tRNA queuosine galactosylation links translational fidelity to cancer vulnerability

The accuracy of protein synthesis mainly relies on a wide range of specific tRNA modifications that fine-tune codon decoding. Among them, galactosyl-queuosine (galQ), a glycosylated derivative of queuosine, is uniquely installed at the wobble position of human tRNATyr, yet its cellular function remains poorly understood. In this work, we investigated the role of galQ in cancer by modulating the expression of QTGAL, the enzyme responsible for its biosynthesis, using siRNA-mediated knockdown in triple- negative breast cancer (TNBC) cell lines. These models were analyzed via molecular assays and high-throughput platforms including RNA-seq, Nano-tRNA-seq, and RiboLace. Our findings show that galQ is essential for translational balance in TNBC. Its loss leads to reduced proliferation, increased stop codon readthrough, and ribosome stalling in 3′-UTRs. Mechanistically, galQ appears to balance the decoding of the two tyrosine codons (UAU and UAC), optimizing fidelity and preventing near cognate misincorporation. These defects are exacerbated in Ribosome Quality Control (RQC)-deficient contexts, such as FOCAD-deleted TNBC, suggesting a synthetic vulnerability. This observations position galQ as a critical, context-dependent modulator of translational fidelity. Its loss not only disrupts proteostasis but may also generate aberrant peptides with immunogenic potential. Targeting QTGAL could thus represent a promising strategy for selectively impairing tumor cell viability and enhancing antigenicity in cancers with defective translational surveillance.