Npa1p, a Component of Very Early Pre-60S Ribosomal Particles, Associates with a Subset of Small Nucleolar RNPs Required for Peptidyl Transferase Center Modification

Abstract

We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p. In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely. Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles. By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor. Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center. Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.

Synthesis of the 40S and 60S ribosomal subunits in eukaryotes is a particularly intricate process that involves the synthesis of four ribosomal RNAs, their assembly with close to 80 ribosomal proteins, and transport of preribosomal particles from the nucleus to the cytoplasm where translation occurs (30, 56, 76, 98). Eukaryotic ribosome synthesis requires the action of RNA polymerases I, II, and III that, respectively, transcribe a long common precursor to the 18S, 5.8S, and 25S rRNAs, the pre-mRNAs of ribosomal proteins, and the precursor to 5S rRNA. The pre-rRNA transcribed by RNA polymerase I contains, in addition to the sequences retained in the mature cytoplasmic ribosomes, long spacer regions that will be removed by a complex series of endo- and exonucleolytic cleavage steps (for a schematic representation of the pre-rRNA processing steps, see Fig. 4C). Moreover, specific nucleotides of rRNAs will undergo posttranscriptional chemical modification. By far the two most common modifications are the conversion of uridines into pseudouridines and the methylation of the oxygen at the 2′ position of ribose moieties. These modifications are carried out by box H/ACA and box C/D small nucleolar RNPs (snoRNPs), respectively (4, 26, 28, 49, 50, 86). Although the precise functions of modified nucleotides in rRNAs are not known, they are believed to significantly contribute to ribosome function since they are present in the most highly conserved and functionally important regions of rRNAs (15). Indeed, M. Fournier and collaborators have recently shown that lack of a single pseudouridine within the peptidyl transferase center, due to alteration of the box H/ACA snoRNP that produces this pseudouridine, is correlated with a substantial reduction in translational activity (48). Likewise, Bonnerot and colleagues have shown that the absence of 2′-O-ribose methylation of U2918 within the peptidyl transferase center increases sensitivity to paromomycin, an A site antibiotic (9).

FIG.4.

Analysis of pre-rRNA processing defects in Npa1p-depleted cells. Levels of various pre-rRNAs or mature rRNAs were assessed by Northern blotting (A) or primer extension analysis (B). Experimental details are identical to those described in the legend of Fig. 1B except that in panel A, left-hand side, RNA samples were separated on a denaturing 1% agarose gel and that in panel B cDNA products were separated on a 6% sequencing gel. Signal intensities were measured by phosphorimager scanning, and values obtained (indicated below each lane) were normalized by using those for MRP RNA (Fig. 1B) as internal standards. (C) Pre-rRNA processing scheme in S. cerevisiae. The primary pre-rRNA transcript is first cleaved, maybe cotranscriptionally, by the Rnt1p endonuclease in the 3′ external transcribed spacer at site B0, producing the 35S pre-rRNA. 35S is cleaved at site A0, producing 33S, which is then cleaved at site A1 corresponding to the 5′ end of mature 18S rRNA, producing 32S. Cleavage of 32S at site A2 in internal transcribed spacer 1 (ITS1) generates 20S, precursor to 18S rRNA, and 27SA2, precursor to 25S and 5.8S rRNAs. 20S is exported to the cytoplasm, where it undergoes endonucleolytic cleavage at site D, generating mature 18S rRNA. 27SA2 can be processed by one of two parallel pathways. Ninety percent of 27SA2 molecules are cut at site A3 by the MRP endonuclease producing 27SA3, the 5′ end of which is digested by the Rat1p and Xrn1p exonucleases up to B1S, producing 27SBS. Ten percent of 27SA2 molecules are processed by an as yet unknown mechanism at site B1L, releasing 27SBL. 27SBS and 27SBL are then processed identically. C2 cleavage releases the 26S and 7SS or 7SL molecules. The 5′ end of 26S is digested by the Rat1p and Xrn1p exonucleases up to the 5′ end of mature 25S rRNA. The ITS2 fragment remaining on 7SS or 7SL molecules is removed in successive steps by several exonucleases. The core exosome intervenes first to produce 5.8S+30S or 5.8S+30L, followed by the Rrp6p exosome component generating 6SS or 6SL molecules. The remaining ITS2 nucleotides are removed by the Rex1p, Rex2p, and Ngl2p exonucleases. The endonucleases acting at sites A0, A1, A2, and C2 are unknown.

Pre-rRNA processing does not occur on naked RNA molecules. Pre-rRNA molecules at all stages of maturation are embedded within so-called preribosomal particles that contain a specific subset of ribosomal and nonribosomal proteins. The existence of preribosomal particles was first demonstrated by pioneering studies from the groups led by J. Warner and R. Planta as far back as the 1960s (89, 90, 93, 94, 101). These teams proposed that the pre-rRNA is first assembled into a 90S preribosomal particle that gives rise to the 43S and 66S preribosomal particles, precursors to the mature 40S and 60S ribosomal subunits, respectively. However, it was not until two years ago, with the use of the tandem affinity purification (TAP) protocol (77) and the advent of very sensitive mass-spectrometric techniques to identify polypeptides, that the composition of various preribosomal particles could start being determined (5, 18, 22, 34, 36, 37, 68, 80-82). Such studies have revealed that well over 120 proteins transiently associate with pre-rRNAs, and they have led to a refined, and certainly incomplete, description of the succession of preribosomal particles (27, 30, 61, 91, 100). Assembly of the 90S preribosomal particle is initiated by the cotranscriptional association of the so-called “U3 processome” with the nascent PolI-transcribed pre-rRNA (18). U3 processome bound to nascent pre-rRNA can be visualized by electron microscopy on Miller spreads of rDNA chromatin as the terminal balls at the tips of pre-rRNA branches (18, 64). Crucial to the association of the U3 processome with the pre-rRNA is the ability of the U3 snoRNA component to base pair with the 5′ external transcribed spacer of the pre-rRNA (6, 7). In addition to the U3 processome, 90S particles also contain other nonribosomal proteins, numerous C/D and H/ACA snoRNPs involved in pre-rRNA cleavage and/or nucleotide modification, and most ribosomal proteins of the 40S ribosomal subunit (34, 36, 82). Strikingly, the 90S particle(s) that has been purified seems to lack most large subunit ribosomal proteins and all nonribosomal factors required for 60S subunit formation, except Rrp5p (which is required for the production of both subunits) and modifying snoRNPs. This finding was rather unexpected although it offered an explanation for the fact that the inactivation of factors involved in 40S ribosomal subunit biogenesis does not block 60S ribosomal subunit synthesis.

