Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes. To comprehensively study the effects of eIF3 remodeling, we contrasted the impact of eIF3d, eIF3e or eIF3h depletion on the translatome of HeLa cells using Ribo-seq. Depletion of eIF3d or eIF3e, but not eIF3h reduced the levels of multiple components of the MAPK signaling pathways. Surprisingly, however, depletion of all three eIF3 subunits increased MAPK/ERK pathway activity. Depletion of eIF3e and partially eIF3d also increased translation of TOP mRNAs that encode mainly ribosomal proteins and other components of the translational machinery. Moreover, alterations in eIF3 subunit stoichiometry were often associated with changes in translation of mRNAs containing short uORFs, as in the case of the proto-oncogene MDM2 and the transcription factor ATF4. Collectively, perturbations in eIF3 subunit stoichiometry exert specific effect on the translatome comprising signaling and stress-related transcripts with complex 5' UTRs that are implicated in homeostatic adaptation to stress and cancer.

• Keywords
• MAPK pathway, eIF3, genetics, genomics, human, ribosomal proteins, ribosome, translation, translational control,
• MeSH
• Eukaryotic Initiation Factor-3 * metabolism   genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• MAP Kinase Signaling System * MeSH
• Protein Biosynthesis MeSH
• Proto-Oncogene Mas * MeSH
• Ribosomal Proteins * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 * MeSH
• MAS1 protein, human MeSH Browser
• Proto-Oncogene Mas * MeSH
• Ribosomal Proteins * MeSH

BACKGROUND: Little is known about the impact of trans-acting genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate the influence of such distant genetic loci on the efficiency of mRNA translation and define their contribution to the development of complex disease phenotypes within a panel of rat recombinant inbred lines. RESULTS: We identify several tissue-specific master regulatory hotspots that each control the translation rates of multiple proteins. One of these loci is restricted to hypertrophic hearts, where it drives a translatome-wide and protein length-dependent change in translational efficiency, altering the stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus across multiple congenic lines points to a translation machinery defect, characterized by marked differences in polysome profiles and misregulation of the small nucleolar RNA SNORA48. Strikingly, from yeast to humans, we observe reproducible protein length-dependent shifts in translational efficiency as a conserved hallmark of translation machinery mutants, including those that cause ribosomopathies. Depending on the factor mutated, a pre-existing negative correlation between protein length and translation rates could either be enhanced or reduced, which we propose to result from mRNA-specific imbalances in canonical translation initiation and reinitiation rates. CONCLUSIONS: We show that distant genetic control of mRNA translation is abundant in mammalian tissues, exemplified by a single genomic locus that triggers a translation-driven molecular mechanism. Our work illustrates the complexity through which genetic variation can drive phenotypic variability between individuals and thereby contribute to complex disease.

• Keywords
• Cardiac hypertrophy, Complex disease, Genetic variation, HXB/BXH rat recombinant inbred panel, Ribosome biogenesis, Ribosome profiling, Ribosomopathy, Spontaneously hypertensive rats (SHR), Translational efficiency, trans QTL mapping,
• MeSH
• Organelle Biogenesis MeSH
• Genetic Variation MeSH
• Peptide Chain Initiation, Translational * MeSH
• Cardiomegaly genetics   metabolism   pathology   MeSH
• Rats MeSH
• Quantitative Trait Loci * MeSH
• RNA, Small Nucleolar genetics   metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Myocardium metabolism   pathology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Rats, Inbred SHR MeSH
• Rats, Transgenic MeSH
• Gene Expression Regulation MeSH
• Ribosomal Proteins genetics   metabolism   MeSH
• Ribosomes genetics   metabolism   pathology   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Sarcomeres metabolism   pathology   MeSH
• Gene Expression Profiling MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Small Nucleolar MeSH
• RNA, Messenger MeSH
• Ribosomal Proteins MeSH

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

• MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• Organisms, Genetically Modified MeSH
• Protein Biosynthesis genetics   MeSH
• Ribosomal Proteins genetics   physiology   MeSH
• Ribosomes metabolism   MeSH
• RNA, Transfer metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   physiology   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Peptide Chain Termination, Translational * genetics   MeSH
• Codon, Terminator metabolism   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• Ribosomal Proteins MeSH
• RNA, Transfer MeSH
• RPS3 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Codon, Terminator MeSH

eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.

• MeSH
• Cryoelectron Microscopy MeSH
• Eukaryotic Initiation Factor-1 chemistry   genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-3 chemistry   genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-5 chemistry   genetics   metabolism   MeSH
• Peptide Chain Initiation, Translational * MeSH
• Ribosome Subunits, Small, Eukaryotic genetics   metabolism   MeSH
• Models, Molecular MeSH
• Protein Domains MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   ultrastructure   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Eukaryotic Initiation Factor-1 MeSH
• Eukaryotic Initiation Factor-3 MeSH
• Eukaryotic Initiation Factor-5 MeSH
• Saccharomyces cerevisiae Proteins MeSH

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

• Keywords
• S. cerevisiae, biochemistry, biophysics, eIF3, initiation, mRNA recruitment, ribosome, structural biology, translation, yeast,
• MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• Guanosine Triphosphate metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• DNA Mutational Analysis MeSH
• Mutant Proteins genetics   metabolism   MeSH
• Protein Subunits genetics   metabolism   MeSH
• Protein Biosynthesis MeSH
• Ribosomes metabolism   MeSH
• RNA, Transfer, Met metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• Guanosine Triphosphate MeSH
• RNA, Messenger MeSH
• Mutant Proteins MeSH
• Protein Subunits MeSH
• RNA, Transfer, Met MeSH

Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.

