Ribosomal RNA 2'-O-methylations regulate translation by impacting ribosome dynamics

Protein synthesis by ribosomes is critically important for gene expression in all cells. The ribosomal RNAs (rRNAs) are marked by numerous chemical modifications. An abundant group of rRNA modifications, present in all domains of life, is 29-O-methylation guided by box C/D small nucleolar RNAs (snoRNAs) which are part of small ribonucleoprotein complexes (snoRNPs). Although 29-O-methylations are required for proper production of ribosomes, the mechanisms by which these modifications contribute to translation have remained elusive. Here, we show that a change in box C/D snoRNP biogenesis in actively growing yeast cells results in the production of hypo 29-O-methylated ribosomes with distinct translational properties. Using RiboMeth-Seq for the quantitative analysis of 29-O methylations, we identify site-specific perturbations of the rRNA 29-O-methylation pattern and uncover sites that are not required for ribosome production under normal conditions. Characterization of the hypo 29-O-methylated ribosomes reveals significant translational fidelity defects including frameshifting and near-cognate start codon selection. Using rRNA structural probing, we show that hypo 29-O-methylation affects the inherent dynamics of the ribosomal subunits and impacts the binding of translation factor eIF1 thereby causing translational defects. Our data reveal an unforeseen spectrum of 29-O-methylation heterogeneity in yeast rRNA and suggest a significant role for rRNA 29-O-methylation in regulating cellular translation by controlling ribosome dynamics and ligand binding.