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: 14.02). 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.

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Available from: Sander Granneman, Sep 28, 2015
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    • "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. "
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    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 · 4.94 Impact Factor
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    • "Depletion of Prp43p leads to a transient accumulation of the 35S pre-rRNA and reduced accumulation of all downstream pre-rRNA intermediates, resulting in impaired production of both the small and large ribosomal subunits. It is likely that Prp43p intervenes at distinct steps of the ribosome biogenesis pathway (22) and could be targeted to and/or activated within different pre-ribosomal particles by different co-factors. Three such potential co-factors termed Ntr1p, Pfa1p and Gno1p were identified using double-hybrid screens, immunoprecipitations or tandem affinity purifications (TAPs) (18,20). "
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    ABSTRACT: We provide evidence that a central player in ribosome synthesis, the ribonucleic acid helicase Prp43p, can be activated by yeast Gno1p and its human ortholog, the telomerase inhibitor PINX1. Gno1p and PINX1 expressed in yeast interact with Prp43p and the integrity of their G-patch domain is required for this interaction. Moreover, PINX1 interacts with human PRP43 (DHX15) in HeLa cells. PINX1 directly binds to yeast Prp43p and stimulates its adenosine triphosphatase activity, while alterations of the G patch abolish formation of the PINX1/Prp43p complex and the stimulation of Prp43p. In yeast, lack of Gno1p leads to a decrease in the levels of pre-40S and intermediate pre-60S pre-ribosomal particles, defects that can be corrected by PINX1 expression. We show that Gno1p associates with 90S and early pre-60S pre-ribosomal particles and is released from intermediate pre-60S particles. G-patch alterations in Gno1p or PINX1 that inhibit their interactions with Prp43p completely abolish their function in yeast ribosome biogenesis. Altogether, our results suggest that activation of Prp43p by Gno1p/PINX1 within early pre-ribosomal particles is crucial for their subsequent maturation.
    Nucleic Acids Research 05/2014; 42(11). DOI:10.1093/nar/gku357 · 9.11 Impact Factor
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    • "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. "
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    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;
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