Reaction of HeLa cell methyl-labelled 28S ribosomal RNA with sodium bisulphite: a conformational probe for methylated sequences.

Goddard, John P.; Maden, B.E.H.

doi:10.1093/nar/3.2.431

Cited by 15 publications

(10 citation statements)

References 16 publications

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“…By contrast, mapping to rRNAs suffered from incomplete cytosine conversion in multiple clusters (top panels in Figure S3C), particularly in 28S rRNA. This clustered non-conversion likely reflects the known sensitivity of the bisulfite reaction to secondary structure (Goddard and Schulman 1972;Goodchild et al 1975;Goddard and Maden 1976) and indicated additional filtering as necessary for highconfidence site calls.…”

Section: Transcriptome-wide Identification Of Candidate M 5 C Sitesmentioning

confidence: 93%

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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Section: Transcriptome-wide Identification Of Candidate M 5 C Sitesmentioning

confidence: 93%

Multiple links between 5-methylcytosine content of mRNA and translation

Schümann

Zhang²,

Sibbritt

et al. 2020

Preprint

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“…0.6 A260 units of PSTV in 500 "l 3 M sodium bisulfite pH 5.9 were incubated for (a) 26 h / 50 C in presence of 20 mM MgCl2, (b) 26 h / 250 C without MgCl2, and (c) 96 h / 250 C without MgCl2. The modified PSTV was obtained after a series of dialyses (20). This material was used for RNase A and RNase T1 digestion, 5'-32P-labelling in vitro, fingerprinting and sequence analysis as described above for unmodified viroid.…”

Section: Methodsmentioning

confidence: 99%

“…Reversible addition of bisulfite to the 5,6-double bond of cytidine at slightly acidic pH causes deamination of this nucleoside to uridine (24, 25,26). This modification is known to depend on the secondary structure of the RNA: cytidine is fully reactive in single-stranded regions, whereas it is resistant against modification in base-paired structures (20,26). Fig.…”

Section: Bisulfite-catalyzed Modification Of Reactive Cytidines Tomentioning

confidence: 99%

Studies on the primary and secondary structure of potato spindle tuber viroid: products of digestion with ribonuclease A and ribonuclease T₁, and modification with bisulfite

et al. 1978

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Potato spindle tuber viroid (PSTV), a small infectios RNA, has been completely digested with RNase T1 and RNase A, and the resulting oligonucleotides have been sequenced using 5'-terminal 32p-labelling with gamma-32p ATP and T4 polynucleotide kinase, fingerprinting and controlled nuclease P1 digestion. Modified nucleotides have not been detected in 5'-positions of these oligonucleotides. PSTV consists of about 359 nucleotides and contains a remarkable stretch of 18 purines, mainly adenosines; there is no AUG initiation triplet present. The established oligonucleotide sequences preclude a perfect intramolecular base complementarity within the covalently closed viroid circle. Therefore, the rigid, rod-like native secondary structure of PSTV, as seen in the electron microscope, must be based on a defective rather than on a homogeneous RNA helix. The detailed analysis of the bisulfite-catalized modification of cytidine to uridine in PSTV revealed a higher reactivity for the majority of the cytidines than would be expected for a perfect helix. Since only cytidines in single-stranded regions are knonw to be fully reactive, this finding provides additional evidence for defects in the helical secondary structure of PSTV.

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“…S3C), particularly in 28S rRNA. This clustered non-conversion likely reflects the known sensitivity of the bisulfite reaction to secondary structure [72][73][74] and indicated additional filtering as necessary for high-confidence site calls.…”

Section: Transcriptome-wide Identification Of Candidate M 5 C Sitesmentioning

confidence: 99%

Multiple links between 5-methylcytosine content of mRNA and translation

et al. 2020

View full text Add to dashboard Cite

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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Reaction of HeLa cell methyl-labelled 28S ribosomal RNA with sodium bisulphite: a conformational probe for methylated sequences.

Cited by 15 publications

References 16 publications

Multiple links between 5-methylcytosine content of mRNA and translation

Multiple links between 5-methylcytosine content of mRNA and translation

Studies on the primary and secondary structure of potato spindle tuber viroid: products of digestion with ribonuclease A and ribonuclease T₁, and modification with bisulfite

Multiple links between 5-methylcytosine content of mRNA and translation

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Reaction of HeLa cell methyl-labelled 28S ribosomal RNA with sodium bisulphite: a conformational probe for methylated sequences.

Cited by 15 publications

References 16 publications

Multiple links between 5-methylcytosine content of mRNA and translation

Multiple links between 5-methylcytosine content of mRNA and translation

Studies on the primary and secondary structure of potato spindle tuber viroid: products of digestion with ribonuclease A and ribonuclease T1, and modification with bisulfite

Multiple links between 5-methylcytosine content of mRNA and translation

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Studies on the primary and secondary structure of potato spindle tuber viroid: products of digestion with ribonuclease A and ribonuclease T₁, and modification with bisulfite