We study the molecular mechanisms involved in assembly and function of translation initiation complexes involved in protein synthesis, using yeast as a model system to exploit its powerful combination of genetics and biochemistry for dissecting complex cellular processes in vivo. The translation initiation pathway produces an 80S ribosome bound to mRNA with methionyl initiator tRNA (tRNAi) base-paired to the AUG start codon. The tRNAi is recruited to the 40S subunit in a ternary complex (TC) with GTP-bound eIF2 to produce the 43S preinitiation complex (PIC) in a reaction stimulated by eIFs 1, 1A, 3 and 5. The 43S PIC attaches to the 5' end of mRNA, facilitated by cap-binding complex eIF4F (comprised of eIF4E, eIF4G, and RNA helicase eIF4A) and PABP bound to the poly(A) tail, and scans the 5 untranslated region (UTR) for an AUG start codon in preferred sequence context. Scanning is promoted by eIFs 1 and 1A, which induce an open conformation of the 40S and binding of TC in a conformation suitable for scanning successive triplets entering the ribosomal P site (P-out), and by eIF4F and other RNA helicases, such as Ded1 and Dbp1, that remove secondary structure in the 5' UTR. AUG recognition leads to tighter binding of TC in the P-in state and evokes irreversible hydrolysis of the GTP bound to eIF2, dependent on the GTPase activating protein (GAP) eIF5, releasing eIF2-GDP from the PIC to leave tRNAi in the P site and allow joining of the 60S subunit to form the 80S initiation complex. Dual roles of initiation factor eIF2beta in regulating scanning and initiation accuracy via distinct interactions with eIF1 and Met-tRNAi. Our previous cryo-EM analysis of partial yeast PICs revealed distinct conformations pertaining to different stages of initiation. A py48S-open complex exhibits upward movement of the 40S head from the body that eliminates interactions of the 40S subunit with Met-tRNAi and mRNA evident in the py48S-closed complex. The py48S-open complex seems well-suited for scanning triplets for complementarity to Met-tRNAi, with TC anchored in the unstable P-out conformation; whereas py48S-closed exhibits the more stable P-in conformation required for start codon selection. eIF1 exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the P-in state, necessitating eIF1 dissociation for start codon selection. The beta-subunit of eIF2 interacts with eIF1, eIF1A and the anticodon stem of tRNAi only in the open complex, which should enable it to stabilize exclusively the scanning conformation of the PIC. Supporting this model, eIF2 and eIF1 substitutions that weaken their interactions in the open complex increased initiation at UUG codons; and compound substitutions also derepressed translation of GCN4 mRNA (the Gcd- phenotype). In the yeast reconstituted system, the compound substitutions impaired TC loading while stabilizing TC binding at PICs assembled on UUG codons, signifying destabilization of the open complex and shift to the closed/P-in state. Remarkably, an eIF1 substitution that should strengthen the eIF2:eIF1 interface had the opposite genetic and biochemical phenotypes. eIF2beta in the open complex is also predicted to clash with Met-tRNAi in the closed state, and substitutions that diminish this clash increased UUG initiation in vivo and stabilized Met-tRNAi binding at UUG codons in vitro, but did not confer a Gcd- phenotype or affect TC loading in vitro. Thus, eIF2beta's clash with Met-tRNAi impedes rearrangement to the closed state without affecting TC binding to the open complex. Therefore, eIF2beta resembles eIF1 in playing a dual role of (i) stabilizing the open/P-out scanning conformation through direct binding to eIF1 and Met-tRNAi, and (ii) impeding transition of Met-tRNAi to the P-in state at non-AUG codons through a clash with tRNAi. Yeast Ded1 promotes 48S translation pre-initiation complex assembly in an mRNA-specific and eIF4F-dependent manner. RNA helicases eIF4A and Ded1 are believed to resolve mRNA structures that impede ribosome attachment or scanning to the start codon, but whether they perform distinct functions has been poorly understood. Previously, we compared the effects of mutations in Ded1 or eIF4A on genome-wide translational efficiencies (TEs) by ribosome profiling. Inactivation of Ded1 substantially reduced the relative TEs of >600 mRNAs, whereas inactivation of eIF4A similarly affected <40 mRNAs. Ded1-dependent mRNAs show greater than average 5UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs (Fig. 7B). Thus, Ded1 is critically required for PIC attachment and scanning through secondary structures, whereas eIF4A promotes a step of initiation common to nearly all mRNAs regardless of their structures. Recently, we measured the kinetics of 48S PIC assembly in the yeast reconstituted system for a panel of native mRNAs identified as being hyper- or hypodependent on Ded1 by ribosome profiling. Ded1 hypodependent mRNAs could be recruited rapidly without Ded1, and addition of Ded1 only moderately accelerated their recruitment. Ded1 hyperdependent mRNAs, by contrast, were recruited poorly in the absence of Ded1, and Ded1 greatly accelerated their recruitment. Eliminating stem-loop structures enhanced Ded1-independent recruitment, and diminished Ded1-acceleration of 48S assembly, on several hyperdependent mRNAs. Moreover, inserting stem-loop structures into a synthetic unstructured mRNA conferred a strong Ded1-requirement for rapid recruitment. Eliminating domains in Ded1 that mediate its association with eIF4A or eIF4G increased the Ded1 concentration required for maximal rate acceleration and, for some mRNAs, also decreased the maximal rate achieved with saturating Ded1. Thus, Ded1 accelerates 48S assembly by resolving mRNA structures in a manner stimulated by its interaction with eIF4F and eIF4A. Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo Scd6 protein family members are evolutionarily conserved components of mRNA granules. Scd6, and two other proteins containing RGG domains, Sbp1 and Npl3, were implicated as general translational repressors in yeast that function by binding to the RNA2 or RNA3 domains of eIF4G. As there was no evidence that Scd6, Sbp1, or Npl3 act as translational repressors of particular native mRNAs in vivo, we examined the effects of tethering MS2 fusions of each protein to a GFP reporter mRNA containing 3UTR MS2 binding sites, in WT cells or mutants lacking DCP2, DHH1, or both. We found that tethering Scd6, but not Sbp1 or Npl3, repressed reporter mRNA abundance via Dcp2 and suppressed reporter mRNA translation via Dhh1, as tethered Scd6 (i) reduced GFP protein and GFP mRNA abundance in WT cells, (ii) reduced GFP protein with little effect on the mRNA level in dcp2 cells, (iii) and had no impact on either GFP protein or mRNA expression in a dcp2 dhh1 double mutant. Repression of both GFP protein and mRNA was enhanced in a ccr4 mutant, lacking the major mRNA deadenylase activity of Ccr4-Not complex, suggesting that Ccr4 interferes with a more efficient repression pathway enlisted by tethered Scd6. Importantly, ribosome profiling of scd6 and dhh1 mutants, and of double mutants also lacking DCP2, suggests that Scd6 cooperates with Dhh1 in translational repression as well as turnover of a group of native mRNAs, and that both processes require DCP2, as the derepression of TEs on deletion of SCD6 or DHH1 was suppressed by dcp2. Thus, it appears that translational repression as well as accelerated mRNA decay conferred on native mRNAs by Scd6 (and Dhh1) depend on mRNA decapping, leading to a pool of uncapped but relatively stable mRNAs incapable of recruiting eIF4F.
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