Suboptimal 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 decoding speed of most codons remains unaffected. However, the translation speed of a codon largely depends on the identity of A- and P-site codons. Stalling at modification-dependent codon pairs induces ribosome collisions, triggering ribosome-associated quality control (RQC) and preventing protein aggregation by degrading aberrant nascent peptides and messenger RNAs. Inactivation of RQC stimulates the expression of molecular chaperones that remove protein aggregates. Our results demonstrate that loss of tRNA modifications primarily disrupts translation rates of suboptimal codon pairs, showing the coordinated regulation and adaptability of cellular surveillance systems. These systems ensure efficient and accurate protein synthesis and maintain protein homeostasis.