Role of eEF2 diphthamide modification in translational fidelity
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA735209 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14735560.fastq.gz | Fastqsanger.gz | |||
| SRR14735561.fastq.gz | Fastqsanger.gz | |||
| SRR14735562.fastq.gz | Fastqsanger.gz | |||
| SRR14735563.fastq.gz | Fastqsanger.gz | |||
| SRR14735564.fastq.gz | Fastqsanger.gz |
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Project description:Diphthamide (DPH) is a conserved amino acid modification on eukaryotic translation elongation factor eEF2. We show that loss of DPH increases -1 ribosomal frameshifting at both programmed, including in SARS-CoV-2, and non-programmed sites. Ribosome profiling of yeast and mammalian cells lacking DPH reveals increased ribosomal drop-off during elongation. The drop-off defect on the long yeast MDN1 gene was suppressed by eliminating out-of-frame stop codons. Our data reveal that loss of DPH impairs the fidelity of translocation during translation elongation resulting in increased rates of ribosomal frameshifting and premature termination at out-of-frame stop codons.
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Project description:Translational quality control is critical for maintaining the accuracy of protein synthesis in all domains of life. Mutations in aminoacyl-tRNA synthetases and the ribosome are known to affect translational fidelity and alter fitness, viability, stress responses, neuron function, and life span. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified 30 nonessential genes that are critical for maintaining the fidelity of stop-codon readthrough. Most of these identified genes have not been shown to affect translational fidelity previously. Intriguingly, we show that several genes controlling metabolism, including cyaA and guaA, unexpectedly enhance stop-codon readthrough. CyaA and GuaA catalyze the synthesis of cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (GMP), respectively. Both CyaA and GuaA increase the expression of ribosomes and tRNAs, allowing aminoacyl-tRNAs to compete with release factors and suppress stop codons. In addition, the effect of guaA deletion on stop-codon readthrough is abolished by deleting prfC, which encodes release factor 3 (RF3). Our results suggest that nucleotide and carbon metabolism is tightly coupled with translational fidelity.
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Project description:A coding sequence-embedded principle governs translational reading frame fidelity
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Project description:Methyl-5-uridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme of m5U conversion at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by TRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with crosslinking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that TRMT2A interacts with proteins involved in both tRNA and rRNA biogenesis. Consistent with this finding, we observed that TRMT2A not only methylates tRNA, but also rRNA. Finally, we addressed the question of the importance of TRMT2A function by showing that its knockdown reduces translational fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards rRNA biogenesis and translational fidelity.
2023-07-04 | PXD036538 | Pride
Project description:Purpose: we aimed to gain a genome-wide view of the dynamics in DNA methylation inheritance and define the factors associated with methylation fidelity. Methods: Using mouse embryonic stem cell (ES-E14TG2a) in both undifferentiated and differentiated states as a model system, we exploited the hairpin bisulfite sequencing approach to generate methylation data for DNA double strands simultaneously at single-base resolution. We generated the genome-wide hairpin bisulfite sequencing data to capture the methylation pattern variation during the stem cell transition from self-renewal to commitment, and integrated with various M-bM-^@M-^\omicsM-bM-^@M-^] data to scrutinize the relationships among DNA methylation inheritance, gene expression, histone modification, transcriptional factor binding and distribution of 5-hydroxylmethylation cytosine. Results and conclusion: Our results indicated that DNA methylation fidelity increases globally during early mouse embryonic stem cell differentiation. Methylation fidelity is remarkably high in promoter regions of actively expressed genes and positively correlated with active histone modification marks and binding of transcriptional factors. Strikingly, methylation fidelity follows a bimodal distribution for the intermediately methylated CpG dyads. In addition, the methylation difference in between two DNA strands rather than different DNA molecules is a major source of the intermediate DNA methylation. Lastly, while 5-hmC and 5-mC tend to coexist, no significant increase in the pairing with unmethylated cytosine was observed. For mouse embryonic stem cell of undifferentiated and spontaneous differentiated states, we determined DNA double strands methylation pattern by hairpin bisulfite sequencing approach and determined gene expression profiles using RNA-seq.
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Project description:Genome-wide screening reveals metabolic regulation of translational fidelity by cyclic AMP
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Project description:Tgt is responsible for the queuosine (Q) modification of the guanine (G) in tRNAs with GUN anti-codon. We have previously identified tgt as being vital for growth in presence sub-lethal concentrations of aminoglycosides in Vibrio cholerae. Here we further explore the role of Q modification in translation fidelity and efficiency of codon decoding upon tobramycin exposure. We characterize the impact on codon decoding efficiency, the overall translation of the bacterial proteome, and the molecular mechanisms underlying the effects of Q modification in antibiotic stress response. Using molecular reporters, we show that Q modification impacts the efficiency of decoding at tyrosine TAT and TAC codons. Proteomics identify the anti-SoxR factor RsxA as better translated in the absence of tgt. RsxA displays a codon bias towards tyrosine TAT and its overabundance leads to decreased expression of genes belonging to SoxR oxidative stress regulon. We also identify conditions which regulate tgt expression. We propose that regulation of Q modification in response to environmental cues leads to translational reprogramming of genes bearing a biased tyrosine codon usage. In silico analysis further identifies candidate genes possibly subjected to such translational regulation, among which DNA repair factors. Such transcripts, which fit the definition of modification tunable transcripts, are plausibly central in the bacterial response to antibiotic stress.
2024-12-11 | PXD035297 | Pride
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:Genomic studies have recently identified RPS15 as a new driver gene in aggressive and chemorefractory cases of chronic lymphocytic leukemia (CLL). RPS15 encodes a ribosomal protein whose conserved C-terminal domain extends into the decoding center of the ribosome. We demonstrate that mutations in highly conserved residues of this domain affect both protein stability —by increasing its ubiquitin-mediated degradation— and cell proliferation rates. On the other hand, we show that mutated RPS15 can be loaded into the ribosomes, directly impacting on global protein synthesis and/or translational fidelity in a mutation-specific manner. Quantitative mass-spectrometry analyses suggest that RPS15 variants may induce additional alterations in the translational machinery as well as a metabolic shift at the proteome level in both HEK293T and MEC-1 cells. These results indicate that CLL-related RPS15 mutations might act following patterns known for other ribosomal diseases, likely switching from a hypo- to a hyperproliferative phenotype driven by mutated ribosomes. In this scenario, loss of translational fidelity causing altered cell proteostasis can be proposed as a new molecular mechanism involved in CLL pathobiology.
2019-09-09 | PXD010924 | Pride
Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.
2022-09-24 | E-MTAB-12178 | biostudies-arrayexpress