A RAC protein-binding site in the internal transcribed spacer 2 of Pre-rRNA transcripts from Schizosaccharomyces...

The interdependence of steps in the processing of the eukaryotic preribosomal rRNA transcripts indicate that rRNA processing, at least in part, acts as a quality control mechanism to help ensure that only functional rRNA is incorporated into mature ribosomes. In search of structural components that underlie this interdependence, we have isolated a large protein complex or RAC that contains an independent binding site for all four of the transcribed spacers in the nascent pre-rRNA. In this study the RAC-binding site in the internal transcribed spacer 2 sequence of Schizosaccharomyces pombe rRNA transcripts was identified, and the influence of this site on rRNA maturation was assessed. Modification exclusion analyses indicate that the protein complex interacts with a helical domain previously shown to contain features common to both the internal transcribed spacer 1 and the 3'-external transcribed spacer. Mutagenic analyses in vitro confirm an interaction with this sequence, and parallel analyses in vivo indicated a critical role in both the maturation of the rRNA components of the large subunit as well as the 18 S rRNA component of the small subunit. Hybridization analyses also indicated greatly elevated levels of unprocessed nascent RNA. These effects are contrasted with mutations in other regions of the secondary structure that resulted in some reduction of plasmid-derived mature rRNA but no elevated levels of the precursor molecules. The significance with respect to rRNA maturation and the interdependences in rRNA processing are discussed.