Molecular Mechanisms of Translational Control

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We study the molecular mechanisms involved in assembly, function, and regulation of translation initiation complexes involved in protein synthesis, using yeast as a model system to exploit its powerful combination of genetics and biochemistry. The translation initiation pathway produces an 80S ribosome bound to mRNA with methionyl initiator tRNA (tRNAi) base-paired to the AUG start codon in the ribosomal P site. 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 poly(A)-binding protein bound to the poly(A) tail, and scans the 5’ 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 by eIF4F and RNA helicase Ded1, which resolve RNA structures in the 5' UTR. AUG recognition stabilizes a closed conformation of the PIC, dissociation of eIF1, highly stable P-site binding of tRNAi, and hydrolysis of the GTP bound to eIF2 stimulated by eIF5. Subsequent release of eIF2-GDP from tRNAi allows joining of the 60S subunit to form the 80S initiation complex. eIF2 function is down-regulated by phosphorylation of its alpha subunit by protein kinase Gcn2 in response to amino acid starvation and other stresses that evoke stalling of ribosomes during translation elongation. Phosphorylation of eIF2 by Gcn2 not only impairs global protein synthesis but stimulates translation of GCN4 mRNA encoding the transcriptional activator GCN4 to up-regulate expression of amino acid biosynthetic genes. Translational control of GCN4 mRNA is mediated by short upstream open reading frames uORFs 1 to 4 via the “delayed reinitiation” mechanism. Decreased assembly of TCs in starved cells allows scanning 40S ribosomal subunits that have translated the first or second uORFs and resumed scanning to bypass the inhibitory uORFs 3-4 and reinitiate at the GCN4 start codon. Distinct uS11/Rps14 interactions with the translation preinitiation complex differentially alter the accuracy of start codon recognition. The eukaryotic 43S pre-initiation complex (PIC), containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP, scans the mRNA leader for an AUG start codon in favorable “ Kozak” context. Recognition of AUG triggers the rearrangement of the PIC from an open scanning conformation to a closed arrested state with more tightly bound Met-tRNAiMet. Cryo-EM reconstructions of yeast PICs suggest remodelling of the interaction between the 40S protein uS11/Rps14 with rRNA and mRNA in the ribosome decoding center between the open and closed states; however, the importance of these conformational changes in start codon recognition was unknown. We found that uS11/Rps14-L137 substitutions disrupting rRNA contacts favoured in the open complex increase initiation at suboptimal start sites, and L137E stabilizes TC binding to PICs reconstituted in vitro with a UUG start codon, all indicating inappropriate rearrangement to the closed state at suboptimal initiation sites. Conversely, uS11/Rps14-R135 and -R136 substitutions perturbing interactions with rRNA exclusively in the closed state conferred the opposite phenotypes of initiation hyperaccuracy, and for R135E, accelerated TC dissociation from reconstituted PICs. These genetic and biochemical findings indicate that distinct interactions of uS11/Rps14 with rRNA stabilize first the open and then the closed conformation of the PIC to influence the accuracy of initiation in vivo. Non-canonical initiation factor eIF2A has a minimal role in translation initiation even in cells with reduced eIF2 function. It has been reported that mammalian eIF2A promotes Met-tRNAi recruitment to the AUG codons of viral mRNAs and enhances initiation at near-cognate codons (NCCs) when TC assembly is impaired by eIF2α phosphorylation. We conducted ribosome profiling on a yeast eIF2AΔ mutant in the presence or absence of eIF2α phosphorylation induced by amino acid starvation, reasoning that mRNAs able to utilize eIF2A might show TE reductions only when eIF2 is impaired. Surprisingly, eIF2AΔ conferred no significant TE reductions for any mRNAs in non-starved cells, and affected only ~30 mRNAs in amino acid-starved cells. We could validate this conditional eIF2A dependence by LUC reporter analysis and polysome profiling of native mRNAs for only several genes. We found no evidence that eIF2AΔ alters the translation of mRNAs containing evolutionarily conserved, inhibitory uORFs. Our findings do not support an important role for yeast eIF2A in a back-up mechanism for Met-tRNAi recruitment when eIF2 function