The early pre-rRNA endonucleolytic cleavages at sites A0 and A1 occur within maturing 90S preribosomal particles, the precise composition of which still remains to be defined. It has been proposed that these cleavages release a substantial number of factors that remain bound to the cleaved off 5′ external transcribed spacer fragments (82). This may, at least in part, explain why the pre-40S particles that evolve following pre-rRNA cleavage at site A2 have lost most of the nonribosomal factors found in 90S particles. A2 cleavage also releases the first pre-60S particle. The composition of the pre-60S particles purified so far is radically different from that of the known 90S particles (5, 22, 37, 68, 80, 81). As they mature, pre-60S particles migrate from the nucleolus to the nucleoplasm (60), and their content of nonribosomal factors becomes simpler (68, 81).

Translocation across the nuclear pores of export-competent pre-60S and pre-40S particles depends upon the Ran GTPase cycle and the export receptor Crm1p/Xpo1p that binds to the nuclear export signal (31, 35, 40, 65, 66). In the case of pre-60S particles, at least one link with Crm1p/Xpo1p is provided by the nuclear export signal-containing Nmd3p protein that interacts with large subunit ribosomal protein Rpl10p (31, 39, 40). Final maturation events take place in the cytoplasm to yield mature functional ribosomal subunits (24, 83, 96, 97).

Although we now have an overall idea of the succession of preribosomal particles, the fine details still need to be worked out. Concerning the early pre-60S pathway, for example, the picture remains blurred because our knowledge is derived from purification experiments performed with bait proteins that interact with several distinct pre-rRNAs that are derived from one another. Hence, so far mostly rather broad mixtures of pre-60S particles have probably been purified.

In this study, we characterized an as yet unknown nucleolar factor, Npa1p (for nucleolar preribosomal-associated), that interacts predominantly with the 27SA2 pre-rRNA. Therefore, we propose that the pre-60S particles purified by using Npa1p as bait correspond to some of the earliest pre-60S particles.

MATERIALS AND METHODS

Strains, media, and plasmids.

Strains GAL::npa1 and GAL::zz-npa1 were obtained as follows. Two gene cassettes flanked on the 5′ side by a segment of the NPA1 promoter and on the 3′ side by the 5′ segment of the NPA1 open reading frame and containing either the HIS3 gene marker and the GAL10 promoter (cassette 1) or the same elements followed by the ZZ-tag sequence (cassette 2) were amplified by PCR by using plasmid pTL26 (55) and oligonucleotides gal-YKL014C/1 (5′-TGTAGACGAAATATGAAAAATTTCAGCAATAAAGCTCATCGCAAAGAATAGTTCCTCTTGGCCTCCTCTAGT-3′) and gal-YKL014C/2 (5′-GAGATCCATAGGCTTCGCTATGATTACTCATTATGTAAGTGCTCCTCTAGTCGAATTCCTTGAATTTTCAAA-3′) (cassette 1) or plasmid pTL27 (55) and oligonucleotides gal-YKL014C/1 and galZZ-YKL014C/2 (5′-GTACTTCTCCCTTCTCTGGTCGCGAGATCCATAGGCTTCGCTATGATTACTCATATTCGCGTCTACTTTCGG-3′) (cassette 2). Cassettes 1 and 2 were integrated into strain YDL402 (55), creating strains GAL::npa1 and GAL::zz-npa1, respectively.

Strains expressing Npa1p-TAP or Npa1p-ZZ used for the immunoprecipitation experiments, the immunolocalization by electron microscopy, and the analysis of the sedimentation profile of Npa1p on a glycerol gradient were produced as follows. Two gene cassettes flanked on the 5′ side by the last 48 nucleotides of the NPA1 open reading frame and on the 3′ side by a segment of the NPA1 terminator and containing either the TAP-tag (cassette 3) or only the ZZ-tag (cassette 4) sequence followed by a TRP1 marker from Kluyveromyces lactis were amplified by PCR by using plasmid pBS1479 (77) and oligonucleotides TAP-YKL014C/1 (5′-GCTAATATTATGGACAGAAGGTGATAGCGACAATGTTGTCAAGAGGCTACGTAAATCCATGGAAAAGAGAAG-3′) and TAP-YKL014C/2 (5′-TTATACATTTCGCACATTATATAGAAAAGTGGACATTTAATTCTTCAAATCTTATTACGACTCACTATAGGG-3′) (cassette 3) or oligonucleotides ZZ-YKL014C/1 (5′-GCTAATATTATGGACAGAAGGTGATAGCGACAATGTTGTCAAGAGGCTACGTAAAGAGCTCAAAACCGCGGC-3′) and TAP-YKL014C/2 (cassette 4). Cassettes 3 and 4 were integrated into strain Y0341 (pra1-1 prb1-1 prc1-1 cps1-3 Δhis3 leu2-3,112 Δura3 Δtrp1::LEU2) creating npa1::TAP and npa1::ZZ, strains respectively.

Strains expressing Krr1p-TAP or Ssf1p-TAP were constructed as described for Npa1p-TAP by using oligonucleotides TAP-Krr1/1 (5′-GCAAAAGATTTCATAGCTCCGGAAGAAGAAGCATACAAGCCAAACCAAAATTCCATGGAAAAGAGAAG-3′), TAP-KRR1/2 (5′-AATTTCTTTCACTTTACAAACATATCTAAGTAAACGAAATGTGTGTGTGTTTCTATACGACTCACTATAGGG-3′), Ssf1-TAP/1 (5′-ATGGTAGCGTACCAGAGGATCTAGATAGTGACTTATTTAGTGAGGTCGAATCCATGGAAAAGAGAAGA-3′), and Ssf1-TAP/2 (5′-TGCGTTGGTGGATAGCCAGGCTTAACTAAAATTTTCTTGGTACCGGAGAATACGACTCACTATAGGG-3′).

A strain expressing Npa1p-green fluorescent protein (GFP) was constructed as previously described (58) by using plasmid pFA6a-GFP(S65T)-TRP1 and oligonucleotides YKL-GFP/1 (5′-GCTAATATTATGGACAGAAGGTGATAGCGACAATGTTGTCAAGAGGCTACGTAAACGGATCCCCGGGTTAATTAA-3′) and YKL-GFP/2 (5′-TTATACATTTCGCACATTATATAGAAAAGTGGACATTTAATTCTTCAAATCTTATGAATTCGAGCTCGTTTAAAC-3′). ThePCR product obtained was transformed into strain Y0341, producing strain Y0341-npa1::GFP.

