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Initiation is the most highly regulated and rate-limiting step of translation that dictates both the quality and quantity of cellular proteins being synthesized. Understanding the regulation of initiation pathway is key to medical advances in treatment of neurological disorders, cancer and metabolic disorders. In eukaryotic translation initiation, the 43S pre-initiation complex (PIC) containing the small ribosomal subunit and methionyl initiator tRNA typically attaches to the 5’end of mRNA and scans the 5' untranslated region (UTR) for the start codon. Resolving mRNA secondary structures in 5' UTR is thus a key regulatory step in both 43S PIC attachment and subsequent scanning of the mRNA. DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving such secondary structures, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast by ribosome profiling. Despite similar reductions in bulk translation, inactivation of Ded1 evoked broader and more extensive changes in TE than did inactivation of eIF4A. Interestingly, Ded1-hyperdependent mRNAs exhibit greater than average 5’ UTR length and propensity for secondary structure. While only a small fraction of mRNAs shows a heightened requirement for eIF4A, dependence on eIF4A is significantly correlated with requirements for Ded1 and features characteristic of Ded1-hyperdependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs and while eIF4A cooperates with Ded1 in this function it also promotes a step of initiation common to virtually all mRNAs. In living cells, eIF4A and Ded1 play distinct but partially overlapping functions in regulating global translational changes.
Using a similar strategy we discovered a role for Dbp1, a paralog of Ded1, in translation initiation. Ribosome footprint profiling revealed substantially reduced TEs of a greater number of mRNAs in the ded1-ts dbp1Δ double mutant versus the ded1-ts or dbp1Δ single mutant, indicating functional overlap between these helicases. Moreover, hundreds of mRNAs become hyperdependent on Dbp1 when Ded1 is impaired; and become hyperdependent on Ded1 when Dbp1 is lacking in the double mutant. These “conditionally Dbp1/Ded1-hyperdependent mRNAs” exhibited properties of Ded1-hyperdependent mRNAs. Since overexpression of Dbp1 in ded1-ts cells substantially rescues the translation of a large fraction of the Ded1-hyperdependent mRNAs, we propose that Dbp1 cooperates with Ded1 in wild-type cells to enhance scanning on many long mRNAs that harbor structured 5’UTRs. Remarkably, we identified a distinct set of Dbp1- hyperdependent mRNAs which exhibit features opposite of those displayed by the mRNAs enjoying functional cooperation between the two helicases. Thus, Dbp1 appears to function uniquely on a subset of highly-translated, short, unstructured mRNAs in addition to acting as a Ded1 paralog on the large cohort of long, structured mRNAs. In summary, my work reveals that the RNA helicases eIF4A, Ded1 and Dbp1 are tacticians of translational control in vivo. Future research directed at elucidating the molecular mechanisms by which these helicases regulate protein synthesis will lead to a more complete understanding of this paramount cellular process. |