5-methylcytidine, m5C, is abundantly found in mRNA and ncRNAs, especially tRNA and rRNAs. In tRNAs, this modification stabilizes the secondary structure and influences anticodon stem-loop conformation. In rRNAs, m5C affects translational fidelity.
Two principles have been used to develop m5C sequencing methods. The first one is antibody-based approach (bisuphite sequencing and m5C-RIP), similar to m6C sequencing. The second is detecting targets of m5C RNA methyltransferases by covalently linking the enzyme to its target, and then using IP specific to the target enzyme to enrich for RNA molecules containing the mark (Aza-IP and miCLIP).