• MeSH
• RNA, Transfer, Amino Acyl metabolism   MeSH
• Peptide Chain Elongation, Translational * MeSH
• Eukaryotic Initiation Factor-3 metabolism   MeSH
• HeLa Cells MeSH
• Peptide Chain Initiation, Translational MeSH
• Yeasts genetics   MeSH
• Humans MeSH
• Paromomycin pharmacology   MeSH
• Gene Expression Regulation MeSH
• Ribosomes drug effects   metabolism   MeSH
• Codon, Terminator * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Transfer, Amino Acyl MeSH
• Eukaryotic Initiation Factor-3 MeSH
• Paromomycin MeSH
• Codon, Terminator * MeSH

The main role of the translation initiation factor 3 (eIF3) is to orchestrate formation of 43S-48S preinitiation complexes (PICs). Until now, most of our knowledge on eIF3 functional contribution to regulation of gene expression comes from yeast studies. Hence, here we developed several novel in vivo assays to monitor the integrity of the 13-subunit human eIF3 complex, defects in assembly of 43S PICs, efficiency of mRNA recruitment, and postassembly events such as AUG recognition. We knocked down expression of the PCI domain-containing eIF3c and eIF3a subunits and of eIF3j in human HeLa and HEK293 cells and analyzed the functional consequences. Whereas eIF3j downregulation had barely any effect and eIF3a knockdown disintegrated the entire eIF3 complex, eIF3c knockdown produced a separate assembly of the a, b, g, and i subunits (closely resembling the yeast evolutionary conserved eIF3 core), which preserved relatively high 40S binding affinity and an ability to promote mRNA recruitment to 40S subunits and displayed defects in AUG recognition. Both eIF3c and eIF3a knockdowns also severely reduced protein but not mRNA levels of many other eIF3 subunits and indeed shut off translation. We propose that eIF3a and eIF3c control abundance and assembly of the entire eIF3 and thus represent its crucial scaffolding elements critically required for formation of PICs.

• MeSH
• Cell Line MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Peptide Chain Initiation, Translational genetics   MeSH
• Humans MeSH
• RNA, Small Interfering MeSH
• Cell Proliferation MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Ribosomal Proteins genetics   metabolism   MeSH
• RNA Interference MeSH
• RNA, Ribosomal genetics   MeSH
• Protein Binding genetics   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• EIF3A protein, human MeSH Browser
• Eukaryotic Initiation Factor-3 MeSH
• RNA, Small Interfering MeSH
• RNA-Binding Proteins MeSH
• Ribosomal Proteins MeSH
• RNA, Ribosomal MeSH

Transfer of genetic information from genes into proteins is mediated by messenger RNA (mRNA) that must be first recruited to ribosomal pre-initiation complexes (PICs) by a mechanism that is still poorly understood. Recent studies showed that besides eIF4F and poly(A)-binding protein, eIF3 also plays a critical role in this process, yet the molecular mechanism of its action is unknown. We showed previously that the PCI domain of the eIF3c/NIP1 subunit of yeast eIF3 is involved in RNA binding. To assess the role of the second PCI domain of eIF3 present in eIF3a/TIF32, we performed its mutational analysis and identified a 10-Ala-substitution (Box37) that severely reduces amounts of model mRNA in the 43-48S PICs in vivo as the major, if not the only, detectable defect. Crystal structure analysis of the a/TIF32-PCI domain at 2.65-Å resolution showed that it is required for integrity of the eIF3 core and, similarly to the c/NIP1-PCI, is capable of RNA binding. The putative RNA-binding surface defined by positively charged areas contains two Box37 residues, R363 and K364. Their substitutions with alanines severely impair the mRNA recruitment step in vivo suggesting that a/TIF32-PCI represents one of the key domains ensuring stable and efficient mRNA delivery to the PICs.

• MeSH
• Alanine genetics   MeSH
• Eukaryotic Initiation Factor-3 chemistry   genetics   metabolism   MeSH
• Phenotype MeSH
• Peptide Chain Initiation, Translational * MeSH
• Ribosome Subunits, Small, Eukaryotic metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Substitution MeSH
• Protein Structure, Tertiary MeSH
• Basic-Leucine Zipper Transcription Factors genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alanine MeSH
• Eukaryotic Initiation Factor-3 MeSH
• GCN4 protein, S cerevisiae MeSH Browser
• RNA, Messenger MeSH
• NIP1 protein, S cerevisiae MeSH Browser
• RPG1 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Basic-Leucine Zipper Transcription Factors MeSH

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.

• MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• Peptide Chain Initiation, Translational physiology   MeSH
• Codon, Initiator genetics   metabolism   MeSH
• Ribosome Subunits, Small, Eukaryotic genetics   metabolism   MeSH
• Multiprotein Complexes genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• Codon, Initiator MeSH
• Multiprotein Complexes MeSH
• NIP1 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.

• MeSH
• Eukaryotic Initiation Factor-3 metabolism   MeSH
• Gene Knockout Techniques MeSH
• Peptide Chain Initiation, Translational * MeSH
• Protein Interaction Domains and Motifs MeSH
• Ribosome Subunits, Small, Eukaryotic metabolism   MeSH
• Protein Subunits metabolism   MeSH
• Ribosomal Proteins genetics   metabolism   physiology   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   physiology   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Two-Hybrid System Techniques MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• Protein Subunits MeSH
• Ribosomal Proteins MeSH
• RPG1 protein, S cerevisiae MeSH Browser
• RPS0A protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH