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 likely evoke stalling of ribosomes engaged in translation elongation. Differential requirements for P stalk components in activating yeast protein kinase Gcn2 by stalled ribosomes during stress. A highly conserved response to amino acid starvation involves activation of protein kinase Gcn2, which phosphorylates eukaryotic initiation factor 2 with attendant inhibition of global protein synthesis and increased translation of yeast transcriptional activator GCN4. Gcn2 contains a domain related to histidyl-tRNA synthetase (HisRS-like domain), and a C-terminal ribosome-binding domain. Previous work indicated that Gcn2 is activated on translating ribosomes by uncharged tRNAs that accumulate in amino acid-starved cells and pair with the cognate codons in the empty ribosomal A, interacting directly with the HisRS-like domain to stimulate kinase activity. Gcn2 can also be activated by conditions that stall elongating ribosomes without reducing aminoacylation of tRNA, but it was unclear whether distinct molecular mechanisms operate in these two circumstances. We identified three regimes that activate Gcn2 in yeast by starvation-independent (SI) ribosome-stalling that leaves an empty A site on the stalled ribosome: (i) treatment with inhibitor tigecycline, which should stall ribosomes at all codons; (ii) deleting the gene encoding tRNAArgUCC, which should stall ribosomes at AGG codons; and depletion of translation termination factor eRF1, which should stall ribosomes at stop codons. Subsequent genetic analysis demonstrated requirements for the HisRS-like and ribosome-binding domains of Gcn2, positive effectors Gcn1/Gcn20, and the tethering of at least one of two P1/P2 heterodimers of the 60S ribosomal P-stalk complex, for detectable activation by SI-ribosome stalling. Remarkably, no tethered P1/P2 proteins were required for strong Gcn2 activation by starvation for various amino acids, indicating that Gcn2 activation has different requirements for the P-stalk depending on how ribosomes are stalled. We propose that accumulation of deacylated tRNAs in starved cells functionally substitutes for the P-stalk in binding to the HisRS-like for eIF2 kinase activation by ribosomes stalled with A sites. Yeast mRNA decapping factors control mRNA abundance and translation to adjust metabolism and cell filamentation to nutrient availability Pat1 and helicase Dhh1 are conserved activators of the mRNA decapping enzyme Dcp1:Dcp2, central to general mRNA decay, and were implicated in repressing translation in glucose-starved cells. Using ribosome profiling and RNA-seq analysis of dhh1, pat1, and dhh1pat1 mutants cultured in rich medium, we identified hundreds of mRNAs up-regulated in a manner indicating cumulative repression by Pat1 and Dhh1. Although the environmental stress response (ESR) is mobilized in these mutants, involving increased expression of stress genes (iESR) and repression of ribosome production and translation factors (rESR); most up-regulated mRNAs are not iESR transcripts. CAGE analyses of capped mRNAs revealed enhanced accumulation of decapped intermediates for the up-regulated transcripts, and ChIP-Seq analysis of RNA Pol II indicated decreased rather than increased transcription of the cognate genes, demonstrating that reduced decapping and 5-3 degradation drives transcript derepression in the mutants. The cumulative contributions of Dhh1 and Pat1 to mRNA decapping are consistent with their independent interactions with distinct segments of Dcp2 involved in its activation, and evidence for distinct decapping complexes containing Dhh1 or Pat1. Although previous work implicated Dhh1 and Pat1 in accelerating degradation of mRNAs enriched for slowly decoded codons, the mRNAs up-regulated in the mutants have average proportions of suboptimal codons. Pat1 and Dhh1 also collaborate to reduce the translational efficiencies (TEs) and protein production of many mRNAs, including highly repressed mRNAs involved in cell adhesion or utilization of the poor nitrogen source allantoin. Pat1/Dhh1 also repress the abundance or TE of transcripts involved in oxidative phosphorylation (OXPHOS), catabolism of non-preferred carbon or nitrogen sources, or in autophagy. We obtained evidence for increased activity of the electron transport chain (ETC) of OXPHOS in the dhh1 and pat1 mutants, and elevated autophagic flux in the pat1dhh1 double mutant. As these genes/pathways are normally repressed in cells growing in rich medium replete, we concluded that Pat1 and Dhh1 function as post-transcriptional repressors of multiple pathways normally activated only during nutrient limitation. Parallel analysis of the dcp2 mutant led to the finding that among the 1300 mRNAs preferentially targeted for degradation by the decapping enzyme, 55% utilize Dhh1/Pat1 or decapping activators Scd6/Edc3 to promote decay, while the remainder employ the Upf factors that mediate nonsense-mediated mRNA decay (NMD). We also found that dcp2 confers a broad reprogramming of translation, wherein well-translated mRNAs exhibit increased TEs at the expense of poorly translated mRNAs, which we could attribute to increased competition for 43S PICs as dcp2 cells contain elevated mRNA levels coupled with decreased ribosome abundance (owing to the ESR response). The increased mRNA/40S ratio and decreased 40S concentration should favor mRNAs with high rates of PIC recruitment at the expense of poorly initiated transcripts. As might be expected, the dcp2 mutation up-regulates many of the same mRNAs required for respiration, utilization of poor carbon/nitrogen sources, and autophagy derepressed in the pat1 and dhh1 mutants, and confers elevated mitochondrial membrane potential and TCA cycle intermediates, indicating increased OXPHOS on glucose-rich medium. dcp2 mutant cells also resemble the decapping activator mutants in showing elevated expression of cell adhesion proteins that function in forming pseudohyphae, and we observed increased filamentation of both dcp2 and pat1 mutant cells on rich medium. As filamentation is normally limited to starvation conditions and is viewed as a strategy for nutrient foraging, this phenotype supports the role of decapping factors in repressing pathways utilized primarily in starved cells.