Nucleolar KKE/D Repeat Proteins Nop56P and Nop58P Interact with Nop1p and Are Required for Ribosome Biogenesis

Abstract

Different point mutations in the nucleolar protein fibrillarin (Nop1p in Saccharomyces cerevisiae) can inhibit different steps in ribosome synthesis. A screen for mutations that are synthetically lethal (sl) with the nop1-5 allele, which inhibits pre-rRNA processing, identified NOP56. An independent sl mutation screen with nop1-3, which inhibits pre-rRNA methylation, identified a mutation in NOP58. Strikingly, Nop56p and Nop58p are highly homologous (45% identity). Both proteins were found to be essential and localized to the nucleolus. A temperature-sensitive lethal mutant allele, nop56-2, inhibited many steps in pre-rRNA processing, particularly on the pathway of 25S/5.8S rRNA synthesis, and led to defects in 60S subunit assembly. Epitope-tagged constructs show that both Nop56p and Nop58p are associated with Nop1p in complexes, Nop56p and Nop1p exhibiting a stoichiometric association. These physical interactions presumably underlie the observed sl phenotypes. Well-conserved homologs are present in a range of organisms, including humans (52% identity between human hNop56p and yeast Nop56p), suggesting that these complexes have been conserved in evolution.Most steps of ribosome biogenesis occur in the nucleolus, a specialized subnuclear structure (for reviews, see references 14, 34, 39, 45, and 53). In eukaryotes including Saccharomyces cerevisiae and humans, a large precursor rRNA transcript (prerRNA) is processed into the mature 18S, 5.8S, and 25S/28S rRNAs. During transcription and processing, these rRNAs associate with approximately 80 ribosomal proteins and with the 5S rRNA. In addition, the mature rRNA regions of the prerRNA undergo extensive covalent nucleotide modification, mainly base modification of uridine to pseudouridine and methylation of the ribose 2Ј-hydroxyl (2Ј-O methylation) (reviewed in reference 27). The large number of concerted reactions occurring during rRNA processing and ribosome assembly has made it difficult to analyze single steps in ribosome synthesis. Over recent years, the analysis of yeast mutants defective in ribosome biogenesis has proved to be a powerful approach (reviewed in reference 51), particularly when combined with in vitro analyses using purified components (8,26,30). Despite this progress, our understanding of the detailed mechanisms of eukaryotic rRNA processing remains poor.The small nucleolar RNAs (snoRNAs) play important roles in the covalent processing of the pre-rRNAs (reviewed in references 2, 28, and 48). With the exception of RNase MRP, which is an endonuclease structurally related to RNase P, the very large numbers of snoRNAs present in eukaryotes can be divided into two groups based on conserved sequence and structural features (3, 12; reviewed in reference 48). Most of the box CϩD snoRNAs direct the site-specific 2Ј-O methylation of the pre-rRNA (19), while most of the box HϩACA snoRNAs select the sites of pseudouridine formation (6,11,33). In addition, a few members of each group of snoRNAs do not appear to select sites of pre-rRNA modification but are re...

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

“…Structure of the pre-rRNA and processing pathway in Saccharomyces cerevisiae. A: Structure of the 35S pre-rRNA+ The 35S operon contains the sequences for the mature 18S, 5+8S, and 25S rRNAs separated by the two internal transcribed spacers (ITS1 and ITS2)+ In addition, two external transcribed spacers, the 59ETS and 39ETS, are present at either end+ B: Pre-rRNA processing pathway+ The 35S pre-rRNA undergoes sequential processing reactions to generate the mature rRNAs+ Cleavage at A0 generates the 33S pre-rRNA+ It is subsequently cleaved at sites A1 at the 59 end of the 18S rRNA giving rise to the 32S pre-rRNA, and A2 in ITS1 yielding the 20S and 27SA2 pre-rRNAs+ The latter two species are precursors for the rRNAs respectively found in the small and large ribosomal subunits+ The 20S pre-rRNA is endonucleolytically cleaved at site D to produce the 18S rRNA+ The 27SA2 intermediate is processed by two alternative pathways+ In the major pathway, 27SA2 is cleaved by RNase MRP at site A3 in ITS1 yielding 27SA3, which is trimmed by 5939 exonucleases Rat1p and Xrn1p to site B1 S , generating the 27SB S precursor+ Approximately 15% of the 27SA2 molecules are processed at site B1 L , producing the 27SB L pre-rRNA+ 27SB S and 27SB L molecules are then processed at sites C1 and C2, yielding mature 25S rRNA and the 7S S and 7S L pre-rRNAs respectively, which are converted to 5+8S S and 5+8S L rRNAs by a complex of 39 59 exoribonucleases, the "exosome+" shifted to their associated proteins+ C/D snoRNPs are now known to contain three common essential proteins, Nop1p/fibrillarin (Tyc & Steitz, 1989, Baserga et al+, 1991Peculis & Steitz, 1994;Ganot et al+, 1997b), Nop56p, and Nop58p/Nop5p (Gautier et al+, 1997;Wu et al+, 1998, Lafontaine & Tollervey, 1999, whereas H/ACA snoRNPs contain at least four, Cbf5p (Jiang et al+, 1993;Cadwell et al+, 1997;, Gar1p (Girard et al+, 1992, Ganot et al+, 1997bHenras et al+, 1998), Nhp2p (Henras et al+, 1998;Watkins et al+, 1998), and Nop10p (Henras et al+, 1998)+ The discovery that Cbf5p, which is highly related to pseudouridine synthases (Koonin, 1996), is an integral snoRNP component strongly suggests that H/ACA snoRNPs not only select, but also modify prerRNA uridine residues )+ C/D snoRNPs also probably catalyze pre-rRNA modifications, as it has recently been noticed that the first characterized snoRNP protein component, Nop1p/ fibrillarin, contains the conserved methyltransferase motifs (Niewmierzycka & Clarke, 1999)+ SnoRNPs need to be able to reversibly interact with the pre-rRNA to catalyze pre-rRNA modifications or to promote pre-rRNA cleavage events+ Little is known about the ways snoRNPs/pre-rRNA interactions are modulated+ In the absence of the Gar1p protein, so-called because it contains two glycine/arginine-rich (GAR) domains at its N-and C-ter...…”

Pol I Transcription and Pre-rRNA Processing Are Coordinated in a Transcription-dependent Manner in Mammalian Cells

“…Nop58 was chosen because immunoprecipitation studies in yeast show that fibrillarin binds to box C/D RNA independent of Nop58 (Gautier et al, 1997;Tollervey, 1999, 2000) and additional site-specific cross-linking analyses confirm and map the independent association of Nop58 and fibrillarin with their respective box C/D elements (Cahill et al, 2002). Therefore, fibrillarin can only be coprecipitated with Nop58 through being a part of snoRNPs.…”

Interaction of the U3-55k protein with U3 snoRNA is mediated by the Box B/C motif of U3 and the WD repeats of U3-55k

“…The RNA is associated with proteins common to all Box C/D snoRNAs, including fibrillarin/Nop1p (34,35), Nop56p (36) and Nop58/Nop5p (37)(38)(39) and proteins specific to the U3 snoRNP, including Sof1p (supressor of fibrillarin; 40), Mpp10 (41,42), Lcp5p (43), Imp3p (44), Imp4p (44) and U3-55k (29,45). Each of these proteins (like U3 RNA) are essential and have been shown to be specifically required for the maturation of 18S rRNA [39][40][41]43,44,46,47 personal communication]. U3-55k, the focus of this work, was initially identified as part of a trimeric complex (p55, p50 and p15) that co-purified from CHO cells with U3 snoRNA.…”