Three distinct 3-methylcytidine (mC) methyltransferases modify tRNA and mRNA in mice and humans.

Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (mC) formation in mammalian RNAs is still poorly understood. However, the recent discovery of as the second gene responsible for mC presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in mC formation in mammals as well. Here, we report the discovery and characterization of three distinct mC-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute mC in specific tRNAs and that METTL8 only contributes mC to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in and null-mutant cells, of mC in total tRNA, and primer extension analysis located METTL2-modified mC at position 32 of tRNA isoacceptors and tRNA We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. , however, modified only mRNA, as determined by biochemical and genetic analyses in null-mutant mice and two human mutant cell lines. Our findings provide the first evidence of the existence of mC modification in mRNA, and the discovery of METTL8 as an mRNA mC writer enzyme opens the door to future studies of other mC epitranscriptomic reader and eraser functions.