Sequence- and structure-selective mRNA m(5)C methylation by NSUN6 in animals

mRNA m(5)C, which has recently been implicated in the regulation of mRNA mobility, metabolism and translation, plays important regulatory roles in various biological events. Two types of m(5)C sites are found in mRNAs. Type I m(5)C sites, which contain a downstream G-rich triplet motif and are computationally predicted to be located at the 5(′) end of putative hairpin structures, are methylated by NSUN2. Type II m(5)C sites contain a downstream UCCA motif and are computationally predicted to be located in the loops of putative hairpin structures. However, their biogenesis remains unknown. Here we identified NSUN6, a methyltransferase that is known to methylate C72 of tRNA(Thr) and tRNA(Cys), as an mRNA methyltransferase that targets Type II m(5)C sites. Combining the RNA secondary structure prediction, miCLIP, and results from a high-throughput mutagenesis analysis, we determined the RNA sequence and structural features governing the specificity of NSUN6-mediated mRNA methylation. Integrating these features into an NSUN6-RNA structural model, we identified an NSUN6 variant that largely loses tRNA methylation but retains mRNA methylation ability. Finally, we revealed a weak negative correlation between m(5)C methylation and translation efficiency. Our findings uncover that mRNA m(5)C is tightly controlled by an elaborate two-enzyme system, and the protein-RNA structure analysis strategy established may be applied to other RNA modification writers to distinguish the functions of different RNA substrates of a writer protein.