Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling
Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18S rRNA and 73 sites in 25S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct, undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and acute environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function. HighlightsO_LImethod enabling single-molecule profiling of RNA modifications is developed and reveals heterogeneous classes of modified ribosomes. C_LIO_LIrRNA 2O methylation and pseudouridylation modifications are independent of each other. C_LIO_LIin functional centers of the ribosome are modified in a concerted fashion. C_LIO_LIof function for RNA helicases Dbp3 and Prp43 produce discrete overlapping subpopulations of incompletely modified ribosomes. C_LIO_LImodification profiles are resilient to rapidly changing nutrient conditions and perturbation of translation C_LI