This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vanrobays, E.
Right arrow Articles by Caizergues-Ferrer, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vanrobays, E.
Right arrow Articles by Caizergues-Ferrer, M.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, March 2003, p. 2083-2095, Vol. 23, No. 6
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.6.2083-2095.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Late Cytoplasmic Maturation of the Small Ribosomal Subunit Requires RIO Proteins in Saccharomyces cerevisiae

Emmanuel Vanrobays, Jean-Paul Gelugne,* Pierre-Emmanuel Gleizes, and Michele Caizergues-Ferrer

Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 31062 Toulouse Cedex, France

Received 1 August 2002/ Returned for modification 13 September 2002/ Accepted 19 December 2002


arrow
ABSTRACT
 
Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.


arrow
INTRODUCTION
 
Seen from a home economics point of view, making ribosomes is the main task of a living cell (36, 37). Synthesis of the ribosome constituents, their processing and assembly into mature particles, and the regulation of the entire process require the participation of numerous factors. Surprisingly, until relatively recently, few players involved in these processes had been identified. However, in the past decade, by means of genetic and biochemical tricks, and thanks to the tractability of the yeast Saccharomyces cerevisiae and, more recently, with in silico searches, dozens of factors (snoRNAs or protein factors) implicated in ribosome biogenesis have been characterized. For most of them, however, their molecular function in this process is not known.

During the past year, systematic analyses of protein complexes in S. cerevisiae by tandem affinity chromatography purification (3, 7, 11, 14, 16) as well as proteomic analysis of the nucleolus of human cells (1) and definition of putative modular transcriptional networks by computer assisted analysis of the transcriptional expression patterns (18, 20) led to a burst of new putative players. Through these approaches, new factors associated with the pre-60S particle, precursor of the large ribosomal subunit (LSU) and thus putatively involved in the maturation-assembly pathway of the LSU, have been identified. Likewise, factors associated with the early processing complexes containing the large 35S pre-rRNA have been characterized (11) and shown to include mostly proteins specifically required for small ribosomal subunit (SSU) production. In contrast, the nonribosomal proteins found in late nucleoprotein complex precursors of the SSU have not been identified yet by these methods. Nevertheless, criss-cross analysis of the different complexes described before (7, 16) allows us to infer a possible role in the processing-assembly pathway of the SSU for a few proteins (http://www.pre-ribosome.de; 27). In S. cerevisiae, the final maturation of the 20S pre-rRNA leading to the mature 18S rRNA of the SSU occurs in the cytoplasm, and few nonribosomal factors specifically involved in this event are known.

We recently reported that Rrp10p/Rio1p is absolutely required for this processing step (35). This protein is the archetype-founder of a family of proteins found in all sequenced archaeal and eukaryotic organisms (see below) (2, 35), which possess a so-called RIO domain overlapping protein kinase motifs. It has recently been demonstrated that Rio1p exhibits protein serine kinase activity in vitro and that mutations abolishing this kinase activity also abrogate Rio1p function in vivo (2).

In a BLAST search through the S. cerevisiae genome, we identified another open reading frame encoding a RIO protein, YNL207W. Considering our previous demonstration of the involvement of Rio1p in the late processing of the SSU, we investigated whether the product of YNL207W (referred to below as Rio2p) is also implicated in the processing of the 20S pre-rRNA. We show in this report that this is indeed the case: depletion of Rio2p prevents 20S pre-rRNA from being processed to 18S rRNA. Furthermore, we show that Rio1p and Rio2p cosediment with pre-40S particles and shuttle from the nucleus to the cytoplasm. Thus, both RIO proteins encoded in the S. cerevisiae genome are required for the processing of the 20S pre-rRNA into mature 18S rRNA.


arrow
MATERIALS AND METHODS
 
Yeast methods. Yeast medium used are standard YP supplemented with 2% glucose (YPD) or 2% galactose plus 2% sucrose (YPG) and YNB supplemented with amino acids and bases as required. Fluoroorotate-resistant clones were selected on supplemented YNB proline medium containing 0.6 g of 5-fluoroorotic acid per liter (26). S. cerevisiae cells were transformed as previously described (9). Escherichia coli DH5{alpha} was used for molecular cloning and propagation of plasmids. For Rio2p depletion, cells growing in YPG medium at 30°C were harvested in early exponential growth phase (optical density, 0.4 to 0.7) by centrifugation, washed, and resuspended in warm YPD or YPG medium. During growth, cells were periodically diluted with warm medium in order to be kept in exponential growth phase.

Plasmids. Plasmids pJPG310 (2µm LEU2 GFP::RRP8) and pEV24 (CEN ARS LEU2 GFP::RIO1) used in this study were described previously (4, 35). New plasmids used in this work were constructed as follows. The RIO2 open reading frame from wild-type genomic DNA was PCR amplified (Pfu polymerase, Promega) with the following oligonucleotides introducing an NcoI restriction site (YNL207W-I-NcoI and YNL207W-II-NcoI) or oligonucleotides introducing a BglII restriction site (YNL207W-III-BglII and YNL207W-IV-BglII) (see Table 2). The 1.3-kb PCR product amplified with oligonucleotides YNL207W-I-NcoI and YNL207W-II-NcoI was digested with NcoI and cloned into NcoI-restricted vector plasmid pNOPPATA1L (CEN ARS LEU2) obtained from K. Hellmuth and E. Hurt, giving plasmid pEV69 (CEN ARS LEU2 RIO2::PROTA).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Oligonucleotides used in this study

The PCR product amplified with YNL207W-III-BglII and YNL207W-IV-BglII was digested with BglII and cloned into BamHI-restricted vector plasmid pHA114 (2µm URA3) (15), leading to plasmid pEV80 (2µm URA3 GAL::RIO2::PROTA). This GAL::RIO2::PROTA allele is thereafter referred to as GAL::RIO2. Plasmids directing the production of green fluorescent protein (GFP)-Rio2p or GFP-Rio1p fusion proteins were constructed as follows. The TEV-RIO2 PstI fragment from pEV69 and the TEV-RRP10/RIO1 PstI fragment from pEV24 (35) were inserted into the PstI site of plasmid pNOPGFPA1L, obtained from K. Hellmuth and E. Hurt, giving plasmids pEV71 (CEN ARS ADE, NOP::GFP::RIO2) and pEV90 (CEN ARS ADE2 NOP::GFP::RIO1), respectively.

