Human TRMT1 catalyzes m²G or m²₂G formation on tRNAs in a substrate-dependent manner

TRMT1 is an N²-methylguanosine (m²G) and N²,N²-methylguanosine (m²₂G) methyltransferase that targets G26 of both cytoplasmic and mitochondrial tRNAs. In higher eukaryotes, most cytoplasmic tRNAs with G26 carry m²₂G26, although the majority of mitochondrial G26-containing tRNAs carry m²G26 or G26, suggesting differences in the mechanisms by which TRMT1 catalyzes modification of these tRNAs. Loss-of-function mutations of human TRMT1 result in neurological disorders and completely abrogate tRNA:m²₂G26 formation. However, the mechanism underlying the independent catalytic activity of human TRMT1 and identity of its specific substrate remain elusive, hindering a comprehensive understanding of the pathogenesis of neurological disorders caused by TRMT1 mutations. Here, we showed that human TRMT1 independently catalyzes formation of the tRNA:m²G26 or m²₂G26 modification in a substrate-dependent manner, which explains the distinct distribution of m²G26 and m²₂G26 on cytoplasmic and mitochondrial tRNAs. For human TRMT1-mediated tRNA:m²₂G26 formation, the semi-conserved C11:G24 serves as the determinant, and the U10:A25 or G10:C25 base pair is also required, while the size of the variable loop has no effect. We defined the requirements of this recognition mechanism as the "m²₂G26 criteria". We found that the m²₂G26 modification occurred in almost all the higher eukaryotic tRNAs conforming to these criteria, suggesting the "m²₂G26 criteria" are applicable to other higher eukaryotic tRNAs.