Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs
1998; Springer Nature; Volume: 17; Issue: 23 Linguagem: Inglês
10.1093/emboj/17.23.7078
ISSN1460-2075
AutoresAnthony K. Henras, Yves Henry, Cécile Bousquet‐Antonelli, Jacqueline Noaillac‐Depeyre, Jean‐Paul Gélugne, Michèle Caizergues‐Ferrer,
Tópico(s)RNA Research and Splicing
ResumoArticle1 December 1998free access Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs Anthony Henras Anthony Henras Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Yves Henry Corresponding Author Yves Henry Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Cécile Bousquet-Antonelli Cécile Bousquet-Antonelli Present address: Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, EH9 3JR UK Search for more papers by this author Jacqueline Noaillac-Depeyre Jacqueline Noaillac-Depeyre Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Jean-Paul Gélugne Jean-Paul Gélugne Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Michèle Caizergues-Ferrer Michèle Caizergues-Ferrer Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Anthony Henras Anthony Henras Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Yves Henry Corresponding Author Yves Henry Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Cécile Bousquet-Antonelli Cécile Bousquet-Antonelli Present address: Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, EH9 3JR UK Search for more papers by this author Jacqueline Noaillac-Depeyre Jacqueline Noaillac-Depeyre Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Jean-Paul Gélugne Jean-Paul Gélugne Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Michèle Caizergues-Ferrer Michèle Caizergues-Ferrer Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France Search for more papers by this author Author Information Anthony Henras1, Yves Henry 1, Cécile Bousquet-Antonelli2, Jacqueline Noaillac-Depeyre1, Jean-Paul Gélugne1 and Michèle Caizergues-Ferrer1 1Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, Cedex 04, France 2Present address: Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, EH9 3JR UK *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:7078-7090https://doi.org/10.1093/emboj/17.23.7078 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site-specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinity-purified complexes containing epitope-tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope-tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively. Both proteins are conserved, essential and present in the dense fibrillar component of the nucleolus. Nhp2p and Nop10p are specifically associated with all H/ACA snoRNAs and are essential to the function of H/ACA snoRNPs. Cells lacking Nhp2p or Nop10p are impaired in global rRNA pseudouridylation and in the A1 and A2 cleavage steps of the pre-rRNA required for the synthesis of mature 18S rRNA. These phenotypes are probably a direct consequence of the instability of H/ACA snoRNAs and Gar1p observed in cells deprived of Nhp2p or Nop10p. Our results suggest that Nhp2p and Nop10p, together with Cbf5p, constitute the core of H/ACA snoRNPs. Introduction Synthesis of ribosomes in all eukaryotes requires extensive modification of a primary rRNA transcript that contains spacer regions flanking the rRNA sequences found in cytoplasmic ribosomes. Maturation of the pre-rRNA involves essentially two types of events: modifications of specific nucleotides confined to regions corresponding to mature rRNAs, and endo- and exonucleolytic cleavages that remove the transcribed spacers (reviewed in Venema and Tollervey, 1995; Sollner-Webb et al., 1996). Most of the nucleotide modifications appear on the pre-rRNA before nucleolytic processing; these are methylations at the 2′-O-hydroxyl position of riboses, and conversions of uridine residues into pseudouridines (Maden, 1990). Interestingly, most of these modifications are clustered in the universal core regions of rRNAs and have been remarkably conserved during evolution, suggesting they play an important role(s) in ribosome