 1 of its possible involvement in RNA structures formed with 5 tif4631-td UTR sequences.

∆TE 0 2 Given the strong, independent contribution of long transcript log -1 length to eIF4B dependence (SI Appendix, Fig. S5B), we con- sidered the alternative hypothesis that hyperdependence of long -2

7 3 4 4 8 3 mRNAs on eIF4B, eIF4A, and Ded1 reflects their relative in- 5 1 36 5 3 3 5 657 804 9 07 575 779 2 1 1224 1389 1 1 206 2493 47 3 1 Maximum CDS size (nt) ability to form the closed-loop intermediate (27), stabilized by mutual interaction of eIF4G with Pab1 bound to the poly(A) tail Fig. 5. mRNAs with heightened eIF4B dependence tend to be longer and and eIF4E bound to the cap. To address this possibility, we in- underenriched in closed-loop–forming initiation factors. (A) Spearman coef- terrogated a published dataset describing the genome-wide as- ficients from correlations between ΔTE values conferred by the indicated − sociation of mRNAs with eIF4E, eIF4G1, eIF4G2, Pab1, and the mutations vs. CDS lengths for ∼5,000 expressed genes (*P < 10 16). Published ΔTE values for tif1-ts and ded1 mutants were used from ref. 19. (B)Notched inhibitory eIF4E-binding Caf20 and Eap1, determined box-plots of ΔTE values in the tif3Δ mutant vs. WT at 37 °C for bins of 400 by sequencing mRNAs immunoprecipitated with these proteins genes of increasing CDS length (except for the last bin, which had 112 genes). (RIP-seq) (28). A subset of 395 mRNAs was found to be enriched Each box depicts the interquartile range containing 50% of the data, inter- for eIF4E, eIF4G1, eIF4G2, and Pab1 and depleted for Caf20 and sected by the median; the notch indicates a 95% confidence interval around Eap1 and thus was considered to have the greatest potential for the median. (C) Cumulative distributions of ΔTE values conferred by tif3Δ at closed-loop formation. Remarkably, we found that this “strong 37 °C for all 2,670 mRNAs or for the 373 SCL mRNAs, characterized for oc- closed-loop” (SCL) set of mRNAs exhibits relatively small re- cupancies of eIF4F-, Pab1-, and eIF4E-binding proteins as shown in ref. 28. The ductions, or even increases, in TE in the tif3Δ mutant at 37 °C (Fig. P value from a Kolmogorov–Smirnov test of statistical significance of the difference between the two distributions is shown. (D) Notched box-plot plots 5C), and similar results were found for tif1-ts, ded1-ts,andded1-cs of ΔTE values in the tif4631-td mutant vs. WT cells constructed as in B using mutants at their nonpermissive temperatures. Thus, mRNAs published data from ref. 29. (E) The same analysis as C except using published considered to be optimized for closed-loop formation tend to be ΔTE values conferred by eIF4G depletion in the tif4631-td mutant (29). hypodependent on eIF4B, eIF4A, and Ded1.

