The tRNA m 1 R 9 methyltransferase (Trm10) family is conserved throughout Eukarya and Archaea. Despite the presence of a single Trm10 gene in Archaea and most single-celled eukaryotes, metazoans encode up to three homologs of Trm10. Several disease states correlate with a deficiency in the human homolog TRMT10A, despite the presence of another cytoplasmic enzyme, TRMT10B. Here we investigate these phenomena and demonstrate that human TRMT10A (hTRMT10A) and human TRMT10B (hTRMT10B) are not biochemically redundant. In vitro activity assays with purified hTRMT10A and hTRMT10B reveal a robust activity for hTRMT10B as a tRNA Asp-specific m 1 A 9 methyltransferase and suggest that it is the relevant enzyme responsible for this newly discovered m 1 A 9 modification in humans. Moreover, a comparison of the two cytosolic enzymes with multiple tRNA substrates exposes the enzymes' distinct substrate specificities, and suggests that hTRMT10B exhibits a restricted selectivity hitherto unseen in the Trm10 enzyme family. Single-turnover kinetics and tRNA binding assays highlight further differences between the two enzymes and eliminate overall tRNA affinity as a primary determinant of substrate specificity for either enzyme. These results increase our understanding of the important biology of human tRNA modification systems, which can aid in understanding the molecular basis for diseases in which their aberrant function is increasingly implicated.

Post‐transcriptional tRNA modifications play a critical role in ensuring a high‐quality pool of tRNA for participation in cellular translation. Despite their importance, important questions remain about the impacts of individual tRNA modifications on tRNA structure and function. Similarly, biological consequences of the absence of tRNA modifications have begun to be characterised in detail only recently. tRNA modifications have important impacts on biology, ranging from important impacts on individual tRNA molecules, to powerful effects on cellular function, and finally important roles in human health and disease. Key Concepts tRNA modifications occur at high frequency and with great chemical diversity. tRNA modifications fine‐tune tRNA structure and function. Through their effects on individual tRNA molecules, the impacts of tRNA modifications propagate to the cellular and organismal levels. tRNA modifications can regulate translation and impact protein homeostasis. Lack of tRNA modifications has been implicated in human diseases, such as neurological disorders, glucose metabolic defects and cancer.