function (Lane et al., 1995). An ever increasing number of trans-acting factors required for pre-rRNA maturation are being characterized (reviewed in Venema and Tollervey, 1995; Sollner-Webb et al., 1996). Several of these are small nucleolar RNAs (snoRNAs), most of which can be classified into two families based on their sharing sequence motifs and displaying structural homologies (Balakin et al., 1996). The members of the family of C/D snoRNAs all contain, as their name implies, two short motifs termed the C (consensus 5′-UGAUGA-3′) and D boxes (5′-GUCUGA-3′). The second family is constituted by snoRNAs which all share the so-called H (consensus 5′-ANANNA-3′) and ACA boxes. C/D and H/ACA snoRNAs display peculiar modes of expression. In metazoans, although some snoRNAs are independently transcribed, the vast majority are produced from introns of pre-mRNAs by 5′→3′ and 3′→5′ exonucleolytic processing (Kiss and Filipowicz, 1995; Caffarelli et al., 1996; Cavaillé and Bachellerie, 1996; Watkins et al., 1996; Ganot et al., 1997a; reviewed in Sollner-Webb, 1993; Tollervey and Kiss, 1997). In yeast and plants, some C/D snoRNAs are processed from di- or polycistronic transcripts by endo- and exonucleolytic activities (Leader et al., 1997; Chanfreau et al., 1998; Petfalski et al., 1998). In all cases examined, and irrespective of whether the snoRNAs were of intronic or non-intronic origin, the C, D, H and ACA conserved boxes, as well as the stem structures present in their vicinity, have been shown to be essential for normal snoRNA processing and accumulation (Terns et al., 1995; Balakin et al., 1996; Caffarelli et al., 1996; Cavaillé and Bachellerie, 1996; Watkins et al., 1996; Ganot et al., 1997a). A few C/D and H/ACA snoRNAs are required for certain pre-rRNA cleavage events. The C/D box snoRNAs U3 (Hughes and Ares, 1991), U14 (Li et al., 1990) and U22 (Tycowski et al., 1994), as well as the H/ACA snoRNAs snR10 (Tollervey, 1987) and snR30 (Morrissey and Tollervey, 1993) are necessary for pre-rRNA processing steps leading to the production of mature 18S rRNA. In contrast, U8 (C/D snoRNA) intervenes in 5.8S and 28S rRNA maturation (Peculis and Steitz, 1993). It was shown recently, however, that the remaining known members of both families are required for site-specific modifications of the pre-rRNA: they function, by way of specific snoRNA/pre-rRNA Watson–Crick base-pairing interactions, as guide RNAs selecting the ribose moieties that will undergo methylation of the 2′ oxygen in the case of C/D snoRNAs (Cavaillé et al., 1996; Kiss-László et al., 1996, 1998; Nicoloso et al., 1996; Tycowski et al., 1996; Cavaillé and Bachellerie, 1998; reviewed in Tollervey, 1996; Bachellerie and Cavaillé, 1997, 1998; Maden and Hughes, 1997; Tollervey and Kiss, 1997) or the uridine residues to be converted into pseudouridines in the case of H/ACA snoRNAs (Ganot et al., 1997b; Ni et al., 1997; reviewed in Smith and Steitz, 1997). In the nucleolus, snoRNAs are not free but found tightly associated with proteins in ribonucleoprotein complexes (snoRNPs) of ∼10–20S in size. It is likely that only the complexes possess correctly regulated biological activity (Bousquet-Antonelli et al., 1997). So far only few proteins of C/D and H/ACA snoRNPs have been characterized. The Nop1p/fibrillarin protein is specifically associated with all C/D snoRNAs (Tyc and Steitz, 1989; Baserga et al., 1991; Peculis and Steitz, 1994; Ganot et al., 1997a) and is required for pre-rRNA methylation and processing (Tollervey et al., 1993), consistent with the involvement of C/D snoRNAs in both processes. The yeast Nop58p/Nop5p protein (Gautier et al., 1997; Wu et al., 1998) has also been shown to interact with all tested C/D snoRNAs (Wu et al., 1998; D.L.J.Lafontaine and D.Tollervey, personal communication) and to be required for their stability (D.L.J.Lafontaine and D.Tollervey, personal communication). In addition, a mouse protein of 65 kDa (Watkins et al., 1998) and a Xenopus laevis protein with an apparent mol. wt of 68 kDa (Caffarelli et al., 1998) have been found to interact with the box C/D-terminal–stem core motif of U14 and U16, respectively, but their identities have not yet been determined. H/ACA snoRNPs are known to contain the proteins Gar1p and Cbf5p. Gar1p is an essential glycine/arginine rich (GAR) domain-containing nucleolar protein that is required both for 18S rRNA production and rRNA pseudouridylation (Girard et al., 1992; Bousquet-Antonelli et al., 1997). The essential nucleolar protein Cbf5p (Jiang et al., 1993; Cadwell et al., 1997) is also necessary for rRNA pseudouridylation (Lafontaine et al., 1998) and 18S rRNA formation (Cadwell et al., 1997; Lafontaine et al., 1998). Cbf5p is strongly homologous to known pseudouridine synthases (Koonin, 1996; Becker et al., 1997) and, therefore, it probably provides the actual catalytic activity. It is highly probable that H/ACA snoRNPs contain other proteins in addition to Gar1p and Cbf5p. Indeed, the snR30-containing snoRNP has been purified, and four proteins of apparent mol. wts 65, 25, 23 and 10 kDa appear to be tightly associated with snR30 (Lübben et al., 1995). Only the 25 kDa protein was positively identified as Gar1p by Western blot analysis (Lübben et al., 1995). In the present study, we sought to characterize the common core protein components of H/ACA snoRNPs by affinity purification of complexes containing epitope-tagged Gar1p. This is clearly a necessary first step in determining the various protein–protein and RNA–protein interactions that govern both the structure and function of these particles. We show that in addition to Cbf5p and Gar1p, all H/ACA snoRNPs contain the Nhp2p protein (Kolodrubetz and Burgum, 1991) and a small novel protein we named Nop10p. These proteins are essential to the synthesis and function of H/ACA snoRNPs and are likely, together with Cbf5p, to constitute their core. Results Gar1p is associated with the Nhp2p and Nop10p proteins We have previously reported that the Gar1p protein, tagged at its C-terminus with two synthetic IgG binding domains (ZZ domains) derived from Staphylococcus aureus protein A, is specifically associated with all H/ACA snoRNAs and fully functional (Ganot et al., 1997a). In an attempt to quickly and efficiently purify H/ACA snoRNPs, we made use of a strain expressing Gar1p–ZZ. An extract prepared from a Gar1p–ZZ expressing strain under nondenaturing conditions was loaded onto an IgG–Sepharose column, and complexes retained along with Gar1p–ZZ were eluted with acetic acid (see Materials and methods for details). Under these conditions, three distinct major polypeptides other than contaminating IgGs are specifically coeluted with Gar1p–ZZ, migrating as proteins of ∼65, ∼22 and ∼10 kDa (Figure 1, lane 1). The broad bands labelled with asterisks correspond to contaminating IgGs. The polypeptide with an apparent molecular weight of 33 kDa (indicated by a dot in Figure 1) is a Gar1p–ZZ degradation product since Western blot analysis revealed that it contains the ZZ tag (data not shown). Given its apparent mobility, the polypeptide migrating just ahead of the 67 kDa marker probably corresponds to Cbf5p (Figure 1, lane 1, unfilled arrow), already shown to be part of H/ACA snoRNPs (Lafontaine et al., 1998). A partial N-terminal amino acid sequence was obtained for the 22 and 10 kDa polypeptides MGKDNKEHK and MHLMYTLGPD, respectively. BLAST analysis of the yeast protein database identified the 22 kDa protein as Nhp2p (Kolodrubetz and Burgum, 1991) (Figure 2A). A protein containing the sequence MHLMYTLGPD could not be found in the protein database. However, a search of the yeast genome against a nucleotide sequence corresponding to this peptide identified a small open reading frame (ORF) on chromosome VIII, situated between ORF YHR072W (ERG7) and YHR073W, encoding a protein of 58 amino acids (Figure 2B). We termed this protein Nop10p. Figure 1.Gar1p is associated with 3 proteins. Purification over an IgG–Sepharose column of complexes containing Gar1p–ZZ (lane 1) or Nhp2p–ZZ (lane 2). Proteins eluted from the columns were resolved by SDS–PAGE on a 15% polyacrylamide gel and revealed by Coomassie staining. Note that the extract used to purify epitope-tagged Nhp2p also contains wild-type Nhp2p. M, molecular weight markers. The identity of the bands is indicated by arrows. The contaminating IgGs are indicated by asterisks, the Gar1p–ZZ degradation product by a dot and the bands probably corresponding to Cbf5p by unfilled arrows. Download figure Download PowerPoint Figure 2.