2'-O-Methylation maintains ribosome structural and translation integrity

In eukaryotic ribosomes, ∼2% of RNA nucleotides undergo 2'-O-methylation, a conserved cellular mechanism thought to be critical for maintaining and regulating translation. Here, we use ribosome profiling, translation assays, proteomics, and high-resolution structural analyses to show that loss of native 2'-O-methylation in yeast ribosomes disproportionally affects the translation of ribosomal protein transcripts, driven by changes in codon usage and recognition of structured RNA. Translation reprogramming by the hypomethylated ribosomes is supported by reduced thermostability and high-resolution evidence of altered ribosomal structures and conformations. Consistent with the roles of the affected proteins in sustaining cellular fitness, hypomethylated ribosomes under stress exhibit a selective loss of downregulated proteins and misassembly associated with methylation loss. Our data provide structural and mechanistic insights into how 2'-O-methylation supports ribosome integrity and mediates cellular stress responses.