Prp43 Bound at Different Sites on the Pre-rRNA Performs Distinct Functions in Ribosome Synthesis

Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK.
Molecular cell (Impact Factor: 14.46). 11/2009; 36(4):583-92. DOI: 10.1016/j.molcel.2009.09.039
Source: PubMed

ABSTRACT Yeast 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.

Download full-text


Available from: Sander Granneman, Aug 20, 2015
  • Source
    • "was performed as previously described (Bohnsack et al. 2009; Granneman et al. 2009; Bohnsack et al. 2012). In brief, UV crosslinking was performed in living cells either in culture medium (in culturo) or pelleted and resuspended in small volume (in vivo), and Rok1-containing complexes were isolated first on IgG sepharose, eluted with TEV protease, and purified on Nickel-NTA. "
    [Show abstract] [Hide abstract]
    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.
    RNA 06/2014; 20(8). DOI:10.1261/rna.044669.114 · 4.62 Impact Factor
  • Source
    • "The RNA–protein complexes were subsequently treated with TEV protease, followed by immobilized metal-ion affinity chromatography (IMAC) as a secondary purification. Using the CRAC method, the Tollervey laboratory identified the binding sites of the probable pre-mRNA-splicing factor ATP-dependent RNA helicase Prp43 [28] and the small nucleolar RNPs (snoRNPs) Nop1, Nop56, Nop58 and rRNA processing 9 (Rrp9) [29] in yeast. "
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA−protein interactions influence many biological processes. Identifying the binding sites of RNA-binding proteins (RBPs) remains as one of the most fundamental and important challenges to the studies of such interactions. Capturing RNA and RBPs via chemical crosslinking allows stringent purification procedures that significantly remove the non-specific RNA and protein interactions. Two major types of chemical crosslinking strategies have been developed to date, i.e., UV-enabled crosslinking and enzymatic mechanism-based covalent capture. In this review, we compare such strategies and their current applications, with an emphasis on the technologies themselves rather than the biology that has been revealed. We hope such methods could benefit broader audience and also urge for the development of new methods to study RNA−RBP interactions.
    Genomics Proteomics & Bioinformatics 01/2014;
  • Source
    • "The unexpected detection of C2ORF3 protein also in the nucleoli suggests a function for C2ORF3 in rRNA processing . As the hPrp43 protein is shown to be involved in rRNA processing (Bohnsack et al. 2009; Lebaron et al. 2009; Pertschy et al. 2009), it is quite conceivable that the C2ORF3 protein would function in this process in collaboration with hPrp43. It would be interesting to determine whether the knockdown of C2ORF3 protein affects ribosomal RNA processing and/or nucleoli structure. "
    [Show abstract] [Hide abstract]
    ABSTRACT: To identify the novel factors involved in the postsplicing intron turnover pathway, we carried out immunoprecipitation with known postsplicing factors, hPrp43 and TFIP11. As an interacting factor, we identified C2ORF3 protein by mass spectrometry. We found that C2ORF3 protein is present in the previously characterized Intron Large (IL) complex with an excised lariat intron. In vitro splicing using C2ORF3-depleted nuclear extracts showed significant repression of splicing, suggesting that C2ORF3 protein is required for pre-mRNA splicing through its presumable role in efficient intron turnover. Interestingly, C2ORF3 protein is localized in both the nucleoplasm and nucleoli, which suggests a potential function in rRNA processing.
    Genes to Cells 12/2013; 19(1). DOI:10.1111/gtc.12114 · 2.86 Impact Factor
Show more