S. cerevisiae strains. Strains used in this work are listed in Table 1. A strain expressing a conditional allele of RIO2 was constructed as follows. A diploid strain heterozygous for a disrupted copy of RIO2, obtained from Euroscarf (strain Y22005), was transformed with pEV80, and selected transformants were sporulated. Two meiotic segregants which were kanamycin resistant and 5-fluoroorotic acid sensitive and unable to grow in glucose-containing medium, YO470 (MATa) and YO471 (MAT{alpha}), were chosen for further functional analyses.


View this table:
[in this window]
[in a new window]
  		TABLE 1. S. cerevisiae strains used in this study

RNA methods. RNA isolation and Northern hybridizations were carried out as previously described (15, 32). The oligonucleotide probes used for Northern hybridizations (numbered 1 to 7 in Fig. 3A) are described in Table 2. Metabolic labeling of RNA was performed as described previously (33). Strain YO471 was grown at 30°C in YPG medium, shifted to YPD medium as described above, grown in this medium for 12 h, and then grown for 7 additional hours in YNB Glu medium without uracil (GAL::RIO2, Glu). The RIO2+ strain (YO511) was grown at 30°C in YNB Glu medium without uracil. Labeling was initiated by addition of 150 µCi of [5,6-3H]uracil to 5-ml samples of these cultures. After 4 min of labeling, 0.6 ml of a 2-mg/ml uracil solution were added. Then 1-ml samples were collected at 1, 3, 5, 10, and 20 min following chase and processed as previously described (4).




View larger version (104K):
[in this window]
[in a new window]
  		FIG. 3. (A) Pre-rRNA processing in S. cerevisiae. In the 35S pre-rRNA, the primary transcript, the sequences of the mature 18S, 5.8S, and 25S rRNAs are flanked by the external transcribed spacers (5' and 3' ETS) and separated by the internal transcribed spacers (ITS1 and ITS2). Cleavage sites are indicated by uppercase letters A to E, and oligonucleotide probes used in Northern blot hybridizations are indicated by the numbers 1 to 7. Pre-rRNA processing pathway: sequential cleavages of the 35S pre-rRNA at sites A0 and A1 generate the 33S and 32S pre-rRNAs. Cleavage of the 32S pre-rRNA at site A2 in ITS1 yields the 27SA2 and 20S pre-rRNAs, which are precursors to the RNA components of the large and small ribosomal subunits, respectively. The 27SA2 precursor is either processed at site A3 by RNase MRP (major pathway) and then to site B1s by the 5'-3' exonucleases Rat1p and Xrn1p, or processed at site B1L (minor pathway), yielding, respectively, the 27SBS and 27SBL pre-rRNAs. These two intermediates follow the same processing pathway to 25S and 5.8SS/L through cleavage at site C2 in ITS2, followed by 3'-5' exonucleolytic digestion of 7SS and 7SL from site C2 to E by the exosome complex, and 5'-3' exonucleolytic digestion to the 5' end of the 25S rRNA by the exonuclease Rat1p. The final maturation of the 20S pre-rRNA by an endonucleolytic cleavage at site D, occurs after pre-SSU export to the cytoplasm, and produces the mature 18S rRNA and a fragment D-A2 (5' ITS1). The D-A2 fragment is then degraded by the 5'-3' exonuclease Xrn1p. (B and C) Depletion of Rio2p specifically affects the steady-state level of mature 18S rRNA and results in 20S pre-rRNA accumulation. YO471 (GAL::RIO2) cells were grown in YPG medium (Gal) or in YPD medium (Glu) for 15 h. Total RNA was extracted and separated in 1% agarose-formaldehyde gels to analyze 35S, 32S, 27SA2, 25S, 20S, and 18S species. Equal amounts of total RNA (5 µg) were loaded in every lane and analyzed. (B) Ethidium bromide staining of the gel. (C) Northern blot analyses of pre-rRNA processing. After transfer to nylon membranes of electrophoresed material, the membranes were hybridized with different probes, as indicated.

Fluorescence in situ hybridization. The 5' part of the ITS1 was detected by fluorescence in situ hybridization with an oligonucleotide probe, as described previously (10).

Glycerol gradients. S. cerevisiae extracts were prepared as previously described (4) except for the method of grinding. From 5 to 20 g of S. cerevisiae cells were ground in an original Waring blender (Osterizer) in the presence of dry ice nuggets. Sedimentation profiles on 10% to 30% glycerol gradients were obtained as described previously (4). A total of 19 fractions (0.6 ml) were collected manually from the top of the gradients.

Immunoprecipitations. Immunoprecipitations and analysis of immunoprecipitated RNAs were done as previously described (35).

Western blotting. Proteins from total extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fractions from glycerol gradients were trichloroacetic acid precipitated, and their protein content was analyzed by SDS-PAGE and Western blotting. Samples were transferred onto nitrocellulose membranes (Hybond C-super; Amersham/Pharmacia). Protein A was detected with the rabbit horseradish peroxidase-conjugated anti-protein A antibody (PAP; Dako SA) diluted to 1:5,000, GFP was detected with a mouse monoclonal anti-GFP antibody (Roche Mannheim) diluted to 1:1,000, ribosomal proteins rpL3, rpL30, and rpS2 were detected with a mouse monoclonal anti-rpL3 antibody diluted to 1:5,000 and a rabbit polyclonal anti-rpL30 antibody cross-reacting with rpS2 (kindly provided by J. Warner) diluted to 1:2,000, followed by enhanced chemiluminescence detection (Amersham/Pharmacia).


arrow
RESULTS
 
RIO protein family. BLAST (40) searches through the S. cerevisiae proteome with Rrp10p/Rio1p as the starting point disclosed that a previously uncharacterized essential gene, YNL207W, encodes a product which shows 19% sequence identity and 43% sequence similarity over its length with Rrp10p/Rio1p. YNL207W has thus been renamed RIO2 and is referred to as such below. Rrp10p/Rio1p and Rio2p proteins show a central well-conserved core of approximately 200 amino acids referenced as the RIO domain in the protein domains database SMART (24) (Fig. 1A). Rio1p and Rio2p homologues were identified throughout archaeal and metazoan species.



