Human TRMT1 catalyzes mG or mG formation on tRNAs in a substrate-dependent manner.

TRMT1 is an N-methylguanosine (mG) and N,N-methylguanosine (mG) methyltransferase that targets G26 of both cytoplasmic and mitochondrial tRNAs. In higher eukaryotes, most cytoplasmic tRNAs with G26 carry mG26, although the majority of mitochondrial G26-containing tRNAs carry mG26 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:mG26 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:mG26 or mG26 modification in a substrate-dependent manner, which explains the distinct distribution of mG26 and mG26 on cytoplasmic and mitochondrial tRNAs. For human TRMT1-mediated tRNA:mG26 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 "mG26 criteria". We found that the mG26 modification occurred in almost all the higher eukaryotic tRNAs conforming to these criteria, suggesting the "mG26 criteria" are applicable to other higher eukaryotic tRNAs.