Saccharomyces cerevisiae strains were grown either in YP medium (1% yeast extract, 1% peptone) supplemented with either 2% galactose, 2% raffinose, 2% sucrose or 2% glucose as carbon sources or in YNB medium [0.17% yeast nitrogen base, 0.5% (NH₄)₂SO₄] supplemented with 2% galactose, 2% raffinose, 2% sucrose, and the required amino acids.

Fluorescence microscopy and immunoelectron microscopy.

Y0341-npa1::GFP cells were treated as previously described (10). Detection of Npa1p-ZZ by immunoelectron microscopy was performed as described by Henras et al. (38).

Immunoprecipitations.

Total cellular extracts were produced from strains expressing either Npa1p-ZZ, Npa1p-TAP, Krr1p-TAP, Ssf1p-TAP, or no tagged protein. Cells frozen in liquid nitrogen were broken with dry ice in a kitchen blender (Osterizer). Aliquots of broken cell powder corresponding to 2 × 10¹⁰ cells were resuspended in 2 ml of 20 mM Tris-HCl (pH 8.0), 5 mM MgAc, 0.2% Triton X-100, 200 mM potassium acetate (KAc), 1 mM dithiothreitol (DTT), 0.5 U of RNasin (Promega) per μl, and protease inhibitors. Extracts were clarified by centrifuging for 10 min at 16,000 × g in a microcentrifuge (Eppendorf 5415D).

For protein analysis, aliquots of extracts corresponding to 200 mg of proteins were added to 200 μl of immunoglobulin G (IgG)-Sepharose beads fast flow (Amersham Biosciences) in a 5-ml final volume of a buffer containing 20 mM Tris-HCl (pH 8.0), 5 mM MgAc, 0.2% Triton X-100, 200 mM KAc, 1 mM DTT, 0.5 U of RNasin (Promega) per μl, and protease inhibitors. Immunoprecipitation was performed at 4°C for 1 h and 30 min on a shaking table. Beads were then washed five times with 10 ml of the buffer used for the immunoprecipitation without RNasin (ice cold), once with 10 ml of TEV cleavage buffer (10 mM Tris-Cl [pH 8.0], 200 mM NaCl, 0.1% NP-40, 0.5 mM EDTA, and 1 mM DTT) and incubated for 2 h at 16°C with 100 U of TEV enzyme (Invitrogen) in 1 ml of TEV cleavage buffer. Eluted proteins were precipitated with trichloroacetic acid, separated on 8, 12, and 15% polyacrylamide-sodium dodecyl sulfate (SDS) gels, and identified by mass spectrometry (MS).

For RNA analysis, aliquots of extracts corresponding to 12 mg of proteins were added to 50 μl of IgG-Sepharose beads (Amersham Biosciences) in a 1-ml final volume of a buffer containing 20 mM Tris-HCl (pH 8.0), 5 mM MgAc, 0.2% Triton X-100, 200 mM KAc, 1 mM DTT, 0.5 U of RNasin (Promega) per μl, and protease inhibitors. Immunoprecipitation was performed at 4°C for 1 h and 30 min on a shaking table. Beads were then washed seven times with 1 ml of the buffer used for the immunoprecipitation (ice cold). A total of 160 μl of 4 M guanidinium isothiocyanate solution, 4 μl of glycogen, 80 μl of 100 mM NaAc (pH 5), 10 mM Tris-HCl (pH 8.0), 1 mM EDTA solution, 120 μl of phenol, and 120 μl of chloroform was added to the beads. The samples were thoroughly mixed, incubated for 5 min at 65°C, and centrifuged for 5 min at 4°C and 16,000 × g in a microcentrifuge (Eppendorf 5415D). The aqueous phases were recovered, mixed with 120 μl of phenol and 120 μl of chloroform, and the samples were centrifuged for 5 min at 4°C and 16,000 × g in a microcentrifuge (Eppendorf 5415D). RNAs from the aqueous phases were then precipitated with ethanol.

MS.

Coomassie blue-stained bands were subjected to in-gel tryptic digestion by using modified porcine trypsin (Promega). The tryptic digests were analyzed by online capillary high-performance liquid chromatography (LC Packings, Dionex, Amsterdam, The Netherlands) coupled to a nanospray LCQ Deca ion trap mass spectrometer (ThermoFinnigan, San Jose, Calif.). Peptides were separated onto a PepMap C18 column (internal diameter, 75 μm; length, 15 cm) (LC Packings) after loading onto a PepMap C18 precolumn (internal diameter, 300 μm; length, 5 mm). The flow rate was set at 150 nl/min. Peptides were eluted by using a 0 to 40% linear gradient of solvent B for 40 min (solvent A was 0.1% formic acid in 5% acetonitrile, and solvent B was 0.1% formic acid in 90% acetonotrile). The mass spectrometer was operated in positive ion mode at a needle voltage of 1.9 kV and a capillary voltage of 30 V. Data acquisition was performed in a data-dependent mode consisting of, alternatively in a single run, a full-scan MS over the range m/z 370 to 2,000 and a full-scan tandem MS (MS/MS) in an exclusion dynamic mode. MS/MS data were acquired by using a three m/z unit isolation window and a relative collision energy of 35%. The SEQUEST Browser software was used for protein identification by searching against S. cerevisiae entries from SwissProt with MS/MS spectra.

Fractionation of yeast extract on glycerol gradient.

A total cellular extract was prepared as described in “Immunoprecipitations” from the strain expressing Npa1p-ZZ. A total of 500 μl of extract corresponding to 5 mg of proteins was loaded on a 10 to 30% glycerol gradient. Preparation of the gradient, loading of the extract, centrifugation, and collection of fractions were performed as described previously (10).

Western analysis.

Proteins from total extracts produced as previously described (17) or obtained from gradient fractions after trichloroacetic acid precipitation or from immunoprecipitated pellets were separated on 12% polyacrylamide-SDS gels and transferred to Hybond-C extra membranes (Amersham Biosciences). ZZ-tagged Npa1p, Gar1p, and Nhp2p and ribosomal proteins L3 and S8 were detected as described elsewhere (17).

RNA extractions, Northern hybridizations, and primer extensions.

RNA extractions were performed as described by Tollervey and Mattaj (88). RNA fractionations by agarose or polyacrylamide gel electrophoresis were performed as described by Henras et al. (38). Primer extensions were performed as described previously (10).

