The Timing of Ribosomal RNA Modification During Eukaryotic Ribosome Biogenesis

Ribosome biogenesis is a highly complex and tightly regulated process that includes the transcription, processing, and modification of ribosomal RNA (rRNA). The rRNA modifications are critical for ribosome function, influencing rRNA stability, structure, and interactions with ribosomal proteins and tRNAs. However, the precise timing of modification installation, especially for pseudouridines, during rRNA maturation remains undetermined. We address this knowledge gap by isolating and analyzing rRNA precursor (pre-rRNA) in Saccharomyces cerevisiae through tandem affinity purification of RNA Polymerase I and III. Direct RNA sequencing using Nanopore technology allows detection of distinct pre-rRNA intermediates corresponding to various stages of the rRNA maturation pathway. Utilizing quantitative RNA modification analyses, we discover that all pseudouridines, along with many other types of rRNA modifications, are installed very early and co-transcriptionally before 20S and 27SA2 pre-rRNAs are separated by RNA cleavage. Notably, we observed that most modifications, particularly those at the 5' end of 18S rRNA, appear to be fully installed in the 35S pre-rRNA. In contrast, certain modifications in the central and 3' regions of 18S rRNA are not completely introduced until later stages of ribosome assembly, either in 33S/32S or 20S pre-rRNAs. These findings provide detailed insights into the timing of rRNA modification during the early stages of rRNA maturation and shed new light on the molecular choreography of eukaryotic ribosome biogenesis.