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

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

BMC biology (2020-04-16)
Ulrike Schumann, He-Na Zhang, Tennille Sibbritt, Anyu Pan, Attila Horvath, Simon Gross, Susan J Clark, Li Yang, Thomas Preiss
摘要

5-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. We 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. Our 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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抗-α-微管蛋白抗体,小鼠单克隆, clone DM1A, purified from hybridoma cell culture
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山羊抗兔IgG抗体,过氧化物酶偶联, 1 mg/mL (after reconstitution), Chemicon®