Substrate diversity of NSUN enzymes and links of 5-methylcytosine to mRNA translation and turnover.

Maps of the RNA modification 5-methylcytosine (m5C) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m5C site calling pipeline. With the resulting union list of 6,393 m5C sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed tRNA:m5C methyltransferases NSUN2 and NSUN6 as the main mRNA m5C "writers," but further showed that the rRNA:m5C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m5C sites and footprints of RNA-binding proteins, we identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. We found that lack of NSUN2 in HeLa cells affected both steady-state levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m5C writers and readers and their effect on mRNA function.