Pre-rRNA precursors, mature rRNAs, and various small RNAs were analyzed by Northern hybridization or primer extensions by using ³²P-labeled oligodeoxynucleotide probes. Sequences of antisense oligonucleotides used to detect these RNAs have been reported previously (16, 17, 38, 75), except oligonucleotides to detect the following: 25.5S (5′-TTAAGAACATTGTTCGCCTA-3′), 6S (5′-TGAGAAGGAAATGACGCT-3′), 5.8S+30 (5′-ACTCACTACCAAACAGAATG-3′), snR3 (5′-CGAATAAGACCGAGTGTTCA-3′), snR35 (5′-CCGATGGACTTGACGCTTATACC-3′), snR52 (5′-GTATCAGAGATTGTTCACGC-3′), snR55 (5′-ATGGTGATGCATGATGTAATCC-3′), snR69 (5′-TTTATAGCATTGTCACTAAG-3′), snR70 (5′-CATCAATTCTCCACTAAAGAAC-3′), and snR73 (5′-GGCGAAATATCATCAAAGTT-3′). Blots were hybridized with 5′ end-labeled oligonucleotide probes and washed as described previously (38).

Pulse-chase analyses.

Cells were grown to an optical density of ∼0.4 in rich (YP) media and were then shifted to minimal (YNB) media for 4 h. To 9-ml samples, 450 μCi [³H]methyl-methionine was added. After 3 min of labeling, 0.9 ml of 0.1 M methionine was added, and 1-ml samples were collected at 1, 2, 5, 10, 20, 40, 60, 90, and 120 min following the addition of cold methionine.

RESULTS

Npa1p is a conserved nucleolar protein required for normal accumulation of 25S and 5.8S rRNAs.

We have been performing database searches for factors that might specifically interact with H/ACA snoRNPs to promote their assembly, trafficking, and/or activity. From this search, we selected two proteins that seemed likely to perform such a function(s), namely Naf1p and a protein encoded by the YKL014c open reading frame. Naf1p was reported to interact in a double-hybrid screen with two components of H/ACA snoRNPs, Cbf5p and Nhp2p (46), and to be coprecipitated with Flag-tagged Cbf5p (41). The protein encoded by the YKL014c open reading frame was reported to be coprecipitated with two tagged components of H/ACA snoRNPs, Nhp2p-TAP and Gar1p-TAP (34). It was also coprecipitated with four components of pre-60S complexes (34). Functional study of Naf1p demonstrated that it is required for the normal accumulation of H/ACA snoRNP components and that it could be involved in early H/ACA snoRNP assembly steps (17, 23, 104). These results prompted us to investigate whether the protein encoded by the YKL014c open reading frame, thereafter termed Npa1p (for nucleolar preribosomal-associated; see below), was also required for normal accumulation of H/ACA snoRNP components. To obtain yeast cells lacking Npa1p, we placed the essential NPA1 gene under the control of the GAL1-10 promoter which is induced in the presence of galactose and repressed when glucose is added to the growth medium. In addition, in order to be able to follow Npa1p depletion, a gene cassette (ZZ) encoding two IgG-binding domains derived from Staphylococcus aureus protein A was inserted in frame just upstream from the NPA1 open reading frame. Npa1p depletion was obtained by transferring GAL::zz-npa1 cells from galactose- to glucose-containing medium. As shown in Fig. 1A, Npa1p becomes almost undetectable by Western blot analysis after 12 h of growth in glucose-containing medium. Npa1p depletion does not lead to a reduction in the levels of Gar1p (Fig. 1A), in contrast to what is observed in cells lacking Naf1p (17). Moreover, H/ACA snoRNA levels are not diminished following Npa1p depletion (Fig. 1B), unlike what is seen in Naf1p-depleted cells (17, 23, 104). In fact, the signals detected for all small RNAs tested seem to increase. As equal amounts of total cellular RNAs have been loaded in all lanes of Fig. 1B, such an effect is probably indicative of the fact that the levels of abundant RNAs, such as rRNAs, are strongly diminished. Strikingly, levels of a ribosomal protein of the large ribosomal subunit rpl3 decrease after prolonged growth of GAL::zz-npa1 cells in glucose-containing medium, suggesting that Npa1p might be involved in the biogenesis and/or stability of the large ribosomal subunit. Consistent with a role of Npa1p in ribosome biogenesis, we find that Npa1p accumulates in the nucleolus (Fig. 2), a finding confirmed in the systematic protein localization study of Huh and colleagues (45). Moreover, the putative human orthologue of Npa1p, the NNP72 protein (unigene identifier Hs18759), is also a nucleolar protein (2) (Fig. 3). Interestingly, a high-resolution study of yeast Npa1p localization by electron microscopy shows that it is found in the immediate vicinity of the dense fibrillar component, i.e., at the interface between this component and the granular component of the nucleolus (Fig. 2B).

FIG. 1.

Levels of H/ACA snoRNP components in Npa1p-depleted cells. GAL::zz-npa1 cells were grown in galactose-containing medium (lanes 1) and transferred to glucose-containing medium (lanes 2 to 5). Culture samples were collected before the transfer (lanes 1) and at 6 (lanes 2), 12 (lanes 3), 24 (lanes 4), and 48 (lanes 5) h after transfer to glucose-containing medium. From these samples, total proteins (A) or RNAs (B) were extracted and subjected to Western blot (A) or Northern blot (B) analysis. In panel A, total proteins were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a cellulose membrane. Specific proteins were detected by enhanced chemiluminescence by using either Dakko rabbit PAP, monoclonal anti-rpl3, or polyclonal anti-rpS8, anti-Nhp2p or anti-Gar1p sera. In panel B, total RNAs were separated by acrylamide gel electrophoresis and transferred to a nylon membrane. Specific RNAs were detected by hybridization with antisense oligonucleotide probes.

FIG. 2.

Npa1p is a nucleolar protein. (A) Detection of Npa1p-GFP by fluorescence microscopy. Green, Npa1p-GFP; blue, DAPI (4′,6′-diaminino-2-phenylindole). The green fluorescence is located in a crescent on one side of the nucleus, opposite the area of DAPI staining, which corresponds to the position of the nucleolus. (B) Detection of Npa1p-ZZ by immunoelectron microscopy. Photographs are centered on the nucleolus. No, nucleolus; Nu, nucleoplasm. Arrowheads point to a section of the dense fibrillar component.

FIG. 3.

Npa1p is a conserved protein. S. cerevisiae Npa1p and its probable orthologues in Saccharomyces paradoxus, Schizosaccharomyces pombe, and human were aligned by using the MultAlin program (12).

