Functional Characterisation of Ribosome Biogenesis Cofactors in Saccharomyces cerevisiae

The biogenesis of eukaryotic ribosomes is a highly complex and dynamic process, which requires a multitude of cofactors. In Saccharomyces cerevisiae, approximately 200 proteins have been identified to function in this pathway, coordinating ribosomal protein assembly and processing of ribosomal RNA (rRNA) precursors. Furthermore, small nucleolar ribonucleoprotein complexes (snoRNPs) introduce numerous nucleotide modifications within the rRNAs, which are guided by basepairing of their RNA component (snoRNA) with the designated target pre-rRNA. SnoRNA binding and pre-rRNA processing involve structural reorganisations of the pre-ribosomal complexes during their maturation. RNA helicases are thought to be key regulators of these events. In yeast, 19 RNA helicases are known to be involved in ribosome biogenesis. However, a detailed characterisation of their molecular functions has been impeded by a lack of information about their target sites. In this study, the recently developed crosslinking technique (CRAC) was used to identify the substrates and binding sites of two RNA helicases, Prp43 and Rok1. All pre-rRNA crosslinking sites of the DEAD box helicase Rok1 were found to cluster on one face of the small ribosomal subunit (SSU). The main binding site, which was confirmed by chemical probing, was found in the eukaryotic expansion segment 6 (ES6), where Rok1 is required for release of the snoRNA snR30. Besides snR30, Rok1 was found to interact also with other snoRNAs involved in pre-rRNA processing. In addition, a search for chimeric sequence reads in the crosslinking data set (CLASH) allowed the identification of several novel basepairing sites of these snoRNAs in the 18S rRNA sequence, close to the Rok1 target sites. New snoRNA interaction sites were discovered mainly in eukaryotic expansion segments of the rRNA sequence, suggesting that these snoRNAs might play important roles in bridging long-range interactions and are part of an extensive interaction network that is important for effective pre-rRNA folding and the coordination of the early processing events during the SSU synthesis. CRAC analysis for the DEAH box helicase Prp43 revealed several crosslinking sites in the pre-rRNA and on box C/D snoRNAs. Interestingly, several box C/D snoRNAs, which guide a cluster of nucleotide modifications in 25S rRNA, were found to accumulate on pre-ribosomal particles after Prp43 depletion. Mapping of the Prp43 crosslinking sites on the LSU structure revealed that they are in close proximity to the modification sites of the affected snoRNAs. CLASH analysis uncovered hybrids containing sequences of the affected snoRNAs and their target sites on 25S rRNA, strongly supporting a direct function of Prp43 in unwinding the basepairing interactions of these snoRNAs. Additionally, Prp43 crosslinks 25S rRNA sequences close to the basepairing sites of two snoRNAs, whose association with pre-ribosomes was decreased after depletion of the helicase. This finding suggests that Prp43 may function in remodelling of pre-ribosomal complexes, enabling these snoRNAs to gain access to their target sites. Similarly, Prp43 was found to crosslink near the 3’-end of the 18S rRNA, where it has been proposed to function in restructuring events that facilitate the final maturation of the 18S rRNA by the endonuclease Nob1. Homologues of yeast Nob1 were identified in higher eukaryotes and archaea. In vitro cleavage assays using Nob1 from Pyrococcus horikoshii or Arabidopsis thaliana demonstrated that the homologs are also able to perform a manganese dependent endonucleolytic cleavage reaction at the cleavage site D of 16S and 18S rRNA, respectively. The availability of the P. horikoshii Nob1 structure allowed the identification and functional analysis of residues important for substrate binding and cleavage within the PIN domain, while the zinc ribbon domain likely functions in anchoring and positioning of the enzyme on the pre-ribosomal complexes near the processing site.