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, researchers at the University of California, Santa Cruz 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. The researchers use their 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.
Pairs of correlated nucleotide changes (nodes) are shown for each mutant (edges) relative to wild type yeast 25S rRNA (A) and 18S rRNA (B). In cases where correlated pairs show differential changes in correlation in different mutants (eg. U24 modifications), node color rings are fragmented with the appropriate mutant edge connecting to either the magenta (negative change in correlation) or black (positive change in correlation) portion of the node.