These results led us to assess the effects of Npa1p depletion on rRNA production and pre-rRNA processing by Northern blotting, primer extensions (Fig. 4), and pulse-chase analyses (Fig. 5). Npa1p depletion leads to significantly reduced 25S and 5.8S rRNA steady-state levels while 5S and 18S rRNA levels are less affected (Fig. 4). Pulse-chase analysis suggests that this is mainly due to reduced 25S and 5.8S synthesis rather than increased turnover (Fig. 5). Pulse-chase analysis also confirms that 25S production is significantly reduced relative to 18S rRNA synthesis in Npa1p-depleted cells. As has been reported for many other factors required for the production of large subunit rRNAs (30, 98), Npa1p depletion leads to a strong accumulation of the 35S pre-rRNA and to an even greater accumulation of the 23S pre-rRNA (Fig. 4 and 5), indicating that early endonucleolytic cleavages at sites A0, A1, and A2 (Fig. 4C) are somewhat delayed. Probably in part as a consequence of A2 cleavage inhibition, 27SA2 pre-rRNA levels are diminished. An accumulation of the 20S pre-rRNA, the immediate precursor to 18S rRNA is also observed, suggesting that cleavage at site D is partially impaired. This may be the cause of the reduction in 18S rRNA levels. The levels of all pre-rRNAs featuring in the processing pathway leading to 5.8S and 25S synthesis, except 27SA2 pre-rRNA, display a transient increase followed by a significant decrease relative to the levels of the control MRP RNA. These data are not straightforward to interpret, but they clearly indicate that the aforementioned processing pathway is perturbed. This is highlighted by the fact that a fragment extending from the A2 to C2 cleavage sites strongly accumulates, which suggests that C2 cleavage occurs prematurely on 27SA2 pre-rRNA.

FIG. 5.

25S and 5.8S rRNA synthesis is reduced in Npa1p-depleted cells. Wild-type YDL402 cells and otherwise isogenic GAL::zz-npa1 cells were grown in galactose-containing medium and transferred for 24 h to a glucose-containing medium. Cells were pulse labeled with 540 μCi of [³H]methyl-methionine for 3 min. An excess of cold methionine was then added, and cell samples were collected at the indicated times after the addition of cold methionine. Total RNAs extracted from these samples were separated on a 1% agarose denaturing gel. Separated RNAs were transferred to a nylon membrane, and labeled RNAs were detected by fluorography.

These data suggest that Npa1p is first and foremost needed for the production of 25S and 5.8S rRNAs and that the less pronounced reduction in 18S rRNA levels detected in Npa1p-depleted cells might be an indirect consequence of the absence of Npa1p.

Npa1p is a component of 27SA2-containing preribosomal particles.

To assess whether Npa1p is present within preribosomal particles, we first analyzed the sedimentation profile of Npa1p-ZZ on a glycerol gradient. The strain used for these experiments expresses Npa1p-ZZ from the endogenous NPA1 promoter. Strikingly, a significant fraction of Npa1p-ZZ cosediments with RNA components of pre-60S ribosomal particles (Fig. 6A). Another significant fraction of Npa1p-ZZ sediments close to the top of the gradient in fractions 4 to 6. In comparison, far less Npa1p-ZZ is present within gradient fractions containing 90S preribosomal particles. To establish whether Npa1p does associate with pre-rRNA(s) found in pre-60S ribosomal particles and, if so, which pre-rRNA(s), Npa1p-ZZ was precipitated from yeast whole-cell extracts, and coprecipitating RNAs were analyzed by Northern blotting (Fig. 6B) and primer extension experiments (Fig. 6C). By far the most efficiently coprecipitating pre-rRNA is the 27SA2 molecule.

FIG. 6.

Npa1p interacts predominantly with the 27SA2 pre-rRNA. (A) Gradient analysis. A total cellular extract was prepared from Npa1p-ZZ-expressing cells and centrifuged through a 10 to 30% glycerol gradient for 10 h at 25,000 rpm in a SW41 Ti rotor. Twenty fractions were collected from the top of the gradient. Proteins and RNAs were extracted from the fractions and subjected to Western (I) and Northern (II) analysis, respectively, as described in previous legends. T, sample from the total nonfractionated extract. (B) Northern analysis of pre-rRNAs and mature rRNAs precipitated with Ssf1p-Tap (lanes 3 and 4), Krr1p-Tap (lanes 5 and 6), Npa1p-ZZ (lanes 7 and 8), or from extracts lacking a tagged protein (lanes 1 and 2). Precipitations were performed by using total cellular extracts and IgG-Sepharose. RNAs were extracted from the pellets obtained after precipitation (lanes IP) or from an amount of total extract corresponding to 1/30 of that used for the precipitation (lanes T). Signal intensities were measured by phosphorimager scanning to derive the percentage of input RNAs precipitated (indicated below each IP lane). bg, background value. (C) Primer extension analysis of pre-rRNAs precipitated with Npa1p-ZZ. Precipitation was performed as described in panel B. The percentage of input RNAs precipitated together with Npa1p-ZZ is indicated on the right of each IP lane.

These data strongly suggest that Npa1p is a specific component of the preribosomal particle(s) that contains the 27SA2 pre-rRNA and no other pre-rRNA. The composition of such a particle(s) has not been determined so far. We reasoned that the majority of Npa1p-associated polypeptides would be components of that particle(s). Hence, to determine the composition of such a particle(s), Npa1p fused to the TAP tag (Npa1p-TAP) was purified over an IgG-Sepharose column, and Npa1p-TAP-containing complexes were released by TEV protease cleavage. A second calmodulin affinity column was not employed because Npa1p-CBP (i.e., the Nap1p protein containing the CBP part of the TAP tag that remains after TEV cleavage) does not seem to bind to this column, maybe because the CBP tag is not accessible. The polypeptides released following TEV cleavage were resolved by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. Coomassie blue-stained bands were excised, and polypeptides contained in these bands were subjected to in-gel trypsin digestion. The resulting peptides were analyzed by online capillary liquid chromatography and nanospray ion trap MS/MS, allowing identification of the proteins from which they were derived (Tables 1 and 2).

TABLE 1.

