Abstract
Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the "foot" region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.
Highlights
The synthesis of cytoplasmic ribosomes in Saccharomyces cerevisiae is initiated by RNA polymerase I-mediated transcription of the 35S ribosomal RNA precursor, which contains the sequences of the mature 18S, 5.8S, and 25S ribosomal RNAs (rRNAs) (Fig. 1A; Henras et al 2008; Thomson et al 2013; Woolford and Baserga 2013)
Initial cleavages (Fig. 1A) in a complex sequence of prerRNA processing and modification events lead to the separation of the biogenesis pathways of the large (LSU, 60S) and small (SSU, 40S) ribosomal subunits
Several RNA helicases are required for the release of individual snoRNAs from pre-ribosomes (Kos and Tollervey 2005; Liang and Fournier 2006; Bohnsack et al 2008), and depletion of the RNA helicase Prp43 leads to the accumulation of several snoRNAs on pre-60S complexes (Bohnsack et al 2009)
Summary
The synthesis of cytoplasmic ribosomes in Saccharomyces cerevisiae is initiated by RNA polymerase I-mediated transcription of the 35S ribosomal RNA precursor (pre-rRNA), which contains the sequences of the mature 18S, 5.8S, and 25S rRNAs (Fig. 1A; Henras et al 2008; Thomson et al 2013; Woolford and Baserga 2013). These RNA helicases have been proposed to act either in the structural remodeling of pre-ribosomal intermediates or in the unwinding of snoRNA–pre-rRNA base-pairing (Ripmaster et al 1992; Venema and Tollervey 1999; Martin et al 2013). We identify the binding sites of the DEAD-box RNA helicase Rok1 on pre-ribosomal RNA by UV cross-linking and analysis of cDNA (CRAC).
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