TRMT1L-catalyzed m(2)(2)G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress.

tRNA modifications are critical for several aspects of their functions, including decoding, folding, and stability. Using a multifaceted approach encompassing eCLIP-seq and nanopore tRNA-seq, we show that the human tRNA methyltransferase TRMT1L interacts with the component of the Rix1 ribosome biogenesis complex and binds to the 28S rRNA as well as to a subset of tRNAs. Mechanistically, we demonstrate that TRMT1L is responsible for catalyzing N2,N2-dimethylguanosine (m(2)(2)G) solely at position 27 of tRNA-Tyr-GUA. Surprisingly, TRMT1L depletion also impaired the deposition of 3-(3-amino-3-carboxypropyl) uridine (acp(3)U) and dihydrouridine on tRNA-Tyr-GUA, Cys-GCA, and Ala-CGC. TRMT1L knockout cells have a marked decrease in tRNA-Tyr-GUA levels, coinciding with a reduction in global translation rates and hypersensitivity to oxidative stress. Our results establish TRMT1L as the elusive methyltransferase catalyzing the m(2)(2)G27 modification on tRNA Tyr, resolving a long-standing gap of knowledge and highlighting its potential role in a tRNA modification circuit crucial for translation regulation and stress response.