Sen et al. PNAS | September 20, 2016 | vol. 113 | no. 38 | 10469 Downloaded by guest on September 26, 2021 As might be expected, the SCL mRNAs constitute one of the eIF4B, eIF4A, and Ded1 (Fig. 5C). Thus, promoting the closed- most highly translated subsets of all yeast mRNAs; however, loop intermediate for SCL genes and achieving the high-level TEs another subset of highly translated mRNAs is enriched only for characterizing these genes in WT cells appears to be the most Pab1 (28). The latter group (designated “group I”) does not critical function of eIF4G in determining TEs in vivo. By contrast, exhibit the increase in TE in tif3Δ cells at 37 °C displayed by the it seems that eIF4B, eIF4A, and Ded1 are not crucial for closed- SCL group of mRNAs (designated “group III”) and instead loop assembly and are more critically required for translation of displays a response more typical of all other yeast genes (SI longer mRNAs with a diminished capacity for closed-loop for- Appendix, Fig. S6A). Thus, robust translation in WT cells does mation and containing longer, more structured 5′ UTRs. not necessarily dictate hypodependence on eIF4B, which ap- pears to be a specific property of the SCL mRNAs. Discussion As recently reported (26), and as is consistent with previous We used a combination of ribosome profiling and reporter analysis in vitro analysis of closed-loop assembly by mRNAs of different to probe in vivo functions of eIF4B in the yeast translatome. Be- lengths (27), the mRNAs in the SCL group have a substantially cause yeast eIF4B was shown to enhance eIF4A helicase activity shorter-than-average median CDS length (SI Appendix, Fig. (10) and stimulate 48S PIC assembly by eIF4A in vitro (13), the S6B). This observation raises the question of whether shorter relative dependence of mRNAs on eIF4B in vivo could be expected length or higher closed-loop potential is the key determinant of to mirror their dependence on eIF4A. However, because eIF4B the hypodependence of SCL mRNAs on eIF4B, eIF4A, and interacts directly with the 40S subunit near the mRNA entry Ded1. To address this question, we divided the SCL mRNAs into channel, and 40S-binding is associated with robust eIF4B function three sets (A, B, and C) based on CDS length and compared (13), we considered that eIF4B could also act independently of their TE changes in tif3Δ cells with length-matched sets of non- eIF4A to promote translation of particular mRNAs. Our results SCL mRNAs (SI Appendix, Fig. S6 D–F). Similar to our findings support the latter possibility but also provide evidence consistent for all SCL mRNAs, in set C also, which contained the longest with eIF4B’s known role as cofactor for eIF4A. mRNAs in the SCL group, the tif3Δ mutation conferred a sig- By ribosome profiling we identified a cohort of >100 mRNAs nificantly smaller reduction in TE in the SCL than in the “all that are hyperdependent on eIF4B for robust translation, mRNA” cohort (SI Appendix, Fig. S6 F vs. C). However, this exhibiting TE reductions of more than twofold in tif3Δ vs. WT difference was not observed for SCL mRNA sets A and B, which cells, and whose 5′ UTRs are significantly longer and more contain the shortest and intermediate CDS lengths, respectively structurogenic than the average 5′ UTR. Considering TE changes (SI Appendix, Fig. S6 compare D and E vs. C). Thus, a short CDS of any magnitude genome-wide also reveals that 5′ UTR length (or some other feature enriched in shorter mRNAs) is sufficient and propensity for structure are inversely correlated with TE to confer reduced dependence on eIF4B, and high closed-loop changes conferred by tif3Δ. Having reached the same conclusion potential does not confer additional independence of eIF4B for from ribosome profiling of tif1-ts cells with impaired eIF4A shorter mRNAs. For longer mRNAs, by contrast, strong closed- function (19), we believe our current results on tif3Δ are consistent loop potential mitigates the impact of longer mRNA lengths with eIF4B acting as a cofactor for eIF4A to resolve 5′ UTR and prevents the marked decline in TE in tif3Δ cells typically structures that impede 43S PIC attachment or scanning. This observed for longer mRNAs (SI Appendix, Fig. S6F). Thus, it notion is further supported by the