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Multiple links between 5-methylcytosine content of mRNA and translation

ABSTRACT: Multiple links between 5-methylcytosine content of mRNA and translation

PROVIDER: PRJNA591627 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10530028_1.fastq.gz	Fastqsanger.gz
	SRR10530028_2.fastq.gz	Fastqsanger.gz
	SRR10530029_1.fastq.gz	Fastqsanger.gz
	SRR10530029_2.fastq.gz	Fastqsanger.gz
	SRR10530030_1.fastq.gz	Fastqsanger.gz

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Multiple links between 5-methylcytosine content of mRNA and translation

Project description:5-methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise. We obtained ~1,000 candidate m5C sites in the wider transcriptome, most of which were found in exonic regions of mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCap and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m5C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in mRNA and noncoding RNA. Candidate m5C sites in mRNAs are enriched in 5’UTRs and near start codons, and commonly embedded in a local context reminiscent of the NSUN2-dependent m5C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that non-conversion levels, at bulk and for many individual sites, were inversely correlated with ribosome association. Altogether, these findings emphasise the major role of NSUN2 in making this mark transcriptome-wide and further substantiate a functional interdependence of cytosine methylation level with mRNA translation

2020-03-09 | GSE140995 | GEO

Multiple links between 5-methylcytosine content of mRNA and translation.

Project description:Background5-Methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA, where it serves incompletely understood molecular functions. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise.ResultsWe obtained ~ 1000 candidate m5C sites in the wider transcriptome, most of which were found in mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCaP and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m5C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in both mRNAs and noncoding RNAs. Candidate m5C sites in mRNAs are enriched in 5'UTRs and near start codons and are embedded in a local context reminiscent of the NSUN2-dependent m5C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that modification levels, at bulk and for many individual sites, were inversely correlated with ribosome association.ConclusionsOur findings emphasise the major role of NSUN2 in placing the m5C mark transcriptome-wide. We further present evidence that substantiates a functional interdependence of cytosine methylation level with mRNA translation. Additionally, we identify several compelling candidate sites for future mechanistic analysis.

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Epitranscriptomic maps of 5-methylcytosine reveal substrate diversity of NSUN methyltransferases and links to mRNA translation and turnover

Project description:Epitranscriptomic maps of 5-methylcytosine reveal substrate diversity of NSUN methyltransferases and links to mRNA translation and turnover

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Epitranscriptomic maps of 5-methylcytosine reveal substrate diversity of NSUN methyltransferases and links to mRNA translation and turnover

Project description:Multiple epitranscriptomic maps of the RNA modification 5-methylcytosine (m5C) have been prepared, often diverging markedly from each other in terms of site abundance and identity. Differences in detection methods, data depth and analysis pipelines, but also biological factors underly much of this disparity. To address this, we re-analysed available datasets from five human cell lines and seven tissues, generated by the prevailing bisulfite RNA sequencing method, with a coherent m5C site calling pipeline. We used the resulting union list of 6,393 m5C sites called across the broader transcriptome to study site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed prior observations such as predominance of m5C sites within mRNA exons, enrichment around start codons, and that the tRNA:m5C methyltransferases (MTases) NSUN2 and 6 are the main m5C ‘writers’ for mRNAs. Each NSUN enzyme recognises mRNA features that strongly resemble their respective canonical substrates. Assessing proximity between mRNA m5C sites and footprints of RNA-binding proteins reconfirmed the mRNA export factor ALYREF as an m5C ‘reader’ and identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. Experiments in HeLa cells lacking NSUN2 showed that this affected both, steady-state level and UPF1-binding to target mRNAs. Our studies emphasise the emerging diversity of both, m5C writers and readers, affecting mRNA function.

2024-07-04 | GSE252369 | GEO

Substrate diversity of NSUN enzymes and links of 5-methylcytosine to mRNA translation and turnover.

Project description:Maps of the RNA modification 5-methylcytosine (m5C) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m5C site calling pipeline. With the resulting union list of 6,393 m5C sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed tRNA:m5C methyltransferases NSUN2 and NSUN6 as the main mRNA m5C "writers," but further showed that the rRNA:m5C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m5C sites and footprints of RNA-binding proteins, we identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. We found that lack of NSUN2 in HeLa cells affected both steady-state levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m5C writers and readers and their effect on mRNA function.

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