In a recent post I discussed the extent of adenosine methylation in RNAs. Meyer et al. found that m6A was found in many mRNAs and showed a bias in its distribution towards the end of coding sequence, stop codons, and the proximal section of 3’UTRs. The main chemically modified base of DNA is 5-methylcytosine. Squires et al. have surveyed the presence of m5C in human RNAs, and find that this modification is also common in tRNAs, rRNAs, mRNAs and ncRNAs.
The principal method for detecting methylated cytosines in nucleic acids is bisulphite sequencing. Bisulphite converts cytosine residues to uracil, but modified cytosines are left unchanged. Hence, when sequenced, C reads as T, and m5C reads as C. When compared to a reference sequence the status of cytosine methylation can be deduced. Squires et al. used bisulphite conversion of RNAs, followed by reverse transcription and high throughput sequencing. A number of other modified forms of cytosine known to be present in some rRNAs, such as N4-methylcytidine (m4C) and N4,2’-O-dimethylcytidine (m4Cm), may also be resistant to bisulphite treatment. With this in mind, Squires et al. termed their detected modified cytosines m5C candidate sites.
Surveying RNAs from HeLa cells, Squires et al discovered 255 modified Cs in tRNAs. This confirmed a number of known sites and identified many new candidate sites, which however generally fitted into a known pattern of modification of residues in specific secondary structural regions – the variable region and the anticodon loop. Modifications in these areas are important in stabilising secondary structure and affect aminoacylation and codon recognition.
Most interestingly, the researchers discovered 10, 275 m5C candidate sites in mRNAs and ncRNAs. Their data covered 10.6% of the total cytosine residues in the transcriptome. m5C seems to be enriched in some classes of ncRNA, but relatively depleted in mRNAs. The majority (83%) however, of their candidate sites were found in mRNAs. Within these transcripts m5C appears to be depleted within protein coding sequences but enriched in 5’ and 3’ UTRs. Further computational analysis showed an association between mRNA m5C sites and binding regions for Argonaute proteins (the proteins that small regulatory RNA molecules complex with to effect post-transcriptional regulation).
Two different methyltransferases are known to catalyse the m5C modification in eukaryotic RNAs, NSUN2 and TRDMT1. Previously these two enzymes had only been shown to methylate a few specific positions in various tRNAs. Squires et al. used RNAi to knockdown NSUN2 and TRDMT1 in HeLa cells and assayed the methylation status of a selected subset of cytosine residues. This showed that a number of m5C sites in mRNAs and ncRNAs are dependent on NSUN2, suggesting that this could be the primary enzyme responsible for cytosine methylation in these classes of RNAs. NSUN2 has been shown to be cell-cycle regulated and a target for the oncogene MYC. Mouse knockouts are small, and have revealed a role in balancing stem cell renewal and differentiation. A recent paper (Khan et al. 2012) has linked mutations in NSUN2 to autosomal-recessive intellectual disability syndrome in humans. It will be interesting to investigate the extent of this enzyme’s role in RNA methylation, and dissect what component of it’s function is responsible for the mouse and human phenotypes.
As with the investigation into m6A, m5C is commonly found in RNAs of many categories, and as with the previous study it is not yet obvious just how important RNA methylation truly is. The phenotypes associated with loss of methyltransferases or demethylases are not that extensive, but neither are they negligible. Some observations are shared between Meyer et al and Squires et al; the enrichments in 3’ UTRs and the correlation between RNA methylation and microRNA/argonaute binding sites (although there were differences in the details of these associations. This investigation by Squires et al into m5C is not on the same level as Meyer et al’s study, in that it lacked the developmental component and wasn’t on the same global scale. On the other hand bisulphite sequencing does pinpoint the exact modified residues, whereas m6A cannot as yet be detected to the same level of accuracy. The methodology used by Squires et al. can be scaled up, and so more global studies of m5C will no doubt appear in the near future. I also look forward to more detailed understanding of the enzymatic pathways involved, and a dissection of their roles in development.
Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, & Preiss T (2012). Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic acids research, 40 (11), 5023-33 PMID: 22344696
Khan MA, Rafiq MA, Noor A, Hussain S, Flores JV, Rupp V, Vincent AK, Malli R, Ali G, Khan FS, Ishak GE, Doherty D, Weksberg R, Ayub M, Windpassinger C, Ibrahim S, Frye M, Ansar M, & Vincent JB (2012). Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability. American journal of human genetics, 90 (5), 856-63 PMID: 22541562
Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, & Jaffrey SR (2012). Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons. Cell, 149 (7), 1635-46 PMID: 22608085