Role of eEF2 diphthamide modification in translational fidelity
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae Cricetulus griseus
PROVIDER: GSE176169 | GEO | 2023/01/10
REPOSITORIES: GEO
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Project description:Codon usage bias, which refers to uneven use of synonymous codons, was shown to associate with mRNA stability from yeast to human. However, the underlying molecular basis is largely unknown. With bioinformatic analyses we unexpectedly found that codon usage bias is inversely correlated to density of out-of-frame stop codons (hidden stop codon or HSC). To understand the physiological function of HSCs, we use the frequency gene of Neurospora crassa to examine the role of HSCs in circadian rhythm. We show that deleting HSCs in the upstream of frequency (frq) open reading frame without altering FRQ sequence resulted in loss of circadian rhythm. Further analyses revealed that HSC deletion of frq resulted in elevated stability of frq mRNA, which consequently causes higher FRQ protein level. Combined various methods, we showed that rare codons in the upstream frq region generates significant amount of aberrant translation at +1 frame, possibly via ribosomal frameshifting. Consistently, at genome wide we showed that both in N. crassa and S. cerevisiae, the codon usage bias combined with numbers of HSC are inversely correlated to mRNA stability. Together, these data indicate that HSCs might act as an intrinsic mechanism to terminate aberrant ribosomal frameshifting events triggered by non-optimal codons, thus fine-tune mRNA stability.
2023-12-31 | GSE245041 | GEO
Project description:Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.
2023-05-01 | GSE220677 | GEO
Project description:Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats,which may expand the coding capacity of mRNA and diversify the regulation in human.
2023-05-01 | GSE220676 | GEO
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
Project description:Programmed ribosomal frameshifting is a process that augments the coding capacity of particular mRNA transcripts. Programmed ribosomal frameshifting may occur during the decoding of chromosomally encoded genes in both prokaryotes and eukaryotes. Although programmed ribosomal frameshifting has been intensively studied, less is known about the occurrence of translation errors that result in frameshifts. Detection of unexpectedly efficient frameshift suppression in E. coli (at levels ranging from 1% to 10% with two unrelated test proteins) led us to investigate the scope of events responsible for restoration of the native frame. To enable a comprehensive approach, we developed a targeted mass-spectrometry method–“translational tiling proteomics”–allowing interrogation of the full set of possible –1 slippage events that could lead to the observed instances of frameshift suppression in vitro and in cells. Our findings point to an unexpectedly high basal level of ribosomal frameshifting, suggest that frameshifted products may contribute more significantly to the proteome than generally assumed, and raise the possibility that particular pseudogenes may specify functional proteins.
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Project description:Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 allele of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.
2017-03-09 | GSE88725 | GEO
Project description:N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E. coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.
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Project description:Many tumors escape by activating multiple cellular pathways that induce immunosuppression. One pivotal immune-suppressive mechanism is the production of tryptophan metabolites along the kynurenine pathway by IFNγ-induced IDO1 enzyme production 4-8. Phase III clinical trials using chemical inhibition of IDO1 in combination with PD1 pathway blockade, however, failed to improve melanoma treatment 9-12. This points at an incomplete understanding of the role of IDO1 and the consequent tryptophan degradation on mRNA translation and cancer progression. Here, we investigated the effects of prolonged IFNγ treatment on mRNA translation in melanoma cells by ribosome profiling. Surprisingly, we observed a massive accumulation of ribosomes ~20 amino acids downstream of tryptophan codons (termed here as W-Bumps) along with their expected stalling at the tryptophan codon itself. This indicated ribosomal bypass of the tryptophan codons in the absence of tryptophan. Detailed examination of W-Bumps position and its corresponding peptide sequences pinpointed towards ribosomal frameshifting events and their effects in the ribosome exit tunnel. In particular, W-Bumps strength was associated with the disorderedness level of potential out-of-frame peptides predicted downstream of tryptophan codons. Indeed, reporter assays demonstrated the induction of ribosomal frameshifting, and the generation of trans-frame proteins and their presentation at the cell surface after IFNγ treatment. Proteomics and immunopeptidomics analyses verified the production of IFNγ-induced trans-frame and out-of-frame aberrant peptides and their presentation on HLA class I molecules. Priming of naïve T cells from healthy donors with aberrant peptides resulted in identification of reactive, peptide-specific T cells. Altogether, our results suggest that IFNγ-induced IDO1-mediated tryptophan depletion plays a role in the immune recognition of melanoma cells by contributing to the diversity of the peptidome landscape, and by inducing the presentation of aberrant peptides.
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