Proteins associated with Npa1p-TAP

Protein	Accession no.a	MWb	Gene deletion phenotypec	Depletion phenotype	Location(s)d	Reference(s)
90S
Kre33p	P53914	119	L	40S decrease	No	36
Enp2p	P48234	81	L	ND	No	36
Kri1p	P42846	68	L	40S decrease	No	79
Utp9p	P38882	65	L	18S decrease	No	18
Nsr1p	P27476	44	V	18S decrease	No, Nu	51
90S and pre-60S
Rrp5p	Q05022	193	L	A0, A1, A2, and A3 inhibition	No	99
Nop58p	Q12499	58	L	A0, A1, and A2 inhibition	No	33
Nop56p	Q12460	56	L	A0, A1, and A2 inhibition	No	33
Cbf5p	P33322	55	L	A0, A1, and A2 inhibition	No	54
Nop1p	P15646	34	L	A0, A1, and A2 inhibition	No	87
Nhp2p	P32495	19	L	A0, A1, and A2 inhibition	No	38, 102
Pre-60S
Dbp3p	P20447	59	V	27SA2 accumulation	No	103
Dbp6p	P53734	70	L	27SA2, 27SB, 7S decrease	No	52
Dbp7p	P36120	83	V	27SA2, 27SB, 7S decrease	No	14
Dbp9p	Q06218	68	L	27SA2, 27SB, 27SA3 decrease	No	13
Drs1p	P32892	85	L	60S decrease	No, Nu	78
Has1p	Q03532	57	L	27SA3, 27SB accumulation	No	20
Mak5p	P38112	87	L	60S decrease	No, Nu	71
Prp43p	P53131	87	L	Splicing defect	No	3
Spb1p	P25582	96	L	27SA2, 27SB accumulation, 7S decrease	No, Nu	53, 74
Erb1p	Q04660	92	L	27SA2, 27SB, 7S decrease	No	73
Nop4p	P37838	78	L	27SA2, 27SB, 7S decrease	No	8, 85
Nop7p	P53261	70	L	27SA3 accumulation, 7S decrease	No	1, 69
Nop2p	P40991	70	L	27SA2, 27SB accumulation	No	42
Nug1p	P40010	58	L	60S decrease	No, Nu	5
Nop8p	Q08287	57	L	27SB decrease	No	106
Ebp2p	P36049	50	L	27SA2, 27SB decrease	No	44, 92
Fpr3p	P38911	46	V	ND	No	84
Rrp1p	P35178	33	L	27SA3 accumulation	No	21, 43
Nop15p	P53927	25	L	27SA3 accumulation	No, Nu	37, 70
Mrt4p	P33201	27	V	60S decrease	No	37, 109
Nip7p	Q08962	20	L	27SB accumulation	No, Nu	107
Noc1p	Q12176	117	L	27SA2, 27SB, 7S decrease	No, Nu	60
Noc2p	P39744	82	L	27SA2, 27SB, 7S decrease	No, Nu	60
Nsa3p	P38779	42	L	25S, 5.8S decrease	No, Nu	68, 25
Npa2p	P47108	135	L	25S, 5.8S decrease	No
Npa3p	P47122	43	L	?	Cyt

^aSwissProt database.

^bMolecular weight.

^cL, lethal; V, viable.

^dNo, nucleolar; Nu, nucleoplasmic; Cyt, cytoplasmic.

TABLE 2.

Additional proteins associated with Npa1p-TAP

Protein type	Protein(s)
Ribosomal large subunit	rpP0, rpL1, rpL2, rpL3, rpL4, rpL5, rpL8, rpL9, rpL10, rpL11, rpL13, rpL19, rpL21, rpL25, rpL26, rpL27, rpL28, rpL30, rpL32, rpL33, rpL35, rpL36
Ribosomal small subunit	rpS0, rpS1, rpS3, rpS4, rpS6, spS7, rpS11, rpS13, rpS17, rpS20, rpS24
Nucleoporin	Nsp1p, Nup2p
Proteosome component	Rpn1p, Pup2p
Cell cycle	Cdc48p
Translation factor	Yef3p, Eft1p, Tef1p, Sui3p, Sis1p, Sro9p

Some of these proteins, such as translation factors and proteasome components, are found frequently in TAPs (34), and therefore the specificity of their interaction with Npa1p is uncertain. Likewise, ribosomal proteins are found with high frequency in large scale TAPs (34). Thus, it is unclear whether the small subunit ribosomal proteins that have been identified by our proteomic approach are genuinely specifically associated with Npa1p. Several nonribosomal Npa1p-associated polypeptides are components of 90S preribosomal particles (see Discussion). However, the identity of the majority of Npa1p-associated proteins is fully consistent with the hypothesis that they are components of a very early pre-60S ribosomal particle(s). Most of these polypeptides are required for 60S biogenesis, and their depletion phenotypes, when known, suggest that almost all are required for 27SA2 processing and/or stability. Moreover, most of these factors are associated with the Nsa3p protein, which has been described by Nissan and coworkers as a component of very early nucleolar pre-60S ribosomal particles (68). Three Npa1p-associated factors, Nop8p, Dbp3p, and Dbp6p, although known to be nucleolar and required for 60S ribosomal subunit biogenesis (52, 103, 106), had never been found in preribosomal particles. Two proteins encoded by the YJR041c and YJR072c open reading frames, which we termed Npa2p and Npa3p, respectively, were totally uncharacterized at the time we obtained the MS data. We therefore studied the localization of these proteins and determined that Npa2p-GFP accumulates in the nucleolus while Npa3p-GFP is concentrated in the cytoplasm (data not shown). These findings were confirmed in the systematic protein localization study (45). Hence, Npa3p may not play a direct role in ribosome biogenesis. In contrast, we found that depletion of Npa2p leads to a strong decrease in the steady-state levels of 25S and 5.8S rRNAs (data not shown), consistent with its predominant nucleolar localization. A role for Npa2p in ribosome biogenesis was also recently inferred from microarray data (72).

Npa1p is associated with a subset of C/D and H/ACA snoRNAs involved in the modification of rRNA residues in the vicinity of the peptidyl transferase center.

In addition to being associated with factors required for the production of large subunit rRNAs, Npa1p is also associated with H/ACA and C/D snoRNP proteins (Table 1). This finding is consistent with the results of Gavin et al. (34), who reported that Npa1p is associated with Nhp2p-TAP and Gar1p-TAP (see Results). Moreover, we have independently confirmed that Npa1p can be coimmunoprecipitated from cell extracts by using anti-Nhp2p antibodies (data not shown). These data suggest that a Npa1p-containing preribosomal particle(s) contains at least a subset of H/ACA and C/D snoRNPs. To assess whether this is the case, the nature of small RNAs coprecipitated with Npa1p-TAP was determined by Northern blot analysis (Fig. 7). We also determined by the same approach the small RNAs coprecipitated with Krr1p-TAP and Ssf1p-TAP. Krr1p is a component of the 90S preribosomal particle (36) (Fig. 7) while Ssf1p is associated mostly with the 27SB pre-rRNAs and to a lesser extent with the 27SA2 pre-rRNA (22) (Fig. 7).