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 1. Rio2p is a member of RIO family and is conserved from archaebacteria to humans. (A) Sequence comparison of the RIO domains of the S. cerevisiae proteins. The E-values of these domains in comparison with the consensus defined in the SMART database are indicated. Black shading highlights amino acids that are identical or similar in all sequences, and dark grey and light grey shading highlights those that are identical or similar in some sequences, respectively. (B) Phylogenetic tree and domain profile of all RIO family members. The phylogenetic tree was established with the ClustalW program and drawn with the Phylodendron program. The RIO domain (SMART) is represented by an oval, and Pfam-B_3091 domain by a circle, and lysine-rich regions by a squared box. Eukaryotes are noted in bold. Accession numbers of members of the RIO1/RRP10 family are: Rio1p_Sc, CAA65511; Rio1p_En, AAC26079; Rio1p_Sp, CAA15723; Rio1p_Ce1, P34649; Rio1p_Ce2, AAC17564; Rio1p_Dm1, AAF50965; Rio1p_Dm2, AAF50033; Rio1p_Ap, BAA79728; Rio1p_Mj, Q57886; Rio1p_Mt, AAB85501; Rio1p_Pa, CAB49514; Rio1p_Ph, BAA30679; Rio1p_At1, Q9FHT0; Rio1p_At2, Q9SK34; Rio1p_Hs1, O14730; Rio1p_Ss, CAC24438; and Rio1p_Hs2, Q9H2L9. Accession numbers of members of the RIO2 family are: Rio2p_Sc, NP_014192; Rio2p_Hs, FLJ11159; Rio2p_Ce; CAB60851.1; Rio2p_Sp, CAB66449.1; Rio2p_Dm, CG11859; Rio2p_At, T45758; Rio2p_Mm, NP_080210; Rio2p_Ap, E72539; Rio2p_Mj, H64433; Rio2p_Pa, B75068; Rio2p_Ph, C71164; and Rio2p_Aj, NP_071248. Species abbreviations: Sc, Saccharomyces cerevisiae; En, Emericella nidulans; Sp, Schizosaccharomyces pombe; At, Arabidopsis thaliana; Hs, Homo sapiens; Ms, Mus musculus; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Ap, Aeropyrum pernix; Mj, Methanococcus jannaschii; Mt, Methanobacterium thermoautotrophicum; Pa, Pyrococcus abyssi; Ph, Pyrococcus horikoshii; Ss, Sulfolobus solfataricus; Af, Archaeoglobus fulgidus. Bold bars on the right side of the figure, 100 amino acids; the bar at the upper right side measures the divergence index.

The sequences of the two yeast proteins and their homologues were aligned and displayed as a phylogenetic tree (Fig. 1B). Based on phylogenetic distances, the proteins cluster in two distinct branches, the RIO1 protein family and the RIO2 family. Together these proteins constitute a larger superfamily, named the RIO family, containing a central RIO domain. The Rio1 proteins are duplicated in higher eukaryotes. Only one of the two copies contains a lysine-rich region at its C terminus, as does the Rrp10p/Rio1p yeast protein. Archaeal Rio1 proteins are smaller and restricted to the RIO domain. A distinguishing feature of the RIO2 protein family is the presence, upstream of the RIO domain, of a conserved 80-amino-acid N-terminal domain (referred as Pfam-B_3091 in the Pfam database) that has no equivalent among other members of the RIO family. A glutamic acid-rich region found at the C terminus (positions 362 to 405 in S. cerevisiae Rio2p) is specific to eukaryotic Rio2 proteins.

Rio2p is required for 18S rRNA production. We recently showed that Rrp10p/Rio1p is an essential cytoplasmic protein required for the processing of 20S pre-rRNA to 18S rRNA (35). Considering the homology between the two proteins, we tested the possible involvement of Rio2p in ribosome biogenesis. Since YNL207W/RIO2 has been shown to be an essential gene (38), a conditional RIO2 mutant allele was constructed (see Materials and Methods). This construct allows expression of the RIO2 open reading frame, tagged at the 3' end with a sequence encoding two protein A epitopes, under the inducible GAL10 promoter (Fig. 2A). Transcription driven by the GAL10 promoter leads to high expression of the gene in culture medium that contain galactose (YPG) but is repressed in culture medium containing glucose (YPD).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 2. RIO2 gene expression under the control of the inducible GAL10 promoter. (A) Schematic representation of the GAL::RIO2::PROTA allele. (B) Growth of strain YO471 carrying the GAL::RIO2::PROTA allele at 30°C. Cells from exponentially growing cultures in YPG medium (Gal) were harvested, washed, and resuspended either in YPD medium (Glu, triangles) or in galactose medium (Gal, crosses), and incubation was pursued for up to 24 h at 30°C. Cell density was measured at regular intervals, and the cultures were periodically diluted to keep them in the exponential phase of growth. The generation time in YPG is similar to that of an isogenic RIO2+ strain. (C) Depletion of Rio2p during growth in YPD medium. Cell extracts of strain YO471 carrying the GAL::RIO2::PROTA allele were prepared from samples harvested at the indicated time points. Rio2p concentration was determined by Western blotting. Equal amounts of total proteins were loaded in every lane, as estimated by Coomassie staining of the gels.

In order to deplete Rio2p, cells of strain YO471 (rio2{Delta}::KAN/pGAL::RIO2) were shifted from galactose medium (YPG) to glucose medium (YPD). This results in a severe reduction of the growth rate of the YO471 strain after 15 h of culture in the presence of glucose (Fig. 2B). An immunoblot, revealing the protein A tag, confirmed that the amount of Rio2p rapidly decreases and becomes barely detectable after 24 h of growth of strain YO471 in YPD (Fig. 2C).