(A) Nhp2p belongs to a family of putative RNA binding proteins. Alignments of S.cerevisiae Nhp2p with related human proteins Nhp2L1p (Saito et al., 1996) and DJ0167F23.5 (DDBJ/EMBL/GenBank accession No. AC004079; genome sequencing centre, Washington University) with L32 ribosomal proteins from yeast (Dabeva and Warner, 1987) and mouse (L30, Wiedemann and Perry, 1984), L7a ribosomal proteins from human (Colombo et al., 1991), S.cerevisiae (Yon et al., 1991) and Methanococcus jannaschii (Bult et al., 1996), and HS6 ribosomal protein from Sulfolobus solfataricus (Sensen et al., 1996). The alignment was obtained with the MultAlin program (Corpet, 1988). Only the region of a given protein displaying significant homologies with all the others is shown. (B) Nop10p is highly conserved. Saccharomyces cerevisiae Nop10p was aligned with its counterpart from C.elegans (Wilson et al., 1994) and mouse/human (EST data). The alignment was obtained with the MultAlin program (Corpet, 1988). ! = I or V; # = D, E, N or Q. Download figure Download PowerPoint To confirm the specific association between Gar1p, Nhp2p and Nop10p, we repeated the affinity purification using an extract prepared from a wild-type strain expressing a Nhp2p protein tagged at its C-terminus with the two synthetic IgG-binding domains derived from S.aureus protein A. This Nhp2p–ZZ fusion rescues perfectly an nhp2::TRP1 knock-out (data not shown) showing that it is functional. Apart from contaminating IgGs (indicated by asterisks next to lane 2 in Figure 1), four major polypeptides are specifically coeluted with Nhp2p–ZZ. One of these was unambiguously identified as Gar1p by Western blot analysis (not shown). The upper polypeptide of apparent mol. wt 65 kDa is very probably Cbf5p (Figure 1, lane 2, unfilled arrow). The polypeptides of 22 and 10 kDa apparent mol. wt precisely comigrate with, and thus almost certainly are, wild-type Nhp2p and Nop10p, respectively (Figure 1, compare lanes 1 and 2). Thus, we conclude that Gar1p, Nhp2p and Nop10p are specifically associated with each other. Nhp2p and Nop10p are essential, conserved proteins present in the dense fibrillar component of the nucleolus Nhp2p was previously identified in a search for novel high mobility group (HMG)-like proteins in Saccharomyces cerevisiae (Kolodrubetz et al., 1988; Kolodrubetz and Burgum, 1991) and its gene was shown to be essential (Kolodrubetz and Burgum, 1991). Even though Nhp2p shares physico-chemical properties with HMG proteins, it displays no significant sequence homology with them (Kolodrubetz and Burgum, 1991). However, these authors noted that Nhp2p displays significant homologies with the L7a ribosomal protein from rat. Moreover, Vilardell and Warner (1997) have noted that Nhp2p shares homologies with the S.cerevisiae L32 ribosomal protein, which is an RNA-binding protein, and predicted that Nhp2p would bind RNA as well. We also conducted a comprehensive BLAST search to identify Nhp2p homologues. The best matches were to two human proteins; one is encoded by the gene WUGSC:H DJ0167F23.5 (genome sequencing, Washington University, St Louis, MO; DDBJ/EMBL/GenBank accession No. AC004079), the other by the NHP2L1 gene (Saito et al., 1996). The former protein displays over a 77 amino acid C-terminal segment 49% identity (72% homology) with yeast Nhp2p. The protein encoded by NHP2L1 displays 38% identity with yeast Nhp2p. Although these proteins are clearly highly related to yeast Nhp2p, we suspect that neither of them correspond to its bona fide human