TRMT1L-catalyzed m² ₂G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under...

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 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 m² ₂G solely at position 27 of tRNA-Tyr-GUA. Surprisingly, TRMT1L depletion also impaired the deposition of acp³U 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 m² ₂G27 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.