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Maps of the RNA modi fication 5-methylcytosine (m 5 C) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisul fite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m 5 C site calling pipeline. With the resulting union list of 6,393 m 5 C sites, we studied site distribution, enzymology, interaction with RNA binding proteins and molecular function. We con firmed tRNA: m 5 C methyltransferases NSUN2 and NSUN6 as the main mRNA m 5 C writers,??but further showed that the rRNA:m 5 C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m 5 C sites and footprints of RNA-binding proteins, we identi fied 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 steadystate levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m 5 C writers and readers and their effect on mRNA function.