counterparts. We have aligned together Nhp2p, its two related human proteins, and HS6, L32 and L7a ribosomal proteins from various organisms (Figure 2A). These proteins display homologies over a region of 53 amino acids, which in S.cerevisiae L32 has been implicated in RNA binding (Vilardell and Warner, 1997). Most notable is the conservation of spacing between blocks of homologous amino acids. These proteins probably constitute a novel family of RNA binding polypeptides. To determine whether the ORF encoding Nop10p is essential, a nop10::TRP1 knock-out allele was integrated in a trp1– diploid strain and tetrads obtained from nop10::TRP1/NOP10 diploids were dissected. In all cases, only two spores from each tetrad gave rise to colonies which were unable to grow on a medium lacking tryptophan (data not shown), showing that the NOP10 gene is essential. To identify Nop10p homologues, we again carried out a comprehensive BLAST search. Nop10p displays 56% identity (74% homology) with a putative protein from Caenorhabditis elegans (Wilson et al., 1994) (Figure 2B). Moreover, numerous protein sequences derived from ESTs from mouse and human sources display 60% identity (70% homology) with yeast Nop10p. The vast majority of mouse and human ESTs code for the same polypeptide and contain start and stop codons. In addition, in the human case, the third in-frame codon upstream of the putative initiator methionine codon is a stop. We therefore believe that the mouse/human protein sequence deduced from ESTs is both correct and full length. We conclude that Nop10p is a highly conserved factor. To determine the subcellular localization of Nhp2p, we made use of the strain expressing Nhp2p–ZZ (see above). For the same purpose, we produced a strain expressing Nop10p tagged at its C-terminus with the two ZZ IgG-binding domains. Immunodetection of Nhp2p–ZZ (Figure 3B) and Nop10p–ZZ (Figure 3C) by electron microscopy shows that both proteins are present in the dense fibrillar component of the nucleolus. Wild-type Gar1p (I.Léger-Silvestre, S.Trumtel, J.Noaillac-Depeyre and N.Gas, submitted) and Gar1p–ZZ (Figure 3A) show the same localization. In addition, some Nhp2p–ZZ and Nop10p–ZZ molecules can also be found in the nucleoplasm. Figure 3.Nhp2p and Nop10p are found in the DFC of the nucleolus. Immunolocalization of Gar1p–ZZ (A), Nhp2p–ZZ (B) and Nop10p–ZZ (C) by electron microscopy. Tagged proteins are detected by treatment with anti-protein A antibodies followed by incubation with colloidal gold conjugated protein A. The positions of some gold particles in the dense fibrillar component of the nucleolus are indicated by arrowheads. No, nucleolus; Nu, nucleoplasm. Download figure Download PowerPoint Nhp2p and Nop10p are specifically associated with H/ACA snoRNAs The fact that Nhp2p and Nop10p are nucleolar proteins specifically associated with Gar1p strongly suggested that they are components of H/ACA snoRNPs. To confirm this, the association of Nhp2p–ZZ and Nop10p–ZZ with H/ACA snoRNAs was tested by immunoprecipitation experiments using IgG–Sepharose. Extracts were prepared under nondenaturing conditions from strains expressing either Nhp2p–ZZ or Nop10p–ZZ and, as controls, from strains expressing ZZ–Nop1p, Gar1p–ZZ, Cbf5p–ZZ or free ZZ domains. RNAs purified from the initial extracts or from the pellets following immunoprecipitations were either 3′ end-labelled with [32P]pCp and separated on a sequencing gel (Figure 4A) or used in Northern blot experiments using probes specific for a number of H/ACA or C/D snoRNAs (Figure 4B). All members of the family of H/ACA snoRNAs are specifically coprecipitated with Nhp2p–ZZ or Nop10p–ZZ at 500 mM KAc (Figure 4A, lanes 8 and 10). This was confirmed by the Northern blot results (see Figure 4B). Phosphoimager quantitation of the Northern blots revealed that the efficiency of precipitation of H/ACA snoRNAs with either tagged protein ranges from ∼25 to 35% of input snoRNA at 500 mM KAc. In contrast, only a weak association of Nhp2p–ZZ or Nop10p–ZZ with