is reduced by stress. Yeast-specific roles for non-canonical initiation factors Tma20/Tma22 in controlling reinitiation on GCN4 mRNA. Evidence suggests that the mammalian heterodimer MCT-1/DENR promotes reinitiation (REI) following translation of short uORFs whose penultimate codons depend on the heterodimer for releasing the cognate deacylated tRNA from post-termination 40S complexes (postTCs), including uORF1 of ATF4 mRNA that mediates the same delayed-REI mechanism governing GCN4 translation. Substituting GCN4 uORF1 with penultimate codons that vary in dependence on the corresponding Tma20/Tma22 heterodimer for 40S recycling, we found little evidence that Tma20/Tma22 enhances REI at GCN4 uORF1 variants equipped with Tma-dependent penultimate codons. Making similar substitutions at uORF4, which differs from uORF1 in being -permissive for REI, we implicated Tma20/Tma22 in blocking, not stimulating REI, by releasing empty post-TCs, in a manner only marginally more pronounced with Tma20/Tma22-dependent penultimate codons. Thus, dissociation of tRNA from postTCs can proceed without Tma20/Tma22 at both uORF1 and uORF4, even at Tma-dependent penultimate codons, and Tma20/Tma22 enhances dissociation of postTCs at uORF4 variants with either class of penultimate codons. The latter function is impeded at uORF1 by its specialized REI-promoting elements. We conclude that Tma20/Tma22 and MCT-1/DENR play distinct roles in the delayed-REI mechanism. Structural evidence that uncharged tRNA is an activator of Gcn2. We collaborated with Jinwei Zhang (NIDDK/NIH) to determine the crystal structure of the HisRS-like domain of Gcn2 from Chaetomium thermophilum. The structure is dramatically similar to authentic HisRS, including an α-helical insertion domain harboring the catalytic center and a separate anticodon binding domain (ABD) that, in authentic HisRS, bind to the acceptor stem and anticodon loop of tRNAHis, respectively. The structural elements for forming histidyl adenylate and aminoacylation of tRNAHis are lacking, consistent with repurposing of the HisRS-like domain as a sensor of amino acid starvation. This model is supported by the location of numerous substitutions in the conserved catalytic core of the HisRS-like domain that hyperactivate or impair Gcn2 function. We further showed that residues in the ABD predicted by the structure to contact the tRNA anticodon loop or connect the ABD to the catalytic core are also crucial for Gcn2 activation in vivo and high-level kinase activity by purified Gcn2 in vitro. The presence of two conserved structural domains for binding different ends of tRNA in the HisRS-like domain, both essential for kinase activation, supports the model that uncharged tRNA directly activates Gcn2 on stalled ribosomes in amino acid-starved cells. Yeast poly(A)-binding protein (Pab1) controls translation initiation in vivo primarily by blocking mRNA decapping and decay. Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in a manner suppressed by deleting the catalytic subunit of decapping enzyme (dcp2), demonstrating that enhanced decapping/degradation is the major driver of reduced mRNA abundance and protein synthesis at limiting Pab1 levels. Remarkably, in contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these TE changes were broadly diminished by dcp2∆, suggesting that reduced mRNA abundance is a major driver of translational reprogramming at limiting Pab1. These results further indicate that differential translation of most transcripts does not require stabilization of the closed-loop mRNP via PABP-eIF4G interaction at normal levels of cellular mRNAs. Our single-molecule poly(A) tail sequencing analysis revealed that Pab1 depletion confers an increased median poly(A) tail length that is nullified by dcp2∆, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. Notably, groups of mRNAs encoding mitochondrial proteins or histones show unusually strong Dcp2-dependent reductions in mRNA abundance on Pab1 depletion despite having average poly(A) tail lengths in wild-type cells, indicating that short tail length is not the sole determinant of enhanced mRNA decay at limiting Pab1. Interestingly, the preferential degradation of histone mRNAs and reduced levels of histone proteins on Pab1 depletion is accompanied by activation of internal cryptic promoters in the manner expected for reduced genic nucleosome occupancies, revealing a new layer of post-transcriptional control of histone gene expression through Pab1 blockage of decapping of histone mRNAs.