TRMT10B is an S-adenosyl-L-methionine-dependent methyltransferase that catalyzes N1-methylation of adenine at position 9 (m1A9) in specific nuclear-encoded tRNAs, making it the first eukaryotic m1A9-specific tRNA methyltransferase 1. Unlike its paralog TRMT10A, which catalyzes m1G9 methylation across multiple tRNA substrates, TRMT10B exhibits highly restricted substrate specificity, targeting primarily tRNAAsp 2. The two enzymes are biochemically non-redundant despite cytoplasmic co-localization, with distinct kinetic properties and tRNA-binding characteristics determining their specificity 2. TRMT10B localization includes both nuclear and mitochondrial compartments, where it localizes to the nucleoplasm and performs tRNA binding 1. Beyond canonical tRNA modification, TRMT10B regulates expression of mitochondrial tRNA-derived fragments (mt-tRFs), suggesting a role in cross-compartment communication between nuclear and mitochondrial gene expression 3. This regulation varies across populations of different ancestries, indicating population-specific modulation of mitochondrial small RNA expression 3. The functional specialization of TRMT10B highlights the complexity of tRNA modification systems in humans and suggests potential disease relevance when this narrow substrate specificity is disrupted.