Global mRNA selection mechanisms for translation initiation

Abstract

BackgroundThe selection and regulation of individual mRNAs for translation initiation from a competing pool of mRNA are poorly understood processes. The closed loop complex, comprising eIF4E, eIF4G and PABP, and its regulation by 4E-BPs are perceived to be key players. Using RIP-seq, we aimed to evaluate the role in gene regulation of the closed loop complex and 4E-BP regulation across the entire yeast transcriptome.ResultsWe find that there are distinct populations of mRNAs with coherent properties: one mRNA pool contains many ribosomal protein mRNAs and is enriched specifically with all of the closed loop translation initiation components. This class likely represents mRNAs that rely heavily on the closed loop complex for protein synthesis. Other heavily translated mRNAs are apparently under-represented with most closed loop components except Pab1p. Combined with data showing a close correlation between Pab1p interaction and levels of translation, these data suggest that Pab1p is important for the translation of these mRNAs in a closed loop independent manner. We also identify a translational regulatory mechanism for the 4E-BPs; these appear to self-regulate by inhibiting translation initiation of their own mRNAs.ConclusionsOverall, we show that mRNA selection for translation initiation is not as uniformly regimented as previously anticipated. Components of the closed loop complex are highly relevant for many mRNAs, but some heavily translated mRNAs interact poorly with this machinery. Therefore, alternative, possibly Pab1p-dependent mechanisms likely exist to load ribosomes effectively onto mRNAs. Finally, these studies identify and characterize a complex self-regulatory circuit for the yeast 4E-BPs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0559-z) contains supplementary material, which is available to authorized users.

eIF4B stimulates translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G

“…Consistent with this model, as reported recently (26) and illustrated in Fig. 5E, genes belonging to the SCL group with the greatest closedloop potential (28) are atypically sensitive to depletion of eIF4G-the opposite of our findings that SCL genes are hypodependent on eIF4B, eIF4A, and Ded1 (Fig. 5C).…”

“…Thus, mRNAs considered to be optimized for closed-loop formation tend to be hypodependent on eIF4B, eIF4A, and Ded1. As might be expected, the SCL mRNAs constitute one of the most highly translated subsets of all yeast mRNAs; however, another subset of highly translated mRNAs is enriched only for Pab1 (28). The latter group (designated "group I") does not exhibit the increase in TE in tif3Δ cells at 37°C displayed by the SCL group of mRNAs (designated "group III") and instead displays a response more typical of all other yeast genes (SI Appendix, Fig.…”

“…S5B), we considered the alternative hypothesis that hyperdependence of long mRNAs on eIF4B, eIF4A, and Ded1 reflects their relative inability to form the closed-loop intermediate (27), stabilized by mutual interaction of eIF4G with Pab1 bound to the poly(A) tail and eIF4E bound to the cap. To address this possibility, we interrogated a published dataset describing the genome-wide association of mRNAs with eIF4E, eIF4G1, eIF4G2, Pab1, and the inhibitory eIF4E-binding proteins Caf20 and Eap1, determined by sequencing mRNAs immunoprecipitated with these proteins (RIP-seq) (28). A subset of 395 mRNAs was found to be enriched for eIF4E, eIF4G1, eIF4G2, and Pab1 and depleted for Caf20 and Eap1 and thus was considered to have the greatest potential for closed-loop formation.…”

“…An obvious possibility for this additional function of eIF4G is promoting closed-loop assembly, because forming this intermediate is favored for eIF4G-hyperdependent mRNAs by their relatively short transcript lengths (27) and high eIF4F and Pab1 occupancies (28). Closed-loop assembly stabilizes eIF4F binding to mRNA (32,34), which should enhance eIF4G functions in recruiting the 43S PIC, through its direct interactions with eIFs 1 and 5 (35), and in recruiting or activating eIF4A and Ded1 (36).…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Using in vitro translation experiments and toe-printing techniques, 48S PIC association with the AUG start codon on a short mRNA was resistant to 59 cap analog (m 7 GDP) in a manner that was dependent on both the 59 cap and poly(A) tail as well as intact eIF4G, Pab1, and the termination factors eRF1 and eRF3 (Amrani et al 2008). Further support for the eIF4E-4G-Pab1-mRNA closed loop comes from the capture of mRNAs bound to each factor from live cells and quantified by RT-PCR or high-throughput sequencing (Archer et al 2015;Costello et al 2015). However, it should be noted that the Pab1-eIF4G interaction is dispensable for cell growth unless the eIF4E-eIF4G interaction is also impaired Park et al 2011).…”