significant correlation between appears that eIF4B function and closed-loop assembly make genome-wide changes in TE in tif3Δ and tif1-ts cells. It is note- overlapping contributions to translation of longer mRNAs. worthy, however, that the magnitude of TE changes is greater in cells lacking eIF4B than in cells lacking eIF4A, even though these eIF4G-Hyperdependent mRNAs Tend to Be Short, Prone to Closed- mutants display similar reductions in bulk translation under our Loop Formation, and Devoid of 5′ UTR Structure. Previously, we profiling conditions. Likewise, only 36 genes were found to be examined the genome-wide effects of depleting eIF4G1 in the hyperdependent on eIF4A, and only three belong to the group of tif4631-td mutant, which lacks the functionally equivalent isoform 111 genes hyperdependent on eIF4B. We reached a similar con- eIF4G2, by analyzing changes in mRNA association with large clusion by comparing changes in ribosome occupancies rather polysomes relative to total mRNA abundance (29). Interestingly, than TE, with only 61 genes exhibiting twofold or greater reduc- we found that mRNAs with heightened dependence on eIF4G1 tions in the tif1-ts mutant; of these 61 genes, only 30 belong to the tend to have relatively short CDS (Fig. 5D), in contrast to the larger group of 246 genes with comparable reductions in ribosome relationship between ΔTE and CDS length noted above for occupancy in tif3Δ(37) cells at 37 °C. Thus, even if all changes in eIF4B, eIF4A, and Ded1 mutants (Fig. 5B and SI Appendix, Fig. ribosome occupancy result from altered translation rates (re- S5A). In addition, we discovered here that TE changes on de- gardless of changes in mRNA abundance), there would still be pletion of eIF4G1 are positively correlated with 5′ UTR length many fewer genes strongly affected by tif1-ts vs. tif3Δ at 37 °C, as and PARS features (SI Appendix, Fig. S7A), indicating that the we concluded from analyzing TE changes. The finding that un- eIF4G1-hyperdependent genes have relatively short, unstructured usually strong dependence on both eIF4A and eIF4B is not 5′ UTRs, as opposed to the relatively long, structured 5′ UTRs of widespread is consistent with the model that eIF4B can function in genes hyperdependent on eIF4B, eIF4A, and Ded1 (Fig. 3D). parallel with eIF4A, e.g., by remodeling the 40S mRNA entry Consistent with their relatively short CDS lengths and short un- channel, in addition to enhancing eIF4A helicase activity. Pre- structured 5′ UTRs, the eIF4G1-hyperdependent genes tend to be sumably both modes of eIF4B function are important for mRNAs translated very efficiently in WT cells (29), as indicated by the with structurogenic 5′ UTRs. Our findings are consistent with strong inverse correlation between changes in TE evoked by previous studies indicating that certain mRNAs with structured 5′ eIF4G1 depletion and TE values in WT cells (SI Appendix, Fig. UTRs exhibit an unusually strong dependence on mammalian S7B). We suggested previously (29) that this trend might be eIF4B (4, 30). explained by proposing that the closed-loop formation, achieved By ribosome profiling of ded1 mutants we previously identified more readily by shorter mRNAs (27), is a key determinant of the a large cohort of Ded1-hyperdependent genes whose 5′ UTRs relatively higher TEs of shorter mRNAs in WT cells and that this also tend to be unusually long and structurogenic (19). Surpris- functional advantage is lost on depletion of eIF4G1. Consistent ingly, the correlations between genome-wide TE changes in ded1 with this model, as reported recently (26) and illustrated in Fig. vs. tif3Δ mutants are similar in magnitude to those observed for 5E, genes belonging to the SCL group with the greatest closed- tif1-ts vs. tif3Δ cells, and the overlap between Ded1- and eIF4B- loop potential (28) are atypically sensitive to depletion of eIF4G— hyperdependent genes, ∼40–70% for various ded1 mutations, is the opposite of our findings that SCL genes are hypodependent on greater than the 3% overlap between genes hyperdependent on

10470 | www.pnas.org/cgi/doi/10.1073/pnas.1612398113 Sen et al. Downloaded by guest on September 26, 2021 eIF4B and eIF4A. Thus, extensive functional cooperation occurs Pab1 occupancies (28). Closed-loop assembly stabilizes eIF4F

