biostudies-literatureWu JGermany's Excellence StrategyGerman Research FoundationSwiss National Science FoundationSFB 1381European Research CouncilPolish Ministry and Higher EducationMarie Sklodowska-Curie Postdoctoral FellowshipEuropean UnionPLGridDutch Research Council (NWO)European Commissiongkaf1311https://www.ebi.ac.uk/biostudies/studies/S-EPMC12721868biostudies-literatureUnknown53(22)Transfer RNA (tRNA) modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding. However, the mechanisms by which tRNA modifications modulate codon optimality and trigger phenotypes remain unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs in wobble uridine modification (U34) mutants. This triggers ribosome collisions and a coordinated hierarchical response of cellular quality control pathways. High-resolution ribosome profiling reveals an unexpected functional diversity of U34 modifications during decoding. For instance, 5-carbamoylmethyluridine (ncm5U) exhibits distinct effects at the A and P sites. Importantly, ribosomes only slow down at a fraction of codons decoded by hypomodified tRNA, and the Nucleic acids researchSuboptimal codon pairs trigger ribosome collisions and cellular quality control responses in tRNA modification mutants.PMC127218682195366691687463404096736871010013944032227021/SOL/2021/2019.162LW.028769065390939984PLG/2020/014009259130777Wu JSin OFufezan CLeidel SAMancilla HKraft CScholer HRMollet FDrexler HCARanff TKoziej LGlatt SBruder JMEggers CfalseSuboptimal codon pairs trigger ribosome collisions and cellular quality control responses in tRNA modification mutants.Transfer RNA (tRNA) modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding. However, the mechanisms by which tRNA modifications modulate codon optimality and trigger phenotypes remain unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs in wobble uridine modification (U34) mutants. This triggers ribosome collisions and a coordinated hierarchical response of cellular quality control pathways. High-resolution ribosome profiling reveals an unexpected functional diversity of U34 modifications during decoding. For instance, 5-carbamoylmethyluridine (ncm5U) exhibits distinct effects at the A and P sites. Importantly, ribosomes only slow down at a fraction of codons decoded by hypomodified tRNA, and the 2025-01-01T00:00:00Z2025 Nov2026-06-04T03:18:19.114Z2026-06-04T03:12:18.022ZS-EPMC127218684142942110.1093/nar/gkaf1311