We made use of this conditional RIO2 allele to determine whether Rio2p is necessary for pre-rRNA processing. As shown in Fig. 3B, depletion of Rio2p (15 h in YPD medium) has no effect on the steady-state level of 25S rRNA but drastically affects 18S rRNA accumulation (Fig. 3B and 3C). To determine which step(s) of 18S rRNA processing is affected, total RNAs extracted from Rio2p-depleted cells were hybridized to specific oligonucleotide probes (Table 2, Fig. 3A). Hybridization to probe 2, complementary to the internal transcribed spacer 1 (ITS1 D-A2), discloses a strong increase of 20S pre-rRNA (Fig. 3C), concomitantly with 18S rRNA reduction. This result clearly shows that, in Rio2p-depleted cells, cleavage at site D is strongly inhibited, as previously observed in Rrp10p/Rio1p-depleted cells (35). Further effects of Rio2p depletion on pre-rRNA processing are a reduction in the level of 27SA2 pre-rRNA (probe 3 complementary to ITS1 A2-A3) and a very slight increase in the level of an aberrant intermediate of approximately 22S/23S detected with probes 2, 3, and 7 (not shown), indicating that this intermediate contains sequences extending from site A0 (or further upstream) to site A3 and results from cleavage of pre-rRNA molecules at site A3 in the absence of cleavage at site A2. The levels of 27SBS and 27SBL are not affected, as revealed by hybridization with probe 5, complementary to the internal transcribed spacer 2 (ITS2 A3-C2) (results not shown).

The kinetics of pre-rRNA processing and rRNA accumulation were analyzed in [3H]uracil pulse-chase labeling experiments (Fig. 4). In a GAL::RIO2 strain grown for 15 h in glucose-containing medium, 20S pre-rRNA is still present after 20 min of chase, while 18S rRNA is only faintly detectable at this time. In contrast, Rio2p depletion does not affect the time course of 27S pre-rRNA conversion to 25S rRNA.



View larger version (66K):
[in this window]
[in a new window]
  		FIG. 4. Depletion of Rio2p-protein A results in reduced synthesis of 18S rRNA. (A) Strain YO511 (RIO2+) was grown at 30°C in YNB Glu without uracil. (B) Strain YO471 (GAL::RIO2) was grown at 30°C in YPG medium (Gal), then shifted for 12 h to YPD medium (Glu), and finally grown for 7 h in YNB Glu without uracil. Cells were labeled for 4 min with [5,6-3H]uracil and chased with a large excess of unlabeled uracil for 1 to 20 min.

Altogether, these data show that depletion of Rio2p strongly (and specifically) affects processing of the 20S pre-rRNA to 18S rRNA. Whether the minor alterations in A1 and A2 cleavages observed reflect the involvement of Rio2p in early processing events or are indirect consequences of this drastic defect in 20S pre-rRNA maturation is not clear.

Cells depleted of Rio2p accumulate 20S pre-rRNA in the cytoplasm. In order to visualize the intracellular fate of the 20S pre-rRNA seen to accumulate in the pulse-chase experiments and in Northern hybridizations, fluorescence in situ hybridization with a probe complementary to the 5' part of the ITS1 (between sites D and A2) was performed on cells depleted of Rio2p (YO470 or YO471, GAL::RIO2, cells grown for 15 h in glucose). As shown in Fig. 5F, a strong ITS1 signal (red) was observed in the cytoplasm compared to the nondepleted cells or to wild-type cells (Fig. 5E and 5A). A comparable result was obtained with cells depleted of Rio1p (Fig. 5C and 5D), as previously shown by electron microscopy (35). Under similar hybridization conditions, ITS1 is detected in large amounts in the nucleus of cells bearing the thermosensitive allele crm1-1 (Fig. 5B), consistent with published results showing the requirement for the exportin Crm1p in the nuclear export of the pre-40S particles (28). Thus, although necessary for the last processing step of 18S rRNA synthesis, Rio2p is not required for the nuclear export of the SSU precursor particles.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 5. Depletion of Rio2p results in accumulation of 20S pre-rRNA in the cytoplasm. The 5' part of ITS1 (red) was detected by fluorescence in situ hybridization in GAL::RIO1 cells (YO296) (C and D) and GAL::RIO2 (YO470) cells (E and F). Cells were grown either in galactose medium or for 15 h in the presence of glucose. The nucleoplasm was counterstained with DAPI (blue). In wild-type cells (A), the ITS1 is mainly present in the nucleolus and is faintly detected in the cytoplasm. In contrast to crm1-1 cells (YO419) shifted for 2 h at 37°C (B), which display a strong signal in the nucleus and no labeling of the cytoplasm, cells depleted of Rio1p or Rio2p show a build-up of the amount of 20S pre-rRNA in the cytoplasm (D and F).

Rio2p is a nuclear and cytoplasmic protein cosedimenting with pre-40S particles. Subcellular localization of Rio2p was investigated with a GFP-Rio2p fusion protein. The GFP-Rio2p fusion was fully functional, as demonstrated by its ability to complement the lethality of the RIO2 invalidation in strain YO566. In this strain expressing a GFP::RIO2 fusion allele, the green fluorescence is detected in the cytoplasm and the nucleus (Fig. 6A). In a strain expressing a GFP::RIO1 allele, the green fluorescence is detected only in the cytoplasm (Fig. 6A), as previously shown (35), and in a strain expressing GFP-Rrp8p, the green fluorescence is detected in the nucleolus (Fig. 6A).



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 6. Sedimentation profile of Rio2p-protein A, localization of GFP-Rio2p, and association of Rio2p with 20S pre-rRNA. (A) Subcellular localization of GFP-tagged proteins: GFP-Rio2p exhibits a cytoplasmic and nuclear pattern of fluorescence. GFP-Rio1p shows a cytoplasmic localization, and for Rrp8p-GFP, a punctate pattern characteristic of nucleolar staining is observed. Positions of nuclei were determined by DAPI staining (blue). Overlay images are shown by superposition of the blue and green stains. (B) Sedimentation profiles of Rio2p-protein A and protein A-Rio1p, small-subunit ribosomal protein rpS2, and large-subunit ribosomal protein rpL3 in glycerol gradients. A total extract was loaded on 10 to 30% glycerol gradients and subjected to centrifugation. Fractions were collected, and proteins in each fraction were precipitated with trichloroacetic acid, separated by SDS-PAGE, and revealed by Western blotting. Fractions containing the peak of 40S and 60S ribosomal subunits and 80S ribosomes are indicated. (C) 20S pre-rRNA selectively copurifies with Rio2-protein A. We probed 1/400 of the clarified cell extract (T) or the bulk of the immunoprecipitated RNA (IP) with a mixture of probes 1 and 6 shown in Fig. 3A (18S and 25S) and a D-A2 probe prepared by multiprime labeling (20S). The fraction of each rRNA or pre-rRNA recovered in the immunoprecipitate was determined by phosphoimager quantification.

