Codon and amino acid content regulate mRNA stability in mammalian cells
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ABSTRACT:
PROVIDER: PRJNA575354 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10215648.fastq.gz | Fastqsanger.gz | |||
| SRR10215649.fastq.gz | Fastqsanger.gz | |||
| SRR10215650.fastq.gz | Fastqsanger.gz | |||
| SRR10215651.fastq.gz | Fastqsanger.gz | |||
| SRR10215652.fastq.gz | Fastqsanger.gz |
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Project description:mRNA degradation is a critical aspect of gene expression which dictates mRNA steady-state levels in conjunction with transcription rate. Previous studies in other model organisms have demonstrated that enrichment of specific codons within the open reading frame (ORF) can influence mRNA stability, primarily by modulating translation elongation speed. Despite advancements in our understanding of mRNA stability regulation by microRNAs and 3'UTR-based factors in mammalian systems, the importance of other mRNA regions such as the ORF sequence on dictating cellular mRNA levels are incompletely understood. To characterize the effects of the coding sequence on mRNA decay in mammals, we analyzed mRNA stability in human and Chinese Hamster Ovary (Cricetulus griseus) cells by both global metabolic labeling and single-gene mRNA reporter transcription shutoffs. In agreement with previous studies, we observed that synonymous codon usage impacts mRNA stability in mammalian cells. Unexpectedly, we also found that amino acid content is a potent determinant of mRNA stability in humans and other mammalian species. Codon and amino acid effects on decay correlate with tRNA levels measured by tRNA-Seq or intracellular amino acid levels measured by HPLC, respectively. These results suggest that both tRNA and amino acid levels have complementary effects on regulation of mRNA stability in mammals, hinting at the possibility of dynamic control of mRNA levels via altered tRNA or amino acid levels.
2020-03-01 | GSE138292 | GEO
Project description:Codon and amino acid identity are major determinants of mRNA stability in humans
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Project description:The ability to genetically encode noncanonical amino acids (ncAAs) has empowered proteins with improved or novel properties. However, existing strategies in mammalian cells rely on the introduction of blank codon for incorporating ncAAs, which is very inefficient and limits their widespread applications. Here, we develop a rare codon recoding strategy that takes advantage of the relative rarity of the TCG codon to achieve highly selective and efficient ncAA incorporation through systematic engineering and big data model predictions. We highlight the broad utility of this strategy for the incorporation of dozens of ncAAs into various functional proteins at the wild-type protein expression levels, as well as the synthesis of proteins with up to 6-site ncAAs or 4 distinct ncAAs in mammalian cells for downstream applications.
2025-01-01 | GSE255465 | GEO
Project description:The ability to genetically encode noncanonical amino acids (ncAAs) has empowered proteins with improved or novel properties. However, existing strategies in mammalian cells rely on the introduction of blank codon for incorporating ncAAs, which is very inefficient and limits their widespread applications. Here, we develop a rare codon recoding strategy that takes advantage of the relative rarity of the TCG codon to achieve highly selective and efficient ncAA incorporation through systematic engineering and big data model predictions. We highlight the broad utility of this strategy for the incorporation of dozens of ncAAs into various functional proteins at the wild-type protein expression levels, as well as the synthesis of proteins with up to 6-site ncAAs or 4 distinct ncAAs in mammalian cells for downstream applications.
2025-01-01 | GSE255464 | GEO
Project description:Messenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in the cell and is a major control point for modulating gene expression. In yeast and other model organisms, codon identity is a powerful determinant of transcript stability, contributing broadly to impact half-lives. General principles governing mRNA stability are poorly understood in mammalian systems. Importantly, however, the degradation machinery is highly conserved, thus it seems logical that mammalian transcript half-lives would also be strongly influenced by coding determinants. Herein we characterize the contribution of coding sequence towards mRNA decay in human and Chinese Hamster Ovary cells. In agreement with previous studies, we observed that synonymous codon usage impacts mRNA stability in mammalian cells. Surprisingly, however, we also observe that the amino acid content of a gene is an additional determinant correlating with transcript stability. The impact of codon and amino acid identity on mRNA decay appears to be associated with underlying tRNA and intracellular amino acid concentrations. Accordingly, genes of similar physiological function appear to coordinate their mRNA stabilities in part through codon and amino acid content. Together, these results raise the possibility that intracellular tRNA and amino acid levels interplay to mediate coupling between translational elongation and mRNA degradation rate in mammals.
