Abstract
SummaryYeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3′ cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.
Highlights
Ribosomes in Saccharomyces cerevisiae consist of 79 ribosomal proteins and four ribosomal RNAs
In strains depleted of Prp43 or expressing only catalytic point mutants, six small nucleolar RNAs (snoRNAs) that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association
Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions
Summary
Ribosomes in Saccharomyces cerevisiae consist of 79 ribosomal proteins and four ribosomal RNAs (rRNAs). Most nucleotide modifications in the rRNA are 20-O-methylation and pseudouridylation at sites selected by base pairing with box C/D and box H/ACA small nucleolar RNAs (snoRNAs), respectively (Kiss-Laszlo et al, 1996; Ganot et al, 1997; Bachellerie et al, 2002). The base pairing of the pre-rRNA with box H/ACA snoRNAs is predicted to be less stable than for box C/D snoRNAs, which have long complementary sequences of up to 19 nucleotides. Both classes of snoRNA can be detected in stable association with 60–90S preribosomes on sucrose density gradients
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