To find out whether Rio2p is associated with preribosomal particles, whole-cell lysates prepared from strains expressing protein A-tagged Rio2p or Rio1p were analyzed by glycerol gradient centrifugation. Presence of Rio2p-protein A, protein A-Rio1p, and ribosomal proteins rpS2 and rpL3 in the different fractions of the glycerol gradients was determined by immunoblotting (Fig. 6B). Rio2p-protein A and protein A-Rio1p peak in fractions of the glycerol gradients in which the 40S ribosomal subunit sediments. A pool of Rio2p-protein A is also found in the upper part of the gradients, which contains soluble proteins.

Detection of Rio2p-protein A around the position of 40S particles suggests that Rio2p-protein A might be associated, at least transiently, with the 43S particle, precursor of the 40S ribosomal subunit. In order to assess such a physical association of Rio2p with pre-SSU particles, we determined if pre-rRNA coimmunoprecipitates with Rio2p. Whole-cell lysates from YO470 (RIO2-PROTA) and YO511 (RIO2+) were incubated with immunoglobulin G-Sepharose beads, and the RNA molecules retained onto the immunoglobulin G beads were extracted, separated in an agarose gel, and probed with 18S, 25S, and ITS1(D-A2) probes. As shown in Fig. 6C, RNA coimmunoprecipitated with Rio2-protein A is selectively enriched for 20S pre-rRNA. No larger pre-rRNA processing intermediate was detected in these experiments (not shown), indicating that Rio2p is not associated with early pre-rRNA processing complexes.

Since both S. cerevisiae RIO proteins associate with 20S-containing pre-SSU and Rio2p has been found in complexes containing Dim1p (7), the protein required for dimethylation of the 18S rRNA adenosine residues A1779 and A1780 (numbering of S. cerevisiae mature 18S rRNA) (22), a late event in 20S pre-rRNA processing (5), we determined if the S. cerevisiae RIO proteins are required for this modification step. Primer extensions experiments with oligonucleotides complementary to ITS1 sequences as primers and total RNA extracted from either wild-type or Rio1p- or Rio2p-depleted cells as templates all revealed the same extension products characteristic of the presence of dimethyladenosine residues at positions A1779 and A1780 of the mature 18S rRNA (not shown). This indicates that this modification of the 20S pre-rRNA can still take place in the absence of either one of these proteins.

Since Rio2p-protein A and protein A-Rio1p are found in the same fractions and 20S pre-rRNA selectively copurifies with Rio1-protein A (35), we attempted to immunoprecipitate GFP-Rio1p with Rio2p-protein A. In strain YO470 transformed with plasmid pEV90 (pNOP::GFP::RIO1 ADE2), expression of the Rio2p-protein A fusion protein constitutes the unique source of Rio2p, while both GFP-Rio1p fusion protein and the endogenous Rrp10p/Rio1p protein are expressed. Under these conditions, Western blotting with a GFP antibody did not reveal any Rrp10p/Rio1p copurifying with Rio2p-protein A on immunoglobulin G-Sepharose.

Rio2p and Rio1p shuttle between the nucleus and the cytoplasm. Detection of Rio2p in the nucleus and the cytoplasm suggests that Rio2p could be a shuttling protein. To test this hypothesis, the distribution of GFP-Rio2p was analyzed in cells carrying the crm1-1 mutation, which is known to affect the nucleocytoplasmic exchanges. Crm1p belongs to the karyopherin superfamily and is involved in the nuclear export of a large range of proteins. In addition, deficiency in Crm1p function results in the accumulation of pre-40S particles in the nucleus (28). In the crm1-1 mutant cells grown at the nonpermissive temperature, a significant nuclear-nucleolar accumulation of GFP-Rio2p is observed (Fig. 7A). The same experiment performed with cells expressing GFP-Rio1p shows that it also accumulates in the nucleus (Fig. 7B), while in the same conditions GFP alone does not (Fig. 7C). These results clearly show that Rio1p and Rio2p are shuttling proteins.



View larger version (50K):
[in this window]
[in a new window]
  		FIG.7. Localization and sedimentation profiles of GFP-Rio1p and GFP-Rio2p in a crm1-1 genetic context. crm1-1 strains used carry plasmids expressing either GFP-Rio2p (A), GFP-Rio1p (B), or GFP (C). Exponentially growing cultures in YNB medium at 25°C were divided in two parts; one part was shifted to 37°C for 2 h, while the other was kept at 25°C, and the subcellular localization of the tagged proteins was analyzed. Subcellular localization of GFP-Rio2p (A) and GFP-Rio1p (B) exhibited a wild-type localization pattern in crm1-1 cells grown at 25°C, in contrast to cells shifted for 2 h at 37°C, in which GFP-RIO proteins display essentially a nuclear and nucleolar pattern of fluorescence. GFP (C) in the crm1-1 strain at either 25°C or 37°C shows a normal cytoplasmic localization pattern. (D) Sedimentation profiles in a glycerol gradient of GFP-Rio2p, GFP-Rio1p, small-subunit ribosomal protein rpS2, and large-subunit ribosomal protein rpL3. A total extract from a crm1-1 strain carrying plasmid pEV24 (LEU2 pNOP::GFP::RIO1) or pEV71 (ADE2 pNOP::GFP::RIO2) and grown at 37°C was loaded on a 10 to 30% glycerol gradient and processed as described in the legend to Fig. 6.

