Human TRMT1 and TRMT1L paralogs ensure the proper modification state, stability, and function of tRNAs

The tRNA methyltransferase 1 (TRMT1) enzyme catalyzes the N2,N2-dimethylguanosine (m2,2G) modification in tRNAs. Intriguingly, vertebrates encode an additional tRNA methyltransferase 1-like (TRMT1L) paralog. Here, we use a comprehensive tRNA sequencing approach to decipher targets of human TRMT1 and TRMT1L. We find that TRMT1 methylates all known tRNAs containing guanosine at position 26, while TRMT1L represents the elusive enzyme catalyzing m2,2G at position 27 in tyrosine tRNAs. Surprisingly, TRMT1L is also necessary for maintaining 3-(3-amino-3-carboxypropyl)uridine (acp3U) modifications in a subset of tRNAs through a process that can be uncoupled from methyltransferase activity. We also demonstrate that tyrosine and serine tRNAs are dependent upon m2,2G modifications for their stability and function in translation. Notably, human patient cells with disease-associated TRMT1 variants exhibit reduced levels of tyrosine and serine tRNAs. These findings uncover unexpected roles for TRMT1 paralogs, decipher functions for m2,2G modifications, and pinpoint tRNAs dysregulated in human disorders caused by tRNA modification deficiency. [Display omitted] •Human TRMT1 catalyzes m2,2G modification in more than half of all tRNAs•Human TRMT1L catalyzes m2,2G in tyrosine tRNAs while modulating acp3U in tRNAs•Tyrosine and serine tRNAs require m2,2G modifications for stability and function•Pathogenic variants in TRMT1 or TRMT1L cause perturbations in tRNA modification Zhang et al. elucidate the targets of a tRNA modification enzyme family and identify unanticipated roles for a duplicated tRNA modification enzyme that can be uncoupled from its ancestral function. These findings uncover the molecular mechanisms by which tRNAs are dysregulated in human disorders caused by tRNA modification deficiency.