C/D snoRNAs was detected at 150 mM KAc, which was even reduced when the salt concentration was increased (Figure 4B, compare lanes 2 and 3, 5 and 6, panels U24 and U14). At 500 mM KAc, at the most, 2% of input C/D snoRNAs are precipitated with either Nhp2p–ZZ or Nop10p–ZZ. Similar results were obtained with Gar1p–ZZ (Ganot et al., 1997a). We conclude that Nhp2p and Nop10p are bona fide components of H/ACA snoRNPs. Figure 4.Nhp2p and Nop10p interact with all H/ACA snoRNAs. (A) Patterns of 3′ end-labelled snoRNAs associated with Nhp2p–ZZ or Nop10p–ZZ. Yeast extracts were obtained from cells expressing ZZ-Nop1p (lanes 1 and 2), Cbf5p–ZZ (lanes 3 and 4), Gar1p–ZZ (lanes 5 and 6), Nhp2p–ZZ (lanes 7 and 8), Nop10p–ZZ (lanes 9 and 10) or the two ZZ domains alone (lanes 11 and 12). RNAs purified from the total extracts (T, lanes 1, 3, 5, 7, 9 and 11) or from the pellets obtained after immunoprecipitations performed in 500 mM KAc (P, lanes 2, 4, 6, 8, 10 and 12) were 3′ end-labelled with [32P]pCp and separated on a 6% sequencing gel. Positions of some of the C/D and H/ACA snoRNAs inferred from their size are indicated. M, molecular weight markers (pBR322 digested with HaeIII–TaqI). (B) Detection by Northern analysis of snoRNAs associated with Nhp2p–ZZ or Nop10p–ZZ. Yeast extracts were obtained from cells expressing Nhp2p–ZZ (lanes 1–3) or Nop10p–ZZ (lanes 4–6). RNAs purified from the total extracts (T, lanes 1 and 4) or from the pellets obtained after immunoprecipitations performed in 150 mM KAc (P150, lanes 2 and 5) or 500 mM KAc (P500, lanes 3 and 6) were separated by PAGE, transferred onto nylon membranes and hybridized with various antisense oligodeoxynucleotide probes detecting various snRNAs. Note that the amount of RNA loaded in the ‘T’ lanes corresponds to one-tenth of the input extract used in the immunoprecitations. Download figure Download PowerPoint Nhp2p and Nop10p are required for pre-rRNA processing The two previously characterized components of H/ACA snoRNPs, Gar1p and Cbf5p, are necessary for pre-rRNA processing (Girard et al., 1992; Cadwell et al., 1997; Lafontaine et al., 1998). This is, at least partly, a consequence of the inactivation of the snR10 and snR30 snoRNPs. If Nhp2p and Nop10p are also essential to the function of H/ACA snoRNPs, they should display pre-rRNA processing defects. To test this, strains conditionally expressing Nhp2p or Nop10p were constructed (see Materials and methods). These strains contain NHP2 or NOP10 chromosomal alleles transcribed from a pGAL1-10/CYC1 hybrid promoter, which is induced by galactose but repressed by glucose. To be able to follow the depletion of either protein, strains containing NHP2–ZZ or NOP10–ZZ chromosomal alleles transcribed from the pGAL1-10/CYC1 promoter were also constructed. The pGAL-NHP2, pGAL-NHP2–ZZ, pGAL-NOP10 and pGAL-NOP10–ZZ strains can be propagated on medium containing galactose. Following transfer to glucose-containing medium, however, their growth was impaired. The doubling time of these strains, which behaved similarly, increased significantly after 16 h of growth on glucose to reach a value of 12–16 h after 48 h, whereas these strains have a doubling time of 3 h on galactose (data not shown). The decline in growth rate was accompanied by the gradual disappearance of Nhp2p–ZZ or Nop10p–ZZ which became almost undetectable after 16 h (data not shown). Northern blot experiments were performed with RNAs prepared from the conditional-lethal strains grown in permissive galactose-containing medium or shifted to non-permissive glucose-containing medium for from 4 h up to 48 h. The Northern blots were hybridized with several oligonucleotide probes detecting mature rRNAs or pre-rRNA processing intermediates (see Figure 5A for a cartoon of the processing pathway, and 5B for the Northern blot autoradiographs). The disappearance of Nhp2p or Nop10p is correlated with a very strong accumulation of the 35S pre-rRNA and the appearance of an aberrant 23S intermediate (Figure 5B) which extends from the 5′ end of