| S-EPMC7018022 | biostudies-literature
Project description:The control of mRNA stability plays a central role in regulating gene expression. In metazoans, the earliest stages of development are driven by maternally supplied mRNAs. The degradation of these maternal mRNAs is critical for promoting the maternal-to-zygotic transition of developmental programs, although the underlying mechanisms are poorly understood in vertebrates. Here, we characterized maternal mRNA degradation pathways in zebrafish using a transcriptome analysis and systematic reporter assays. Our data demonstrate that ORFs enriched with uncommon codons promote deadenylation by the CCR4-NOT complex in a translation-dependent manner. This codon-mediated mRNA decay is conditional on the context of the 3â² UTR, with long 3â² UTRs conferring resistance to deadenylation. These results indicate that the combined effect of codon usage and 3â² UTR length determines the stability of maternal mRNAs in zebrafish embryos. Our study thus highlights the codon-mediated mRNA decay as a conserved regulatory mechanism in eukaryotes. zebrafish embryonic mRNA profile at 2 different stages (2 hpf and 6 hpf) in wildtype and 3 additional conditions (miR-430 inhibition, RNApol II inhibition and CNOT7 inhibition) at 6 hpf. All experiments are performed as triplicates
2016-03-17 | E-GEOD-71609 | biostudies-arrayexpress
Project description:Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that limitation for the amino acid arginine causes a selective loss of tRNA charging, which regulates translation through ribosome pausing at two of six arginine codons. Interestingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation led to loss of arginine tRNA charging and ribosome pausing. Codon-specific ribosome pausing decreased protein production and triggered premature ribosome termination without significantly reducing mRNA levels. Together, our results suggest that amino acids which are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on synonymous codon usage.
2018-07-19 | GSE113751 | GEO
Project description:Background Cells regulate protein synthesis in response to fluctuating nutrient availability through mechanisms that affect both translation initiation and elongation. Branched-chain amino acids, leucine, isoleucine, and valine, are essential nutrients. However, how their depletion affects translation remains largely unclear. Here, we investigate the immediate effects of single, double, and triple branched-chain amino acid deprivation on translational dynamics in NIH3T3 cells using RNA-seq and ribosome profiling. Results All starvation conditions increased ribosome dwell times, with pronounced stalling at all valine codons during valine and triple starvation, whereas leucine and isoleucine starvation produced milder, codon-specific effects. Notably, stalling under isoleucine deprivation largely decreased under triple starvation. Positional enrichment of valine codons near the 5′ end and downstream isoleucine codons potentially contributes to these patterns, suggesting a possible elongation bottleneck that influences translational responses under branched-chain amino acid starvation. The presence of multiple valine stalling sites was associated with decreased protein levels. Finally, codon-specific dwell time changes correlated strongly with patterns of tRNA isoacceptor charging. Conclusions Together, these findings suggest that differential ribosome stalling under branched-chain amino acid starvation reflects a balance between amino acid supply, tRNA charging dynamics, codon position, and stress-response signaling.
2025-09-02 | GSE291653 | GEO
Project description:Background: Cells regulate protein synthesis in response to fluctuating nutrient availability through mechanisms that affect both translation initiation and elongation. Branched-chain amino acids, leucine, isoleucine, and valine, are essential nutrients. However, how their depletion affects translation remains largely unclear. Here, we investigate the immediate effects of single, double, and triple branched-chain amino acid deprivation on translational dynamics in NIH3T3 cells using RNA-seq and ribosome profiling. Results: All starvation conditions increased ribosome dwell times, with pronounced stalling at all valine codons during valine and triple starvation, whereas leucine and isoleucine starvation produced milder, codon-specific effects. Notably, stalling under isoleucine deprivation largely decreased under triple starvation. Positional enrichment of valine codons near the 5' end and downstream isoleucine codons potentially contributes to these patterns, suggesting a possible elongation bottleneck that influences translational responses under branched-chain amino acid starvation. The presence of multiple valine stalling sites was associated with decreased protein levels. Finally, codon-specific dwell time changes correlated strongly with patterns of tRNA isoacceptor charging. Conclusions: Together, these findings suggest that differential ribosome stalling under branched-chain amino acid starvation reflects a balance between amino acid supply, tRNA charging dynamics, codon position, and stress-response signaling.
2025-09-02 | GSE291652 | GEO
Project description:Mass spectromery for DiZSeK residue specific incorporation on TCG codon.
2023-10-29 | PXD046483 |