Defective queuosine and i6A/ms2i6A modification of tRNATyr cause frameshifting and protein aggregation.

Queuosine (Q) modification at the wobble position (Q34) of tRNAs fine-tunes translational speed but is not essential for viability, leaving its physiological role unclear. In bacteria, Q34 is synthesized de novo, whereas eukaryotes obtain queuosine (Q) or its precursor queuine (q) from external sources. Q34 uniquely co-occurs with N6-isopentenyladenosine (i6A) or its derivative 2-methylthio-N6-isopentenyladenosine (ms2i6A) at position 37 of tRNATyr. We show that loss of Q34 (∆tgt) causes a severe growth defect in Escherichia coli lacking ms2i6A due to deletion of the MiaA isopentenyltransferase (∆miaA), which is rescued by tRNATyr overexpression. Simultaneous absence of Q34 and ms2i6A37 increases +1 frameshifting at tyrosine codons and promotes protein aggregation, indicating impaired tRNATyr function. This functional interplay is evolutionarily conserved: Q34 deficiency aggravates the growth defect of Schizosaccharomyces pombe lacking the isopentenyltransferase Tit1 and thus i6A. In S. pombe, Q34 enhances tRNATyr abundance in tit1∆ cells and reduces i6A37 levels in wild-type, revealing reciprocal regulation. Together, these findings demonstrate a synergistic role of Q34 and (ms2)i6A37 in maintaining translational fidelity and proteostasis, with potential implications for human health when Q availability is limited.