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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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Single-nucleotide resolution mapping of m5C candidate sites in RNA. HeLa cell RNA preparations were spiked with a trace amount of in vitro transcribed R-Luc RNA and bisulfite-converted as detailed in the ‘Materials and Methods’ section. (A) Negative control R-Luc and (B) endogenous tRNA(AspGUC) as a positive control were first analyzed by conventional sequencing to establish the efficacy of bisulfite conversion (top panels; columns signify cytosine positions along the RNA sequence, rows represent individually sequenced alleles, open boxes indicate cytosine to uracil conversion read as thymidine in cDNA and filled boxes indicate a retained cytosine). Numbers below refer to cytosine positions in the primary RNA sequence. Nucleotide positions highlighted in red designate previously identified m5C sites in tRNA(AspGUC). Dual axis charts (bottom panels) display next-generation sequencing data mapped at 2 mm for the same control RNAs. Blue bars represent bisulfite-induced cytosine conversion, while red lines represent read coverage across individual residues. Top and bottom panels are aligned to each other by interrogated cytosine residues.
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gks144-F1: Single-nucleotide resolution mapping of m5C candidate sites in RNA. HeLa cell RNA preparations were spiked with a trace amount of in vitro transcribed R-Luc RNA and bisulfite-converted as detailed in the ‘Materials and Methods’ section. (A) Negative control R-Luc and (B) endogenous tRNA(AspGUC) as a positive control were first analyzed by conventional sequencing to establish the efficacy of bisulfite conversion (top panels; columns signify cytosine positions along the RNA sequence, rows represent individually sequenced alleles, open boxes indicate cytosine to uracil conversion read as thymidine in cDNA and filled boxes indicate a retained cytosine). Numbers below refer to cytosine positions in the primary RNA sequence. Nucleotide positions highlighted in red designate previously identified m5C sites in tRNA(AspGUC). Dual axis charts (bottom panels) display next-generation sequencing data mapped at 2 mm for the same control RNAs. Blue bars represent bisulfite-induced cytosine conversion, while red lines represent read coverage across individual residues. Top and bottom panels are aligned to each other by interrogated cytosine residues.

Mentions: To develop a method for single nucleotide mapping of m5C sites in a cellular transcriptome we adapted an RNA bisulfite conversion protocol, originally devised for primer extension-based detection of m5C (40), for use with a sequencing-based readout (see ‘Materials and Methods’ section for details). As in DNA bisulfite sequencing, sites of m5C in RNA will be read as cytosine in cDNA sequence, while unmodified cytosines will appear as thymidine. Although some other types of modified cytosine can also be resistant to bisulfite treatment (see ‘Discussion’ section), for simplicity we refer in the following to sites of non-conversion as ‘m5C candidate sites’. We chose to analyse RNA preparations from HeLa cells (a human cervical cancer cell line) and used both positive and negative control RNAs to monitor success of the procedure. Our positive control was the endogenous tRNAAsp(GUC), which harbours three previously identified m5C sites at structural positions 38, 47 and 48 (7,31,32). Our negative control was in vitro transcribed Renilla luciferase (R-Luc) mRNA lacking m5C, which was spiked into the cellular RNA sample prior to bisulfite treatment. An aliquot of this RNA was used for conventional bisulfite sequencing reactions to examine the expected m5C patterns in our controls. The R-Luc negative control RNA exhibited virtually complete cytosine conversion (Figure 1A; 99.8% conversion overall), while all three known m5C sites in tRNAAsp(GUC) selectively displayed low levels of conversion (Figure 1B; position 38 and 48: 0%, position 47: 12.5% conversion). These results showed that our RNA conversion protocol was efficient and accurate detection of m5C sites is achieved.Figure 1.


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)

Single-nucleotide resolution mapping of m5C candidate sites in RNA. HeLa cell RNA preparations were spiked with a trace amount of in vitro transcribed R-Luc RNA and bisulfite-converted as detailed in the ‘Materials and Methods’ section. (A) Negative control R-Luc and (B) endogenous tRNA(AspGUC) as a positive control were first analyzed by conventional sequencing to establish the efficacy of bisulfite conversion (top panels; columns signify cytosine positions along the RNA sequence, rows represent individually sequenced alleles, open boxes indicate cytosine to uracil conversion read as thymidine in cDNA and filled boxes indicate a retained cytosine). Numbers below refer to cytosine positions in the primary RNA sequence. Nucleotide positions highlighted in red designate previously identified m5C sites in tRNA(AspGUC). Dual axis charts (bottom panels) display next-generation sequencing data mapped at 2 mm for the same control RNAs. Blue bars represent bisulfite-induced cytosine conversion, while red lines represent read coverage across individual residues. Top and bottom panels are aligned to each other by interrogated cytosine residues.
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Related In: Results  -  Collection

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gks144-F1: Single-nucleotide resolution mapping of m5C candidate sites in RNA. HeLa cell RNA preparations were spiked with a trace amount of in vitro transcribed R-Luc RNA and bisulfite-converted as detailed in the ‘Materials and Methods’ section. (A) Negative control R-Luc and (B) endogenous tRNA(AspGUC) as a positive control were first analyzed by conventional sequencing to establish the efficacy of bisulfite conversion (top panels; columns signify cytosine positions along the RNA sequence, rows represent individually sequenced alleles, open boxes indicate cytosine to uracil conversion read as thymidine in cDNA and filled boxes indicate a retained cytosine). Numbers below refer to cytosine positions in the primary RNA sequence. Nucleotide positions highlighted in red designate previously identified m5C sites in tRNA(AspGUC). Dual axis charts (bottom panels) display next-generation sequencing data mapped at 2 mm for the same control RNAs. Blue bars represent bisulfite-induced cytosine conversion, while red lines represent read coverage across individual residues. Top and bottom panels are aligned to each other by interrogated cytosine residues.
Mentions: To develop a method for single nucleotide mapping of m5C sites in a cellular transcriptome we adapted an RNA bisulfite conversion protocol, originally devised for primer extension-based detection of m5C (40), for use with a sequencing-based readout (see ‘Materials and Methods’ section for details). As in DNA bisulfite sequencing, sites of m5C in RNA will be read as cytosine in cDNA sequence, while unmodified cytosines will appear as thymidine. Although some other types of modified cytosine can also be resistant to bisulfite treatment (see ‘Discussion’ section), for simplicity we refer in the following to sites of non-conversion as ‘m5C candidate sites’. We chose to analyse RNA preparations from HeLa cells (a human cervical cancer cell line) and used both positive and negative control RNAs to monitor success of the procedure. Our positive control was the endogenous tRNAAsp(GUC), which harbours three previously identified m5C sites at structural positions 38, 47 and 48 (7,31,32). Our negative control was in vitro transcribed Renilla luciferase (R-Luc) mRNA lacking m5C, which was spiked into the cellular RNA sample prior to bisulfite treatment. An aliquot of this RNA was used for conventional bisulfite sequencing reactions to examine the expected m5C patterns in our controls. The R-Luc negative control RNA exhibited virtually complete cytosine conversion (Figure 1A; 99.8% conversion overall), while all three known m5C sites in tRNAAsp(GUC) selectively displayed low levels of conversion (Figure 1B; position 38 and 48: 0%, position 47: 12.5% conversion). These results showed that our RNA conversion protocol was efficient and accurate detection of m5C sites is achieved.Figure 1.

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