between Ded1 and eIF4B that is comparable to, or exceeds, that binding to mRNA (32, 34), which should enhance eIF4G func- INAUGURAL ARTICLE between eIF4A and eIF4B. Perhaps eIF4B performs an un- tions in recruiting the 43S PIC, through its direct interactions suspected cofactor function for Ded1; alternatively, many mRNAs with eIFs 1 and 5 (35), and in recruiting or activating eIF4A and could require independent contributions of eIF4B and Ded1 to Ded1 (36). The diminished capacity of many long mRNAs to form enhance PIC attachment or scanning through structured 5′ the closed-loop and thereby stabilize eIF4G binding should render UTRs, as we suggested previously to explain the functional them particularly sensitive to mutations in eIF4A, Ded1, or eIF4B overlap between eIF4A and Ded1 (19). that reduce helicase activity or eIF4B function in PIC recruitment Our analysis of LUC reporters with unstructured 5′ UTRs or scanning, thus helping to explain the tendency of long mRNAs harboring SL insertions revealed that a strong cap-proximal SL to be hyperdependent on the helicases/eIF4B. Closed-loop as- confers a nearly complete dependence on eIF4B at the stage of sembly also might facilitate reinitiation by ribosomes recycled at PIC recruitment to the mRNA. Previously, we found that a cap- the of the mRNA and delivered to the 5′ end of the proximal SL moderately increased the requirement for Ded1 but same transcript (37, 38); this function could be particularly im- did not increase the dependence on eIF4A for efficient trans- portant for achieving the high-level translation of small mRNAs lation compared with that seen for a reporter with unstructured with strong closed-loop potential. By eliminating the multiple 5′ UTR (19). We speculated that eIF4A is either ineffective in advantages of closed-loop assembly in PIC recruitment and rein- resolving stable SL structures or dispensable for this reaction in itiation, depletion of eIF4G should disproportionately impair cells containing Ded1. To explain the relatively stronger effect of translation of the short, highly efficient mRNAs optimized for eliminating eIF4B on a LUC reporter with a stable cap-proximal closed-loop assembly and have relatively lesser effects on the SL, it could be proposed that eIF4B supports the overlapping longer, more structured mRNAs that form the closed loop poorly functions of Ded1 and eIF4A in resolving such structures or that at WT levels of eIF4G. The resulting strong reduction in trans- eIF4B acts independently of the helicases to overcome the in- lation of the former highly efficient mRNAs should allow the hibitory effect of a stable SL on PIC attachment. longer, more structured mRNAs to compete better for limiting An inverse correlation exists between transcript length and TE PICs, offsetting the diminished PIC recruitment evinced by eIF4G in WT yeast (25), which might reflect an increased probability that depletion and attendant diminished eIF4A and Ded1 activity for 5′ UTRs of longer mRNAs engage in secondary structures in- these less efficient mRNAs. If, as seems plausible, the helicases/

volving the extended CDS of these mRNAs. Indeed, we found that eIF4B are not critically required for closed-loop assembly, then GENETICS shorter mRNAs tend to contain short, unstructured 5′ UTRs and helicase/eIF4B inactivation should produce the observed small they harbor nonstructurogenic sequences surrounding their start reductions in the translation of highly efficient short mRNAs, codons (31). The inefficient translation of longer mRNAs also could which are also depleted of 5′ UTR structures. Their relative TEs reflect their decreased ability to form the closed-loop intermediate frequently increase in the helicase/eIF4B mutants because of re- stabilized by eIF4G interactions with eIF4E and Pab1 (27). In- duced competition from the longer, more structured mRNAs that terestingly, we found that longer mRNAs tend to be unusually have greater dependence on helicase/eIF4B activities. dependent on eIF4B, eIF4A, and Ded1. Although mRNA length, In conclusion, although eIF4G is a cofactor for eIF4A and also ′ ′ 5 UTR length, and 5 UTR structure all contribute independently appears to regulate Ded1 (36), its most critical role