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Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA.

Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T - Nucleic Acids Res. (2012)

Bottom Line: We confirmed 21 of the 28 previously known m(5)C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions.We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs.Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW, 2010, Australia.

ABSTRACT
The modified base 5-methylcytosine (m(5)C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m(5)C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m(5)C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10,275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.

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Validation of novel m5C candidate sites. Conventional bisulfite sequencing data is shown for three novel sites, (A) residue 48 in tRNA(LysCUU), (B) residue 174 in the RNase P RNA component H1 (RPPH1), and (C) residue 748 in cyclin-dependent kinase 2 interacting protein mRNA (CINP). Top panels display results for endogenous transcripts. Data for spiked-in in vitro transcribed negative controls harboring the same sequence flanked by unique priming sites are also shown (middle panels) as are corresponding next-generation sequencing results (lower panels). Numbering of cytosine positions is as described in Figure 1, positions highlighted in red designate m5C sites identified by next- generation sequencing. See Supplementary Figure 3 for additional validation data.
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gks144-F3: Validation of novel m5C candidate sites. Conventional bisulfite sequencing data is shown for three novel sites, (A) residue 48 in tRNA(LysCUU), (B) residue 174 in the RNase P RNA component H1 (RPPH1), and (C) residue 748 in cyclin-dependent kinase 2 interacting protein mRNA (CINP). Top panels display results for endogenous transcripts. Data for spiked-in in vitro transcribed negative controls harboring the same sequence flanked by unique priming sites are also shown (middle panels) as are corresponding next-generation sequencing results (lower panels). Numbering of cytosine positions is as described in Figure 1, positions highlighted in red designate m5C sites identified by next- generation sequencing. See Supplementary Figure 3 for additional validation data.

Mentions: To validate our global site mapping data, we verified three candidate sites from mRNAs, eight from tRNAs, and two from other non-coding RNAs by conventional bisulfite sequencing (Figure 3 and Supplementary Figures S2 and S3). Among the selected sites there were six that had been previously identified as m5C in other animal homologs (tRNAGly(UCC), 3 sites; tRNALys(CUU), 1 site; mitochondrial tRNAGlu(UUC), 1 site; 12S mitochondrial rRNA, 1 site). The three sites in mitochondrial tRNASer(GCU) were entirely novel, as those were chosen in protein-coding mRNAs (cyclin-dependent kinase 2 interacting protein, CINP; nicotinate phosphoribosyltransferase domain containing 1, NAPRT1; NADH dehydrogenase 1 beta subcomplex 7 mRNA, NDUFB7; 1 site each) and a non-coding RNA (RPPH1, Ribonuclease P RNA component H1). To assess potential non-conversion due to local RNA structure, we designed and prepared in vitro transcribed negative control transcripts mimicking the sequence context of five of the sites to be verified. These negative controls were spiked into independently prepared HeLa cell RNA prior to bisulfite treatment. None of these negative controls showed any evidence of non-conversion at the test sites, whereas all sites except position 47 in tRNAGly(UCC were convincingly confirmed (Figure 3 and Supplementary Figure S3). This demonstrated that most of the novel m5C candidate sites predicted by our next-generation sequencing-based mapping are independently verifiable.Figure 3.


Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA.

Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T - Nucleic Acids Res. (2012)

Validation of novel m5C candidate sites. Conventional bisulfite sequencing data is shown for three novel sites, (A) residue 48 in tRNA(LysCUU), (B) residue 174 in the RNase P RNA component H1 (RPPH1), and (C) residue 748 in cyclin-dependent kinase 2 interacting protein mRNA (CINP). Top panels display results for endogenous transcripts. Data for spiked-in in vitro transcribed negative controls harboring the same sequence flanked by unique priming sites are also shown (middle panels) as are corresponding next-generation sequencing results (lower panels). Numbering of cytosine positions is as described in Figure 1, positions highlighted in red designate m5C sites identified by next- generation sequencing. See Supplementary Figure 3 for additional validation data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367185&req=5

gks144-F3: Validation of novel m5C candidate sites. Conventional bisulfite sequencing data is shown for three novel sites, (A) residue 48 in tRNA(LysCUU), (B) residue 174 in the RNase P RNA component H1 (RPPH1), and (C) residue 748 in cyclin-dependent kinase 2 interacting protein mRNA (CINP). Top panels display results for endogenous transcripts. Data for spiked-in in vitro transcribed negative controls harboring the same sequence flanked by unique priming sites are also shown (middle panels) as are corresponding next-generation sequencing results (lower panels). Numbering of cytosine positions is as described in Figure 1, positions highlighted in red designate m5C sites identified by next- generation sequencing. See Supplementary Figure 3 for additional validation data.
Mentions: To validate our global site mapping data, we verified three candidate sites from mRNAs, eight from tRNAs, and two from other non-coding RNAs by conventional bisulfite sequencing (Figure 3 and Supplementary Figures S2 and S3). Among the selected sites there were six that had been previously identified as m5C in other animal homologs (tRNAGly(UCC), 3 sites; tRNALys(CUU), 1 site; mitochondrial tRNAGlu(UUC), 1 site; 12S mitochondrial rRNA, 1 site). The three sites in mitochondrial tRNASer(GCU) were entirely novel, as those were chosen in protein-coding mRNAs (cyclin-dependent kinase 2 interacting protein, CINP; nicotinate phosphoribosyltransferase domain containing 1, NAPRT1; NADH dehydrogenase 1 beta subcomplex 7 mRNA, NDUFB7; 1 site each) and a non-coding RNA (RPPH1, Ribonuclease P RNA component H1). To assess potential non-conversion due to local RNA structure, we designed and prepared in vitro transcribed negative control transcripts mimicking the sequence context of five of the sites to be verified. These negative controls were spiked into independently prepared HeLa cell RNA prior to bisulfite treatment. None of these negative controls showed any evidence of non-conversion at the test sites, whereas all sites except position 47 in tRNAGly(UCC were convincingly confirmed (Figure 3 and Supplementary Figure S3). This demonstrated that most of the novel m5C candidate sites predicted by our next-generation sequencing-based mapping are independently verifiable.Figure 3.

Bottom Line: We confirmed 21 of the 28 previously known m(5)C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions.We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs.Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW, 2010, Australia.

ABSTRACT
The modified base 5-methylcytosine (m(5)C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m(5)C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m(5)C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10,275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.

Show MeSH