Queuosine glycosylation occurs after anticodon loop methylations and protects against stress-induced tRNA fragmentation

Abstract

Queuosine, present at position 34 (Q34) of vertebrate tRNAAsp and tRNATyr, is mannosylated and galactosylated by QTMAN and QTGAL, respectively. However, the interplay between these glycosylations and other anticodon loop modifications, and their impact on tRNA fragmentation, have remained unknown. Here, we define hierarchical modification pathways for the anticodon loop modifications of the two human glycoQ34-containing tRNAs. Our data reveal bidirectional interdependence of Q34 and m5C38 in tRNAAsp with subsequent Q34 mannosylation occurring in an m5C38-independent manner. Anticodon loop modifications of tRNATyr are installed independently, but sequentially in a pathway in which TRMT5-mediated m1G37 formation precedes queuosinylation and galactosylation. Q34 glycosylation enhances protein synthesis but alongside their canonical function in translation, tRNAs are precursors of tRNA-derived fragments that can regulate diverse aspects of gene expression. Our data reveal that Q34 mannosylation protects tRNAAsp from stress-induced cleavage and modulates cleavage site selection, thus expanding the relevance of Q glycosylation beyond translation.