Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans

Chemical RNA modifications are central features of epitran-scriptomics, highlighted by the discovery of modified ribo-nucleosides 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 (m(3)C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of trm141 as the second gene responsible for m(3)C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m(3)C formation in mammals as well. Here, we report the discovery and characterization of three distinct m(3)C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m(3)C in specific tRNAs and that METTL8 only contributes m(3)C to mRNA. MS analysis revealed that there is an similar to 30-40% and similar to 10-15% reduction, respectively, in METTL2 and -6 null-mutant cells, of m(3)C in total tRNA, and primer extension analysis located METTL2-modified m(3)C at position 32 of tRNA(Thr) isoacceptors and tRNA(Arg(CCU)). 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. METTL8, however, modified only mRNA, as determined by biochemical and genetic analyses in Mettl8 null-mutant mice and two human METTL8 mutant cell lines. Our findings provide the first evidence of the existence of m(3)C modification in mRNA, and the discovery of METTL8 as an mRNA m(3)C writer enzyme opens the door to future studies of other m(3)C epitranscriptomic reader and eraser functions.