Cancer associated mutants of eIF1A impair Rps3/Rps10 binding and enhance scanning of cell cycle genes [Ribo-Seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE116981 | GEO | 2018/07/13
REPOSITORIES: GEO
Similar Datasets
Project description:The Ribo-seq analysis demonstrated that eIF1A is predominantly essential for translation of genes with long 5'UTR genes including cell proliferation and cell cycle progression genes. eIF1A depletion causes broad stimulation of initiation in 5’UTRs at near-cognate AUG codons that diminshes the translation initiation fidelity
2018-07-13 | GSE116982 | GEO
Project description:The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context, and AUG recognition stabilizes a closed PIC conformation. The unstructured N-terminal tail (NTT) of yeast eIF1A deploys five basic residues to contact tRNAi, mRNA, or 18S rRNA exclusively in the closed state. Interestingly, EIF1AX mutations altering the human eIF1A NTT are associated with uveal melanoma (UM). We found that substituting all five basic residues, and seven UM-associated substitutions, in yeast eIF1A suppresses initiation at near-cognate UUG codons and AUGs in poor context. Ribosome profiling of NTT substitution R13P reveals heightened discrimination against unfavorable AUG context genome-wide. Both R13P and K16D substitutions destabilize the closed complex at UUG codons in reconstituted PICs. Thus, electrostatic interactions involving the eIF1A NTT stabilize the closed conformation and promote utilization of suboptimal start codons. We predict UM-associated mutations alter human gene expression by increasing discrimination against poor initiation sites.
2017-12-21 | GSE108334 | GEO
Project description:The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The textbook knowledge of eukaryotic translation initiation depicts 5’→3’ unidirectional migration of the pre-initiation complex (PIC) along the 5’UTR. In probing translation initiation from ultra-short 5’UTR, we report that an AUG triplet near the 5’ end can be selected via PIC backsliding. The bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5’ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5’ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.
2021-11-24 | GSE176058 | GEO
Project description:Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation is regulated differently from canonical translation is poorly understood. We thus used start codon-selective reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of non-AUG reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA are resistant to cycloheximide (CHX), a translation inhibitor which slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes cause queuing of scanning pre-initiation complexes (PIC), preferentially enhancing otherwise poor recognition of non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. Moreover, PIC queuing can cause translation from an AUG codon in a poor context to become less sensitive to CHX. We further find that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence, but not those targeting newly initiated ribosomes. In total, these data indicate that ribosome queuing enables mRNAs with poor initiation context, namely those from non-AUG start codons, to be resistant to pharmacological inhibitors.
2019-06-05 | GSE125086 | GEO
Project description:So far, the annotation of translation initiation sites (TISs) has been based mostly upon bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic cell line. A neural network was trained on the ribosomal footprints at previously annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs in 5062 transcripts with sufficient sequence coverage. Functional interpretation suggested 2994 novel upstream open reading frames (uORFs) in the 5´ UTR (924 AUG, 2070 near-cognate codons), 1406 uORFs overlapping with the coding sequence (116 AUG, 1290 near-cognate) and 546 N-terminal protein extensions (6 AUG, 540 near-cognate). The TIS detection method was validated on the basis of previously published alternative TISs and uORFs. On average, TICs in newly annotated TISs were significantly more conserved among primates than control codons, both for AUGs (p<10-10) and near-cognate codons (p=3.8×10-3). The derived transcriptome-wide map of novel candidate TISs will help to explain how human proteome diversity is influenced by alternative translation initiation and regulation. Examination of translational initiation in human cell lines using ribosomal footprinting
2012-08-08 | E-GEOD-39561 | biostudies-arrayexpress
Project description:The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at the NCCs initiating N-terminal extensions on GRS1 and ALA1 mRNAs, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.
2019-10-18 | GSE138599 | GEO
Project description:So far, the annotation of translation initiation sites (TISs) has been based mostly upon bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic cell line. A neural network was trained on the ribosomal footprints at previously annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs in 5062 transcripts with sufficient sequence coverage. Functional interpretation suggested 2994 novel upstream open reading frames (uORFs) in the 5´ UTR (924 AUG, 2070 near-cognate codons), 1406 uORFs overlapping with the coding sequence (116 AUG, 1290 near-cognate) and 546 N-terminal protein extensions (6 AUG, 540 near-cognate). The TIS detection method was validated on the basis of previously published alternative TISs and uORFs. On average, TICs in newly annotated TISs were significantly more conserved among primates than control codons, both for AUGs (p<10-10) and near-cognate codons (p=3.8×10-3). The derived transcriptome-wide map of novel candidate TISs will help to explain how human proteome diversity is influenced by alternative translation initiation and regulation.
2012-08-08 | GSE39561 | GEO
Project description:Genomic analyses in budding yeast have helped to define the foundational principles of eukaryotic gene expression, but have systematically excluded specific classes of potential coding regions, including those with non-AUG start codons. Without methods to define coding regions empirically, the prevalence of these non-canonical coding regions has been impossible to assess. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified a class of 149 genes that encode N-terminally extended alternate protein isoforms that result from translation initiation at non-AUG codons upstream of the annotated AUG start codon. These alternate isoforms are produced in concert with canonical isoforms and are translated with a high degree of specificity, resulting from initiation at only a small subset of possible start codons in 5’ leader regions. Their translation is enriched during meiosis, and is induced by low eIF5A levels, which are observed in this context. These findings reveal widespread production of non-canonical protein isoforms and, more generally, show unexpected complexity to the rules by which the budding yeast genome is decoded.
2020-07-24 | GSE150375 | GEO
Project description:Cancer associated mutants of eIF1A impair Rps3/Rps10 binding and enhance scanning of cell cycle genes [Ribo-Seq]
| PRJNA480786 | ENA
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