FIG. 7.

A subset of modification guide snoRNAs is associated with Npa1p-TAP. Krr1p-TAP (lanes 1 and 2), Npa1p-TAP (lanes 3 and 4), and Ssf1p-TAP (lanes 5 and 6) proteins were precipitated by using IgG-Sepharose. RNAs were extracted from the pellets obtained after precipitation (lanes IP) or from an amount of total extract corresponding to 1/30 of that used for the precipitation (lanes T). Pre-rRNAs and small RNAs were analyzed by primer extension and Northern blotting, respectively, as previously described. Signal intensities were measured by phosphorimager scanning to derive the percentage of input RNAs precipitated together with tagged proteins (indicated on the right of each panel). bg, background.

As expected, three small RNA molecules that are known to base pair with the 35S pre-rRNA, U3 (7), U14 (57, 62), and snR30 (63), are efficiently coprecipitated with Krr1p-TAP but only extremely weakly with Npa1p-TAP or Ssf1p-TAP. In contrast, a subset of the H/ACA and C/D snoRNAs that are involved in the modification of residues in the vicinity of the peptidyl transferase center is very efficiently coprecipitated with Npa1p-TAP. The two small RNAs that are most efficiently coprecipitated with Npa1p-TAP are snR37 and snR42. Strikingly, these two RNAs are very weakly coprecipitated with Krr1p-TAP, suggesting that they may not significantly associate with 35S pre-rRNA. The efficiency with which small RNAs involved in the modification of residues close to the peptidyl transferase center are coprecipitated with Ssf1p-TAP is variable. Small RNAs such as snR37, snR52, snR69, and snR73 are significantly associated with Ssf1p-TAP. In contrast, snR42 may dissociate from a pre-60S ribosomal particle(s) prior to the recruitment of Ssf1p.

Our results suggest that a subset of the H/ACA and C/D snoRNPs involved in nucleotide modifications can be present within pre-60S ribosomal particles. These snoRNPs may sometimes perform their nucleotide modification function within these pre-60S ribosomal particles and/or their presence within pre-60S particles may be needed for other functions such as promoting proper folding of preribosomal RNAs.

DISCUSSION

Npa1p, a newly identified nucleolar factor required for 60S ribosomal subunit formation and pre-rRNA processing.

Our study clearly demonstrates that Npa1p is a nucleolar protein required for optimum function of the pre-rRNA processing pathway leading to 25S and 5.8S synthesis. Npa1p performs a conserved function in all eukaryotes since we found its probable human orthologue, which is also nucleolar. Despite its large size, we were unable to find any motif of known function within the sequence of Npa1p that might have provided clues to its precise molecular role. Npa1p is almost certainly not a nuclease, at least not an essential one, since no single pre-rRNA processing step is totally inhibited in cells lacking Npa1p and since pulse-chase analysis shows that 25S and 5.8S synthesis still occurs with reasonable efficiency. In the absence of Npa1p, the accumulation of 27SA2 is reduced, and a cleavage fragment extending from sites A2 to C2 accumulates. This suggests that in Npa1p-depleted cells, C2 cleavage can occur directly on the 27SA2 pre-rRNA prior to processing at sites A3 and B1. Accumulation of the A2-C2 fragment was also observed in Ssf1p-depleted cells. Whether Npa1p (and indeed Ssf1p) plays a direct role in preventing premature C2 cleavage or whether this is merely a consequence of the improper assembly of 27SA2-containing pre-60S particles remains to be determined.

At least two nucleolar factors needed for large subunit rRNA synthesis, Nop7p and Noc3p, are required for the initiation of DNA replication (19, 108). Moreover, four other components of pre-60S particles, Nog1p, Nop5p, Nsa3p, and Rpf2p, seem to be associated with the Orc1p protein that binds to the origins of DNA replication (19). Whether this is a property shared by many other protein components of pre-60S particles remains an intriguing possibility. Npa1p may not be a component of the complex that assembles on the origins of DNA replication, because we failed to detect Npa1p in complexes purified when TAP-tagged Orc1p or Mcm5p was used as bait (data not shown). However, the fact that Npa1p interacts with Cdc28p in a double-hybrid assay (95) may indicate that Cdc28p phosphorylates Npa1p (which contains consensus Cdc28p phosphorylation sites) in a cell cycle-dependent manner, as has been shown for several proteins required for DNA replication (47, 67). Determining whether Npa1p is indeed phosphorylated by Cdc28p, and, if so, at what stage of the cell cycle and for what purpose constitutes a challenge for future research.

Composition and localization of 27SA2-containing pre-60S particles.

Information concerning the protein composition of pre-60S particles has been gathered from TAP experiments by using proteins known to be required for 60S ribosomal subunit formation as baits (5, 22, 37, 68, 80, 81). The proteomic data resulting from these experiments, although rich and useful, are difficult to interpret for two main reasons. The first is that these data are most probably always incomplete (sometimes significantly so) due to the fact that some factors may be lost during the purification procedure and/or escape detection by MS. We know, for example, that the box H/ACA snoRNP proteins Gar1p and Nop10p as well as the box C/D protein Snu13p must feature among the Npa1p-associated proteins, although we failed to detect them. The second reason is that most TAP experiments have so far been conducted by using bait proteins that interact strongly with several different pre-rRNAs that stand in precursor-product relationships. Therefore, the panels of proteins obtained probably correspond to the composition of a rather broad spectrum of pre-60S particles. From these lists, it is difficult to derive the precise timing of association and dissociation of any given factor. Defining or purifying a given pre-60S particle is probably impossible, since pre-60S particles may undergo continual changes in conformation and protein content. However, we could improve the resolution of our models if we knew the partners of bait proteins that interact mainly with only one pre-rRNA.

