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 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. Overall design: We sequenced 33 samples of 18S and 25S ribosomal rRNA. 3 replicates of wild type yeast and 3 replicates of wild type shifted to cold. We sequenced 2 replicates of snr80 KO, Prp43 mutant, Dbp3 KO and Pxr1 KO.