Sequence- and structure-selective mRNA m5C methylation by NSUN6 in animals

mRNA m⁵C, 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 m⁵C sites are found in mRNAs. Type I m⁵C 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 m⁵C 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 m⁵C 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 m⁵C methylation and translation efficiency. Our findings uncover that mRNA m⁵C 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.