Formation and determination of the oxidation products of 5-methylcytosine in RNA

Huang, Wei; Lan, Meng-Dan; Qi, Changxing; Zheng, Shu-Jian; Wei, Shaozhong; Yuan, Bi-Feng

doi:10.1039/c6sc01589a

Cited by 113 publications

(105 citation statements)

References 32 publications

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“…Two 'readers' of m 5 C have been reported, the mRNA export adapter ALYREF (Aly/REF export factor) [49] and the DNA/RNA-binding protein YBX1 (Y-box binding protein 1) [47,51], implying certain molecular functions (see below). Finally, a potential route to 'erase' m 5 C from RNA is indicated by the presence of 5-hydroxymethylcytosine (hm 5 C), 5-formylcytosine and 5-carboxylcytosine in RNA, which represent intermediates in an oxidative demethylation pathway initiated by ten-eleven translocation (TET) dioxygenases [52][53][54][55].…”

Section: Introductionmentioning

confidence: 99%

Multiple links between 5-methylcytosine content of mRNA and translation

et al. 2020

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Background: 5-Methylcytosine (m 5 C) 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 m 5 C 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.Results: We obtained~1000 candidate m 5 C 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 m 5 C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in both mRNAs and noncoding RNAs. Candidate m 5 C sites in mRNAs are enriched in 5′UTRs and near start codons and are embedded in a local context reminiscent of the NSUN2-dependent m 5 C 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. Conclusions: Our findings emphasise the major role of NSUN2 in placing the m 5 C 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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Section: Introductionmentioning

confidence: 99%

Multiple links between 5-methylcytosine content of mRNA and translation

et al. 2020

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“…Two 'readers' of m 5 C have been reported, the mRNA export adapter ALYREF (Aly/REF export factor) (Yang et al 2017) and the DNA/RNAbinding protein YBX1 (Y-box binding protein 1) Yang et al 2019b), implying certain molecular functions (see below). Finally, a potential route to 'erase' m 5 C from RNA is indicated by the presence of 5-hydroxymethylcytosine (hm 5 C), 5-formylcytosine and 5-carboxylcytosine in RNA, which represent intermediates in an oxidative demethylation pathway initiated by Ten-eleven translocation (TET) dioxygenases (Delatte et al 2016;Huang et al 2016;Miao et al 2016;Zhang et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Multiple links between 5-methylcytosine content of mRNA and translation

Schümann

Zhang²,

Sibbritt

et al. 2020

Preprint

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Abstract5-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 ∼1,000 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 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 modification 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.

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“…There are differences, however, in the occurrence of m 5 C in specific RNA types in different species. For example, m 5 C appears not to be present in tRNA and mRNA from bacteria, while it has been found in eukaryal and archeal tRNA and mRNA (Amort et al, ; Cui et al, ; David et al, ; Dubin & Taylor, ; Edelheit et al, ; Fu et al, ; Huang et al, ; Huber et al, ; Motorin, Lyko, & Helm, ; Salditt‐Georgieff et al, ; Squires et al, ; Yang et al, ).…”

Section: ‐Methylcytosine In Abundant Rna Speciesmentioning

confidence: 99%

The dynamic RNA modification 5‐methylcytosine and its emerging role as an epitranscriptomic mark

2018

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It is a well‐known fact that RNA is the target of a plethora of modifications which currently amount to over a hundred. The vast majority of these modifications was observed in the two most abundant classes of RNA, rRNA and tRNA. With the recent advance in mapping technologies, modifications have been discovered also in mRNA and in less abundant non‐coding RNA species. These developments have sparked renewed interest in elucidating the nature and functions of those “epitransciptomic” modifications in RNA. N6‐methyladenosine (m6A) is the best understood and most frequent mark of mRNA with demonstrated functions ranging from pre‐mRNA processing, translation, miRNA biogenesis to mRNA decay. By contrast, much less research has been conducted on 5‐methylcytosine (m5C), which was detected in tRNAs and rRNAs and more recently in poly(A)RNAs. In this review, we discuss recent developments in the discovery of m5C RNA methylomes, the functions of m5C as well as the proteins installing, translating and manipulating this modification. Although our knowledge about m5C in RNA transcripts is just beginning to consolidate, it has become clear that cytosine methylation represents a powerful mechanistic strategy to regulate cellular processes on an epitranscriptomic level. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Processing > tRNA Processing RNA Turnover and Surveillance > Regulation of RNA Stability

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Formation and determination of the oxidation products of 5-methylcytosine in RNA

Abstract: Chemical labeling coupled with LC-MS enables the sensitive and simultaneous detection of the oxidative products of 5-methylcytosine. With this method, we can determine 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in RNA of mammals.

Cited by 113 publications

References 32 publications

Multiple links between 5-methylcytosine content of mRNA and translation

Multiple links between 5-methylcytosine content of mRNA and translation

Multiple links between 5-methylcytosine content of mRNA and translation

The dynamic RNA modification 5‐methylcytosine and its emerging role as an epitranscriptomic mark

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