To find out whether, in these conditions, Rio2p and Rrp10p/Rio1p are still associated with preribosomal particles, whole-cell lysates derived from crm1-1 cells expressing GFP-tagged Rio2p or Rio1p and grown at the nonpermissive temperature were analyzed on glycerol gradients. The presence of GFP-Rio2p, GFP-Rio1p, and ribosomal proteins rpS2 and rpL3 in the different fractions of the glycerol gradients was examined by immunoblotting (Fig. 7D). GFP-Rio2p and GFP-Rio1p peak in fractions of the glycerol gradients in which the rpS2 protein sediments without rpL3. Accumulation of pre-40S particle is revealed by the larger amount of the rpS2 protein detected in these fractions (compare detection of rpS2 in Fig. 6B and Fig. 7D). These results indicate that when export of the SSU precursors is inhibited, both RIO proteins stay in the nucleus. Together, the nuclear localization of GFP-Rio1p and GFP-Rio2p in crm1-1 cells at the nonpermissive temperature, their cosedimentation with pre-40S particles, and association with 20S pre-rRNA suggest that these proteins associate with precursors of the SSU in the nucleus.


arrow
DISCUSSION
 
The functional analysis of Rio2p described above demonstrates that Rio2p is found in both the nucleus and the cytoplasm and that Rio2p-depleted cells, like Rio1p-depleted cells, exhibit a strong accumulation of 20S pre-rRNA. However, the level of 20S pre-rRNA accumulated does not reflect the 18S rRNA deficit (in samples from Rio2p-depleted cells, 20S pre-rRNA is barely detectable in ethidium bromide-stained gels, Fig. 3B). Thus, most of the unprocessed 20S pre-rRNA does not lead to immature SSU but is degraded. Such an accumulation of 20S pre-rRNA might stem from (i) defects of pre-40S subunit assembly impairing either its export from the nucleus or its final processing or both; (ii) alteration of elements of the export machinery, as previously shown for prp20-1 and yrb1-1 mutants (28); or (iii) direct inhibition of the endonucleolytic cleavage at site D.

Our data indicate that, as previously observed in Rio1p-depleted cells (35), 20S pre-rRNA is found in the cytoplasm of Rio2p-depleted cells, indicating that neither Rio1p nor Rio2p is required for the export of the pre-40S particles. We also show that in crm1-1 cells grown at the nonpermissive temperature, in which the export of pre-40S particles is blocked, GFP-Rio2p and GFP-Rio1p are found mainly in the nucleus. Moreover, in extracts prepared from CRM1+ cells as well as in extracts from crm1-1 cells grown at the nonpermissive temperature, both Rio1p and Rio2p cosediment with pre-40S particles and 20S pre-rRNA is selectively coimmunoprecipitated with tagged Rio1p (35) and Rio2p (Fig. 6C). Taken together, these data show that both Rio1p and Rio2p are shuttling proteins required for the cytoplasmic processing of 20S pre-rRNA and suggest that they associate in the nucleus with pre-40S particles and are exported from the nucleus as elements of these particles.

Although depletion of Rio2p or Rrp10p/Rio1p causes the same phenotype, i.e., cytoplasmic accumulation of 20S pre-rRNA, they do not have overlapping functions: (i) both genes are essential for viability, and (ii) RIO2 disruption is not complemented by overexpression of Rio1p or vice versa (our unpublished observations). In Rio2p-depleted cells, besides 20S pre-rRNA accumulation, minor effects on pre-rRNA processing are also observed: a slight accumulation of the aberrant 23S pre-rRNA and a small decrease in the level of the 27SA pre-rRNA. We attribute these defects to slowed processing at the A0, A1, and A2 cleavage sites. This could be related to the association of Rio2p with proteins present in the 90S preribosomes (Table 3) (7, 11). Such an effect is not seen in Rrp10p/Rio1p-depleted cells (35).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Ynl207p/Rio2p-associated factors

Localization of the two proteins is also somewhat different: in normal conditions, GFP-Rio1p is found exclusively in the cytoplasm, while Rio2p localizes in the nucleus and the cytoplasm. This nuclear localization is in good agreement with the recent identification of Ynl207p/Rio2p in different complexes (7). In tandem affinity purification tag experiments (30), with tagged Rio2p as the bait, different factors, some of them clearly involved in the biogenesis of the SSU, such as Tsr1p and Dim1p (8, 22) (Table 3), have been affinity purified (7). The functions and cellular localization of other ones are unknown, but some of them, like Enp1p, are found in 90S preribosomes (11). Rio2p as well as Enp1p, Ltv1p, Yor145p, and Rrp12p were also found in a multiprotein complex purified with tagged Tsr1p (Ydl060p) as the bait, a protein known to be required for SSU biogenesis (8). Furthermore, Ltv1p, a protein required for viability at low temperatures, which is found in these complexes, interacts in two-hybrid screens with rpS3, a protein of the SSU, and Crm1p, a protein required for the export of pre-40S particles. All these data clearly suggest that Rio2p is present in the nucleolus and the nucleus in complexes required for maturation and assembly of the SSU, as proposed in a recent review (6).

Certainly, due to its low abundance and its steady-state cytoplasmic localization, Rrp10p/Rio1p has not been referenced yet in these systematic purifications of multiprotein complexes. Compilation of all these results suggests that Rio2p may associate with pre-40S particles in the nucleus and earlier than Rio1p or with complexes of longer half-lives.

The RIO protein family has also been described by Koonin and coworkers (23) in an attempt to evaluate the entire repertoire of potential protein kinases in bacteria and archaea. These authors extensively scanned the sequences of available complete genomes for proteins with similarity to protein kinases. Retrieved putative protein kinases have been assigned to five distinct families, among them the RIO superfamily. More recently, by means of profile-matching procedures, conservation of important residues, and fold recognition techniques, Krupa and Srinivasan reported the occurrence of RIO-like proteins in eubacteria as well (21). They suggest that lipopolysaccharide kinases encoded in the genome of gram-negative bacteria are related to RIO proteins in eubacteria, archaea, and eukaryotes, pointing to the RIO domain as a possible evolutionary link between eukaryotic protein kinase-like sequences in prokaryotes and bona fide eukaryotic protein kinases.

