Queuosine glycosylation protects against stress-induced tRNA fragmentation and modulates cleavage site selection

Alongside their canonical function as adaptors in translation, tRNAs are precursors of tRNA-derived fragments that can regulate diverse aspects of gene expression. Queuosine (Q), present at position 34 of eukaryotic tRNA ^{Asn/Asp/His/Tyr} , has been implicated in suppressing tRNA fragmentation. In vertebrates, Q ₃₄ of tRNA ^Asp and tRNA ^Tyr is further modified by mannosylation and galactosylation, respectively, catalyzed by QTMAN and QTGAL. However, the interplay between these glycosylations and other anticodon loop modifications, and their impact on tRNA fragmentation, have remained unclear. Here, we define a modification circuit in human tRNA ^Asp in which Q ₃₄ stimulates DNMT2-dependent m ⁵ C ₃₈ formation, while subsequent Q ₃₄ mannosylation does not impact m ⁵ C ₃₈ installation; reciprocally, m ⁵ C ₃₈ inhibits Q ₃₄ incorporation by the tRNA-guanine transglycosylase TGT. By contrast, anticodon loop modifications of tRNA ^Tyr are installed independently, although our data support a hierarchical pathway in which TRMT5-mediated m ¹ G ₃₇ formation precedes queuosinylation and galactosylation. Alongside demonstrating that Q ₃₄ glycosylation enhances protein synthesis, our data reveal that mannosylation of Q ₃₄ protects tRNA ^Asp from stress-induced cleavage, thus expanding the relevance of Q glycosylation beyond translation.