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EIF1A residues implicated in cancer stabilize translation preinitiation complexes and favor suboptimal initiation sites in yeast

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE108334 | GEO | 2017/12/21

SECONDARY ACCESSION(S): PRJNA423281

REPOSITORIES: GEO

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eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide

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

Cancer associated mutants of eIF1A impair Rps3/Rps10 binding and enhance scanning of cell cycle genes [Mars-seq]

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

Cancer associated mutants of eIF1A impair Rps3/Rps10 binding and enhance scanning of cell cycle genes [Ribo-Seq]

2018-07-13 | GSE116981 | GEO

Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors

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

Bi-directional ribosome scanning controls the stringency of start codon selection

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

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

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

Altered proteome in translationinitiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage

Project description:The recognition of AUG start codon and selection of the open reading frame (ORF) is fundamental to protein biosynthesis. Defect in the start codon selection adversely affectproteome and have a pleiotropic effect on cellular function. Using proteomic techniques, we identified differential protein expression patterns in the translation initiation defectivehyper GTPaseeIF5G31R mutant cells. Consistently, we observed altered proteome involved in protein catabolism, nucleotide metabolism, fatty acid metabolism, glycolysis, and autophagy.The utilization of the upstream UUG codons by the eIF5G31R mutation caused downregulation of uridylate kinase expression and DNA damage. The eIF5G31R mutant cells showed lower Glutathione levels, high ROS activity, and sensitivity to H2O2.

2022-05-19 | PXD029460 | Pride

Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting

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

EIF1AX-A113 splice and RAS mutations cooperate to drive thyroid tumorigenesis through ATF4 and c-MYC

Project description:Translation initiation in higher eukaryotes is orchestrated by the tight regulation of the cap binding and the 43S pre-initiation complexes (PIC). The PIC component eukaryotic initiation factor 1A (EIF1A), encoded on human chromosomes X and Y by EIF1AX and EIF1AY, respectively, is essential for recruitment of the ternary complex and for assembling the 43S PIC, which after recruitment onto capped mRNAs scans their 5’UTR and localizes the AUG to initiate translation. Recurrent EIF1AX mutations are found in several cancers, including ~1% of papillary thyroid cancers in a mutually exclusive manner with other drivers (BRAF, RAS, and oncogenic fusions). They are enriched in advanced thyroid cancers (11%) where they display a striking co-occurrence with RAS, which we show cooperate to induce tumorigenesis in mouse models and in isogenic cell lines. The C-terminal tail EIF1AX-A113splice mutation is the most prevalent in advanced thyroid cancer, and private to this disease. We found that EIF1AX-A113spl variants stabilize the PIC and induces ATF4 expression, a sensor of cellular stress, which is co-opted to suppress EIF-2α phosphorylation, thus enabling a general increase in protein synthetic rate. RAS stabilizes c-MYC, an effect augmented by EIF1AX-A113spl. ATF4 and c-MYC induce expression of aminoacid transporters, and enhance sensitivity of mTOR to aminoacid supply. These mutually reinforcing events generate therapeutic vulnerabilities to MEK, BRD4 and mTOR kinase inhibitors. The RNA-Seq and Ribosome Profiling samples provided here were used to support the claim of increased translational efficiency of ATF4.

2019-01-28 | GSE113695 | GEO

A coding sequence-embedded principle governs translational reading frame fidelity

Project description:Upon initiation at an AUG start codon, the ribosome must maintain the correct reading frame for hundreds of codons in order to produce functional proteins. Although some sequence elements are able to trigger programmed ribosomal frameshifting (PRF), very little is known how the ribosome normally prevents spontaneous frameshift errors. Using high resolution ribosome profiling data sets, we discovered that the translating ribosome uses the 3’ end of 18S rRNA to scan the AUG-like codons after the decoding process. The internal mRNA:rRNA interaction not only contributes to predominant translational pausing, but also provides a post-decoding mechanism to safeguard the ribosome in the correct reading frame. Partially eliminating the AUG-like “sticky” codons in the reporter message leads to increased +1 frameshift errors. Remarkably, mutating the highly conserved CAU triplet of 18S rRNA globally changes codon “stickiness”. Further supporting the role of “sticky” sequences in reading frame maintenance, the codon composition of open reading frames is highly optimized across eukaryotic genomes by minimizing the appearance of AUG-like codons in the frame 2. These results suggest an important layer of information embedded within the protein coding sequences that instructs the ribosome to ensure reading frame fidelity during translation.

2016-04-12 | GSE80156 | GEO

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