The RIO domain defined in the SMART database (24) overlaps a putative kinase domain containing the key sequence motifs that are associated with catalytic activity and conserved among most protein kinases (corresponding to the canonical domains I to IX): the ATP binding site (conserved G, K, and E residues, positions 85 to 87 in Rio1p and 104 to 106 in Rio2p) and the catalytic D and N residues (D244 and N249 in Rio1p and D229 and N234 in Rio2p). Quite recently, a kinase activity has been demonstrated in vitro for Rrp10p/Rio1p (2). Moreover, mutations of conserved amino acid residues required for catalytic activity (i.e., within the ATP binding motif or catalytic domain) abrogate the kinase activity of the recombinant protein extracted from E. coli and Rio1p in vivo function.

Cytological analyses of Rio1p-depleted cells and of a strain carrying a weak RRP10/RIO1 allele (D244E) suggest that Rrp10p/Rio1p could be involved in cell cycle progression. Likewise, in Emericella nidulans, mutations of sudD, the RIO1 homologue, lead to an abnormal nuclear morphology (17). A recent systematic genetic analysis reveals that loss-of-function mutations or, in the case of essential genes, heterozygosity for loss-of-function mutations of numerous genes involved in ribosome biogenesis lead to altered average cell size. Further analysis disclosed that expression of most of these genes depends on the transcription factor Sfp1p (20). Some of these Sfp1p-regulated factors have established functional links to either Rio1p (Gar1p [35]) or Rio2p (Enp1p and Rrp12p [7]). Taken together, these data connect the involvement of Rio1p and Rio2p in ribosome biogenesis, as revealed by the drastic block in 20S pre-rRNA processing resulting from Rio1p or Rio2p depletion (35; this study), to cell cycle defects observed in Rio1p-depleted cells (2), or in sudD mutants of E. nidulans (17).

In this study we have shown that the RIO proteins in S. cerevisiae are shuttling proteins required for 20S pre-rRNA maturation. From our previous data (35) and the results reported herein, we infer that these proteins associate with pre-40S particles in the nucleus/nucleolus and are exported to the cytoplasm as part of 20S pre-rRNA containing precursors of the SSU. Recent proteomics data are in good agreement with this hypothesis (for a review, see reference 6). Several factors involved in preribosome assembly and pre-rRNA processing are phosphoproteins. Srp40p (Nopp140) interacts with and is phosphorylated by casein kinase II (25), and Nsr1p/nucleolin and nucleophosmin/B23 are also phosphorylated by casein kinase II and cdc2 (13, 25, 29), suggesting that the phosphorylation status of some trans-acting factors is involved in the control of ribosomes biogenesis. However, so far no kinase activity essential for ribosome biogenesis has been described among all the factors recently identified.

Considering their putative kinase activity, RIO proteins could themselves act as functional regulators of factors associated with the pre-40S complexes or required for 20S pre-rRNA maturation. In this respect, identifying the substrates of these putative protein kinases and disclosing how their activity and/or localization could be modulated should shed light on their function(s) in the late SSU processing pathway. The occurrence of RIO proteins or RIO-related proteins in eubacteria and archaea argues that these proteins are involved in highly conserved and ancient molecular processes.


arrow
ACKNOWLEDGMENTS
 
We are grateful to J. Warner, Ed Hurt, and A. Tartakoff for providing us with antibodies, plasmids, and S. cerevisiae strains. We thank members of the Ferrer laboratory for helpful discussions and Y. Henry for critical reading of the manuscript. We also thank Y. de Préval for synthesis of oligonucleotides, D. Villa for the photographs, and A. Rivals for technical support.

This work was supported by the CNRS and the Université Paul Sabatier and grants from the Programme de Recherches Fondamentales en Microbiologie et Maladies Infectieuses et Parasitaires du Ministère de Education Nationale (MENRT) to M.C.F. E.V. was supported by grants from the MENRT and the Fondation pour la Recherche Médicale.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: LBME du CNRS, 118 route de Narbonne, 31062 Toulouse cedex, France. Phone: 33 5 61 33 59 58. Fax: 33 5 61 33 58 86. E-mail: jpg{at}ibcg.biotoul.fr. Back


