5-methylcytosine (m⁵C) in eukaryotic RNA has been known to exist for decades, however, laborious detection methods have limited the understanding of its role. With the availability of high-throughput sequencing techniques, these drawbacks have been overcome, revealing non-random distribution of internal methylation in a wide variety of RNA biotypes [1]. Recently, we implemented a bisulfite sequencing-based technique for transcriptome-wide (bsRNA-seq) detection of m⁵C and mapped thousands of m⁵C sites in the human transcriptome including in mRNA and non-coding RNA [2]. Biased distribution of m⁵C within mRNAs e.g. enrichment in the untranslated regions, is consistent with a role in post-transcriptional gene regulation. We have investigated the enzymes responsible for modifying RNA by coupling RNAi-mediated knockdown with bsRNA-seq identifying hundreds of candidate m⁵C sites that are targeted by the two RNA methyltransferases, NSUN2 and TRDMT1. Interestingly, both RNA methyltransferases have a link to cancer; TRDMT1 is inhibited by the anti-cancer drug, 5-azacytidine, and NSUN2 is overexpressed in tumours. As such, we are investigating the role of RNA methylation in cancer by comparing the m⁵C profiles of normal prostate cells (PrEC) and metastatic prostate cancer cells (LNCaP). Analysis of the recorded patterns of m⁵C sites in RNA shows many transcripts are differentially methylated between each cell line. We are currently consolidating and extending the potential link of m⁵C to post-transcriptional gene regulation and cancer, as well as addressing its molecular function.

[1] Sibbritt T., et al., Mapping and significance of the mRNA methylome. WIREs RNA, 4(4): p. 397–422, 10.1002/wrna.1166

[2] Squires, J. E., et al., Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res, 2012. 40(11): p. 5023-5033, 10.1093/nar/gks144