the 5′ external transcribed spacer to cleavage site A3 in internal transcribed spacer 1. In contrast, the levels of the 32S and 27SA2 intermediates are strongly diminished (Figure 5B). Steady-state levels of the 20S intermediate are also strongly reduced in Nop10p-depleted cells but less so in Nhp2p-depleted cells (Figure 5B, panel 20S, compare lanes 6–9 with lanes 15–18). Probably as a direct consequence of this, mature 18S rRNA levels are more diminished in the former than in the latter case (Figure 5B, panel 18S, compare lanes 6–9 with lanes 15–18). Contrary to what is seen for the 32S, 27SA2 and 20S intermediates, no obvious reduction in the levels of the 27SB and 7S species can be detected in cells lacking Nhp2p or Nop10p (Figure 5B). Primer extensions were performed with the RNA samples used for the Northern blot experiments. Results of primer extension experiments show that cleavages at sites A0, A3, B1(L) and B1(S) occur at the normal positions (data not shown). They also confirm the disappearance of the 27SA2 intermediate as indicated by the progressive reduction in the levels of the cDNA products extending to site A2 (data not shown). Figure 5.pre-rRNA processing defects detected in cells lacking Nhp2p or Nop10p. (A) Schematic representation of the pre-rRNA processing pathway. The structure of the 35S pre-rRNA is depicted above. The positions of the oligonucleotide probes used in Northern analyses and primer extension experiments are indicated by small filled boxes. In wild-type cells, the 35S pre-rRNA is first cleaved at site A0 within the 5′ external transcribed spacer (5′ ETS) producing intermediate 33S which is very rapidly cleaved at site A1, the 5′ end of 18S rRNA, to produce intermediate 32S. 32S is then cleaved at site A2 within the internal transcribed spacer 1 (ITS1) releasing 20S, the immediate precursor to 18S rRNA, and 27SA2. 27SA2 is then processed via two alternative pathways. It is either cut at site A3 to produce 27SA3, which is then trimmed by 5′ to 3′ exonucleases up to site B1(S), producing 27SB(S). Alternatively, it can be processed into 27SB(L) by an as yet unknown mechanism. 27SB(S) and 27SB(L) are then processed in the same manner to produce 25S and 5.8S(S) or 5.8S(L), respectively. In cells lacking Nhp2p or Nop10p, cleavages at sites A1 and A2 are inhibited, as revealed by the diminished levels of the 32S, 27SA2 and 20S intermediates. 35S is instead directly cut at site A3, producing 23S which is degraded. For other details and references, see the review by (Venema and Tollervey, 1995). (B) Detection by Northern analysis of pre-rRNA processing intermediates produced in cells progressively depleted of Nhp2p or Nop10p. Total RNAs were extracted from wild-type cells (WT, lanes 1–3 and 10–12) or from cells possessing NHP2 (GAL::nhp2, lanes 4–9) or NOP10–ZZ (GAL::nop10, lanes 13–18) alleles transcribed from a hybrid GAL-CYC1 promoter. Cells were grown in galactose- (Gal, lanes 1, 4, 10 and 13) or in glucose-containing medium (Glu, lanes 2, 3, 5–9, 11, 12, 14–18) for the times indicated. Total RNAs were separated on 1% agarose–formaldehyde gels to detect the 35S, 32S, 27SA2, 27SB, 25S, 23S, 20S and 18S species, or on 8% polyacrylamide gels to detect the 7S(L), 7S(S), 5.8S(L) and 5.8S(S) species. The 35S and 32S species were revealed by probe b, 27SA2 and 27SB by probe e, 23S and 20S by probe b, 25S by probe f, 18S by probe a, 7S(L) and 7S(S) by probe e, and 5.8S(L) and 5.8S(S) by probe d. Download figure Download PowerPoint Taken together, these results indicate that the main pre-rRNA processing defects occurring in cells depleted of Nhp2p or Nop10p are inhibitions of cleavages at sites A1 and A2, whose consequences are an inhibition of 18S rRNA production. Cleavage at site A0 is also at least delayed since we observe an accumulation of the 23S intermediate. Cells depleted of Nhp2p or Nop10p are defective in pre-rRNA pseudouridylation Most H/ACA snoRNAs function as guides selecting uridi
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