Mis-targeted methylation in rRNA can severely impair ribosome synthesis and activity

Eukaryotic rRNAs contain scores of two major types of nucleotide modifications, 2'-O-methylation (Nm) and pseudouridylation (Psi). Both types are known to alter the stability and dynamics of RNA folding. In Eukaryotes, these modifications are created by small nucleolar RNPs (snoRNPs) with site-specificity provided by the snoRNA component. Little is yet known about the influence of such modifications on ribosome synthesis or activity, although in a few cases depletions of natural modifications have impaired ribosome function. Our previous work showed that targeting Nm modifications to non-natural sites in yeast rRNA can severely impair cell growth, however, the underlying basis of the interference effects were not described. Here, we show that targeting Nm formation to several individual sensitive sites in the peptidyl transferase center (PTC) strongly impairs ribosome accumulation and activity. Methylation was detected for all sites targeted, suggesting that the non-natural modification is the basis of the interference effects. For certain sensitive sites, the translation rate was reduced by 70-100%, due to: (1) a marked decrease (28-50%) in ribosomal subunits caused by slower pre-rRNA processing and mainly faster rRNA turn over and, (2) impaired activity of the surviving ribosomes. This last finding infers that the mis-targeted methylations compromise PTC function. The discovery that a new methylation can trigger robust rRNA degradation indicates that modification effects are monitored for quality control. These findings imply that nucleotide modifications can serve as evolutionary constraints and that snoRNP mutations expected to occur in nature can cause human disease.