In this study, we have identified the protein partners of Npa1p. Npa1p fulfills the above criterion since it is predominantly associated with the 27SA2 pre-rRNA at steady state, although it probably also transiently interacts with late 90S particles (see below). We have compared the panels of proteins associated with Npa1p with those associated with other bait proteins that also accumulate in the nucleolus and are present in 27SA2-containing particles, namely Nsa3p, Ssf1p, and Nop7p. In contrast to Npa1p, Nsa3p and Ssf1p partition between 27SA2, 27SB, and 7S-containing particles (22, 68). Nop7p is also present in these three types of particles although it is clearly much more abundant in the latter two than in the former (37). From our data and the above-mentioned comparison, we can draw the following conclusions. It is most probable that all 25 Npa1p-associated proteins listed in Table 1 known to be required for 60S ribosomal subunit biogenesis or to be present in pre-60S particles are components of 27SA2-containing particles. Strikingly, our results strongly suggest that 27SA2-containing particles are associated with at least 8 different putative RNA-dependent helicases (Table 1), underscoring the highly complex nature of conformational rearrangements that are likely to take place immediately after A2 cleavage. Two of these putative helicases, Dbp6p and Dbp9p, are most probably present at some stage together in 27SA2-containing preribosomal complexes because a yeast two-hybrid assay performed with these proteins is positive (13). We were surprised to find Prp43p, a putative helicase involved in the release of the lariat-intron from the spliceosome (3, 59), among the factors associated with Npa1p. Prp43p is certainly not a contaminant for the following reasons. It was also found associated with Nsa3p (68), it accumulates in the nucleolus (45), and its human orthologue is found both in speckles and in the nucleolus (2, 29). Oddly, although Nsa3p and Nop7p were purified with tagged Npa1p, this protein was not detected in TAP experiments that used these former two proteins as baits. Such discrepancies have already been observed between the results of TAP experiments with Ssf1p and Nop7p as baits, for example (see discussion in reference 22), and most probably are due to experimental shortcomings (see above). Npa1p was also not detected in one TAP experiment performed with Ssf1p (22). However, Ssf1p was also not detected among the Npa1p-TAP-associated proteins (this study). This result, too, may be due to experimental problems since in the large-scale TAP study, Npa1p was identified among the proteins interacting with Ssf1p-TAP (34).

Interestingly, Npa1p is also associated with a small subset of proteins that are thought to be present (Nsr1p) or have been detected in 90S particles (36, 82). One of these, Rrp5p, would be expected to be present in 27SA2-containing pre-60S particles, since it is required not only for A0, A1, and A2 cleavages and 40S subunit synthesis but also for A3 cleavage (99). Nsr1p, Kre33p, Enp2p, Kri1p, and Utp9p, on the other hand, have never been reported to be required for some aspect of 60S ribosomal subunit synthesis. These proteins could, nevertheless, be present in Npa1p-containing pre-60S particles. Another possibility is that Npa1p is recruited to late 90S preribosomes (i.e., containing the 32S pre-rRNA) just prior to A2 cleavage by a very transient interaction with a subset of late 90S proteins, maybe Nsr1p, Kre33p, Enp2p, Kri1p, and/or Utp9p. This possibility would be compatible with the observation that a fraction of Npa1p sediments above the 27SA2-27SB peak on a glycerol gradient. However, we have never been able to detect the 32S pre-rRNA in the material precipitated with Npa1p (data not shown).

The Nsa3p protein has also been found associated with a subset of 90S proteins (68). Except for Rrp5p, these (Nop14p, Rrp8p, Rrp9p, and Utp10p) are different from the 90S proteins found interacting with Npa1p. Hence, Nsa3p could be recruited to 90S preribosomal particles by transiently interacting with a different subset of late 90S proteins. More surprising is the observation that seven proteins (Nop8p, Fpr3p/Nip46p, Dbp3p, Dbp6p, Dbp7p, Dbp9p, and Nhp2p) have been found associated with Npa1p and not with Nsa3p. Indeed, as just discussed, Nsa3p probably associates with preribosomal particles prior to A2 cleavage, and we would therefore predict that it is associated with all 27SA2-containing particles since it interacts with 27SA2, 27SB, 7S, and 25S and is still detected in the export-competent Arx1p-containing pre-60S particles. One explanation for this discrepancy could be that these seven proteins are more loosely bound to preribosomal complexes and could have dissociated during the second affinity chromatography of the Nsa3p TAP, which was not employed during our purification of tagged Npa1p.

A high-resolution study of Npa1p localization in the nucleolus by electron microscopy shows that this protein is present immediately adjacent to the dense fibrillar component of the nucleolus. This suggests that at least a subset of 27SA2-containing pre-60S ribosomal particles is located adjacent to the dense fibrillar component. This is fully consistent with the general view that pre-rRNA transcription takes place at the boundary between the fibrillar centers and the dense fibrillar component and that the very early processing steps occurring within 90S particles take place inside the dense fibrillar component (reviewed in reference 32). Our hypothesis also fits well with the finding that the Rlp7p protein which associates with slightly more mature pre-60S particles than Npa1p is found within the granular component (32).

Modifying snoRNPs can associate with Npa1p, a marker protein of early pre-60S particles.

The timing of 2′-O-ribose methylations of rRNAs in yeast was first studied by RNase fingerprinting of pre-rRNAs (11). All 2′-O-methyl groups were detected on what was then known as the 37S pre-rRNA (i.e., 35S pre-rRNA), except those added on U2918 and G2919 in the peptidyl transferase center which appear later on the “29S” pre-rRNA (i.e., 27S pre-rRNA; the distinction between various 27S species was not made at the time). Recently, Bonnerot and colleagues have shown that the late U2918 methylation requires the presence of either the Spb1p methyltransferase or the snR52 snoRNA, strongly suggesting that this modification is carried out either by Spb1p or the C/D snoRNP containing snR52 (9). This is consistent with our finding that both Spb1p and snR52 are associated with Npa1p, which we believe reflects their common presence in early 27SA2-containing pre-60S particles. Strikingly, a subset of other C/D as well as H/ACA snoRNAs involved in the modification of the peptidyl transferase center are also very efficiently coprecipitated with Npa1p, suggesting that the corresponding snoRNPs might be present within early pre-60S particles. The efficiency with which some of these RNAs, in particular snR37 and snR42, are coprecipitated with Npa1p is comparable to the efficiency with which 27SA2 pre-rRNA is coprecipitated. Hence, the coprecipitation data of snR37 and snR42 with Npa1p cannot be attributed to the extremely weak interaction of that protein with the 35S pre-rRNA. The presence of modifying snoRNPs in pre-60S particles may reflect the fact that some modifications are introduced at the 27S stage only (as seems to be the case for the methylations of U2918 and G2919) or that there can be some flexibility in the timing of the modifications relative to the cleavage events, i.e., modification at a given site may occur at the 35S stage on some transcripts and at a later stage on others, as has been demonstrated in Xenopus oocytes (105). In addition, some snoRNPs might be required in early pre-60S particles to promote proper folding of 27SA2 pre-rRNA.