in yeast to eIF4B hyperdependence, mRNA length is the strongest de- appears to be stabilizing closed-loop assembly. As a result, de- terminant by far. We also observed that mRNAs with the strongest pleting eIF4G most strongly impairs the translation of short, potential for closed-loop assembly (SCL/group III) (28) tend to unstructured mRNAs with the highest closed-loop potential, be hypodependent on eIF4B/eIF4A/Ded1. The SCL group has whereas eliminating eIF4B or inactivating eIF4A and Ded1 most shorter-than-average transcript lengths (26), however, and we strongly impairs mRNAs with exactly the opposite features. deduced that belonging to the SCL group imparts a translational advantage in cells lacking eIF4B only for longer genes with CDS Methods lengths >700 nt. Thus, eIF4B and the helicases are particularly Construction of Yeast Strains and Plasmids. Yeast strains FJZ046 (MATa his3Δ1 important for translation of mRNAs with both longer length and leu2Δ0lys2Δ0met15Δ0ura3Δ0TIF3) and FJZ052 (MATa his3Δ1leu2Δ0 lys2Δ0 reduced capacity for closed-loop formation, whereas shorter met15Δ0ura3Δ0tif3Δ::hisG) were described previously (13). Plasmids used are mRNAs are relatively independent of eIF4B/eIF4A/Ded1 re- listed in SI Appendix,TableS1, and generated as described in the SI Appendix. gardless of their closed-loop potential. The tendency toward short, unstructured 5′ UTRs also contributes to the relative in- Yeast Biochemical Methods. Methods for analysis of bulk polysome profiles, dependence of short mRNAs on eIF4B and the helicases. assays of LUC reporters in whole cell extracts (WCEs) (19), and association of Interestingly, interrogating data from our previous study (32) reporter mRNA with native 40S subunits (13) were described previously; revealed that yeast eIF4G is most critically required for mRNAs details are provided in the SI Appendix. that are relatively short, have a high probability for closed-loop ′ Ribosome Footprint Profiling and RNA-Seq. Ribosome profiling was conducted assembly (26), have relatively short and unstructured 5 UTRs, and essentially as described (39, 40), as detailed in the SI Appendix, on isogenic exhibit greater-than-average TEs in WT cells. Similarly, depletion tif3Δ (FJZ052) and WT (FJZ046) cells cultured in synthetic complete (SC) of eIF4G was found to diminish translation of short vs. long genes medium and treated with 100 ug/mL cycloheximide for 2 min before har- preferentially in , a condition that is exploi- vesting. Statistical analysis of differences in ribosome footprint, RNA-seq ted via down-regulation of eIF4G expression in starved animals to read counts, or TE values between WT and mutant samples was conducted up-regulate long, stress-response genes (33). The remarkable fact using DESeq (41) (tabulated in Dataset S1). Genes with fewer than 128 total that depleting eIF4G preferentially impairs the translation of the mRNA reads in the four samples combined (two replicates of both WT and categories of mRNAs least impacted by inactivating eIF4B, eIF4A mutant strains) were excluded from the calculation of TE values. or Ded1 implies that eIF4G carries out a function beyond stimu- ′ ACKNOWLEDGMENTS. We thank members of the A.G.H. laboratory and the lating the unwinding of 5 UTR structures by eIF4A or Ded1 and Dever and Lorsch groups for many helpful suggestions; Wendy Gilbert and that this additional function is required for the robust translation of colleagues for critical insights about the closed-loop potential of short eIF4G- short mRNAs with strong closed-loop potential. dependent genes; Sumit Sen, Razvan Chereji, and Tingfen Yan for help with An obvious possibility for this additional function of eIF4G bioinformatics; and Philip McQueen and Peter Munson for advice on statistical analysis. N.T.I. was supported by Searle Scholars Program Grant is promoting closed-loop assembly, because forming this in- 11-SSP-229 and National Institute of Environment Health Sciences Grant R21 termediate is favored for eIF4G-hyperdependent mRNAs by ES22575-01. This work was supported in part by the Intramural Research their relatively short transcript lengths (27) and high eIF4F and Program of the National Institutes of Health.

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