Translational fidelity screens reveal eIF3 as a key regulator of start codon selectivity [RNA-seq]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE207330 | GEO | 2023/05/05
REPOSITORIES: GEO
Similar Datasets
Project description:The translation initiation machinery and the ribosome orchestrate a highly dynamic scanning process to distinguish proper start codons from surrounding nucleotide sequences. Here, we performed genome-wide CRISPRi screens in human K562 cells to systematically identify modulators of the frequency of translation initiation at near-cognate start codons. We observed that depletion of any eIF3 core subunit promoted near-cognate start codon usage, though sensitivity thresholds of each subunit to sgRNA-mediated depletion varied considerably. Double sgRNA depletion experiments suggested that enhanced near-cognate usage in eIF3D depleted cells required canonical eIF4E cap-binding and was not driven by eIF2A or eIF2D-dependent leucine tRNA initiation. We further characterized the effects of eIF3D depletion and found that the N-terminus of eIF3D was strictly required for accurate start codon selection, whereas disruption of the cap-binding properties of eIF3D had no effect. Lastly, depletion of eIF3D activated TNFα signaling via NF-κB and the interferon gamma response. Similar transcriptional profiles were observed upon knockdown of eIF1A and eIF4G2, which also promoted near-cognate start codon usage, suggesting that enhanced near-cognate usage could potentially contribute to NF-κB activation. Our study thus provides new avenues to study the mechanisms and consequences of alternative start codon usage.
| S-EPMC10325891 | biostudies-literature
Project description:Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1A, eIF1, eIF2–GTP–Met-tRNAiMet, eIF3, eIF4A and eIF4B. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.
2022-06-09 | PXD031985 | Pride
Project description:Translational fidelity screens reveal eIF3 as a key regulator of start codon selectivity [RNA-seq]
| PRJNA854886 | ENA
Project description:The eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection.
| S-EPMC8599844 | biostudies-literature
Project description: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.
| S-EPMC6868437 | biostudies-literature
Project description:Sequences within 5' UTRs dictate the site and efficiency of translation initiation. In this study, an unbiased screen designed to interrogate the 5' UTR-mediated regulation of the growth-promoting gene MYC unexpectedly revealed the ribosomal pause relief factor eIF5A as a regulator of translation initiation codon selection. Depletion of eIF5A enhances upstream translation within 5' UTRs across yeast and human transcriptomes, including on the MYC transcript, where this results in increased production of an N-terminally extended protein. Furthermore, ribosome profiling experiments established that the function of eIF5A as a suppressor of ribosomal pausing at sites of suboptimal peptide bond formation is conserved in human cells. We present evidence that proximal ribosomal pausing on a transcript triggers enhanced use of upstream suboptimal or non-canonical initiation codons. Thus, we propose that eIF5A functions not only to maintain efficient translation elongation in eukaryotic cells but also to maintain the fidelity of translation initiation.
| S-EPMC6917043 | biostudies-literature
Project description:Translation initiation factor eIF1A stimulates preinitiation complex (PIC) assembly and scanning, but the molecular mechanisms of its functions are not understood. We show that the F131A,F133A mutation in the C-terminal tail (CTT) of eIF1A impairs recruitment of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex to 40S subunits, eliminating functional coupling with eIF1. Mutating residues 17-21 in the N-terminal tail (NTT) of eIF1A also reduces PIC assembly, but in a manner rescued by eIF1. Interestingly, the 131,133 CTT mutation enhances initiation at UUG codons (Sui(-) phenotype) and decreases leaky scanning at AUG, while the NTT mutation 17-21 suppresses the Sui(-) phenotypes of eIF5 and eIF2beta mutations and increases leaky scanning. These findings and the opposite effects of the mutations on eIF1A binding to reconstituted PICs suggest that the NTT mutations promote an open, scanning-conducive conformation of the PIC, whereas the CTT mutations 131,133 have the reverse effect. We conclude that tight binding of eIF1A to the PIC is an important determinant of AUG selection and is modulated in opposite directions by residues in the NTT and CTT of eIF1A.
| S-EPMC1829380 | biostudies-literature
Project description: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.
| S-EPMC4446449 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2019-12-03 | GSE132010 | GEO
Project description:eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNA(i)(Met)) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE1) and SE2, was found to stimulate recruitment of Met-tRNA(i)(Met) in the ternary complex (TC) with eIF2.GTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI1 and SI2, reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNA(i)(Met) in the P site for AUG selection.
| S-EPMC2802195 | biostudies-literature