arrow
REFERENCES
 
    1
  1. Andersen, J. S., C. E. Lyon, A. H. Fox, A. K. Leung, Y. W. Lam, H. Steen, M. Mann, and A. I. Lamond. 2002. Directed proteomic analysis of the human nucleolus. Curr. Biol. 12:1-11.[CrossRef][Medline]
    2. 2
  2. Angermayr, M., A. Roidl, and W. Bandlow. 2002. S. cerevisiae Rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression. Mol. Microbiol. 44:309-324.[CrossRef][Medline]
    3. 3
  3. Bassler, J., P. Grandi, O. Gadal, T. Lessmann, E. Petfalski, D. Tollervey, J. Lechner, and E. Hurt. 2001. Identification of a 60S preribosomal particle that is closely linked to nuclear export. Mol. Cell 8:517-529.[CrossRef][Medline]
    4. 4
  4. Bousquet-Antonelli, C., E. Vanrobays, J. P. Gelugne, M. Caizergues-Ferrer, and Y. Henry. 2000. Rrp8p is a S. cerevisiae nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2. RNA 6:826-843.[Abstract]
    5. 5
  5. Brand, R. C., J. Klootwijk, T. J. M. Van Steenbergen, A. J. De Kok, and R. J. Planta. 1977. Secondary methylation of S. cerevisiae ribosomal precursor RNA. Eur. J. Biochem. 75:311-318.[Medline]
    6. 6
  6. Fatica, A., and D. Tollervey. 2002. Making ribosomes. Curr. Opin. Cell Biol. 14:313-318.[CrossRef][Medline]
    7. 7
  7. Gavin, A. C., M. Bosche, R. Krause, P. Grandi, M. Marzioch, A. Bauer, J. Schultz, J. M. Rick, A. M. Michon, C. M. Cruciat, M. Remor, C. Hofert, M. Schelder, M. Brajenovic, H. Ruffner, A. Merino, K. Klein, M. Hudak, D. Dickson, T. Rudi, V. Gnau, A. Bauch, S. Bastuck, B. Huhse, C. Leutwein, M. A. Heurtier, R. R. Copley, A. Edelmann, E. Querfurth, V. Rybin, G. Drewes, M. Raida, T. Bouwmeester, P. Bork, B. Seraphin, B. Kuster, G. Neubauer, and G. Superti-Furga. 2002. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141-147.[CrossRef][Medline]
    8. 8
  8. Gelperin, D., L. Horton, J. Beckman, J. Hensold, and S. K. Lemmon. 2001. Bms1p, a novel GTP-binding protein, and the related Tsr1p are required for distinct steps of 40S ribosome biogenesis in yeast. RNA 7:1268-1283.[Abstract]
    9. 9
  9. Gietz, R. D., R. H. Schiestl, A. R. Willems, and R. A. Woods. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355-360.[CrossRef][Medline]
    10. 10
  10. Gleizes, P. E., J. Noaillac-Depeyre, I. Leger-Silvestre, F. Teulieres, J. Y. Dauxois, D. Pommet, M. C. Azum-Gelade, and N. Gas. 2001. Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex. J. Cell Biol. 155:923-936.[Abstract/Free Full Text]
    11. 11
  11. Grandi, P., V. Rybin1, J. Baßler, E. Petfalski, D. Strauß, M. Marzioch, T. Schäfer, B. Kuster, H. Tschochner, D. Tollervey, A. Gavin, and E. Hurt. 2002:90S. Preribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. Mol. Cell 10:105-115.[CrossRef][Medline]
    12. 12
  12. Grava, S., P. Dumoulin, A. Madania, I. Tarassov, and B. Winsor. 2000. Functional analysis of six genes from chromosomes XIV and XV of Saccharomyces cerevisiae reveals YOR145c as an essential gene and YNL059c/ARP5 as a strain-dependent essential gene encoding nuclear proteins. Yeast 16:1025-1033.[CrossRef][Medline]
    13. 13
  13. Gulli, M. P., M. Faubladier, H. Sicard, and M. Caizergues-Ferrer. 1997. Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin. Chromosoma 105:532-541.[Medline]
    14. 14
  14. Harnpicharnchai, P., J. Jakovljevic, E. Horsey, T. Miles, J. Roman, M. Rout, D. Meagher, B. Imai, Y. Guo, C. J. Brame, J. Shabanowitz, D. F. Hunt, and J. L. Woolford. 2001. Composition and functional characterization of yeast 66s ribosome assembly intermediates. Mol. Cell 8:505-515.[CrossRef][Medline]
    15. 15
  15. Henras, A., Y. Henry, C. Bousquet-Antonelli, J. Noaillac-Depeyre, J. P. Gelugne, and M. Caizergues-Ferrer. 1998. Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs. EMBO J. 17:7078-7090.[CrossRef][Medline]
    16. 16
  16. Ho, Y., A. Gruhler, A. Heilbut, G. D. Bader, L. Moore, S. L. Adams, A. Millar, P. Taylor, K. Bennett, K. Boutilier, L. Yang, C. Wolting, I. Donaldson, S. Schandorff, J. Shewnarane, M. Vo, J. Taggart, M. Goudreault, B. Muskat, C. Alfarano, D. Dewar, Z. Lin, K. Michalickova, A. R. Willems, H. Sassi, P. A. Nielsen, K. J. Rasmussen, J. R. Andersen, L. E. Johansen, L. H. Hansen, H. Jespersen, A. Podtelejnikov, E. Nielsen, J. Crawford, V. Poulsen, B. D. Sorensen, J. Matthiesen, R. C. Hendrickson, F. Gleeson, T. Pawson, M. F. Moran, D. Durocher, M. Mann, C. W. Hogue, D. Figeys, and M. Tyers. 2002. Syst. identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180-183.[CrossRef][Medline]
    17. 17
  17. Holt, C. L., and G. S. May. 1996. An extragenic suppressor of the mitosis-defective bimD6 mutation of Aspergillus nidulans codes for a chromosome scaffold protein. Genetics 142:777-787.[Abstract]
    18. 18
  18. Ihmels, J., G. Friedlander, S. Bergmann, O. Sagir, Y. Ziv, and N. Barkai. 2002. Revealing modular organization in the yeast transcriptional network. Nat. Genet. 31:370-377.[CrossRef][Medline]
    19. 19
  19. Jiang, P. S., J. H. Chang, and B. Y. Yung. 2000. Different kinases phosphorylate nucleophosmin/B23 at different sites during G(2) and M phases of the cell cycle. Cancer Lett. 153:151-160.[CrossRef][Medline]
    20. 20
  20. Jorgensen, P., J. L. Nishikawa, B. J. Breitkreutz, and M. Tyers. 2002. Syst. identification of pathways that couple cell growth and division in yeast. Science 297:395-400.[Abstract/Free Full Text]
    21. 21
  21. Krupa, A., and N. Srinivasan. 2002. Lipopolysaccharide phosphorylating enzymes encoded in the genomes of Gram-negative bacteria are related to the eukaryotic protein kinases. protein Sci. 11:1580-1584.[CrossRef][Medline]
    22. 22
  22. Lafontaine, D., J. Vandenhaute, and D. Tollervey. 1995. The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast. Genes Dev. 9:2470-2481.[Abstract/Free Full Text]
    23. 23
  23. Leonard, C. J., L. Aravind, and E. V. Koonin. 1998. Novel families of putative protein kinases in bacteria and archaea: evolution of the eukaryotic; protein kinase superfamily. Genome Res. 8:1038-1047.[Abstract/Free Full Text]
    24. 24
  24. Letunic, I., L. Goodstadt, N. J. Dickens, T. Doerks, J. Schultz, R. Mott, F. Ciccarelli, R. R. Copley, C. P. Ponting, and P. Bork. 2002. Recent improvements to the SMART domain-based sequence annotation resource. Nucleic Acids Res. 30:242-244.[Abstract/Free Full Text]
    25. 25
  25. Li, D., U. T. Meier, G. Dobrowolska, and E. G. Krebs. 1997. Specific interaction between casein kinase 2 and the nucleolar protein Nopp140. J. Biol. Chem. 272:3773-3779.[Abstract/Free Full Text]
    26. 26
  26. McCusker, J. H., and R. W. Davis. 1991. The use of proline as a nitrogen source causes hypersensitivity to and allows more economical use of 5FOA in Saccharomyces cerevisiae. Yeast 7:607-608.[CrossRef][Medline]
    27. 27
  27. Milkereit, P., H. Kühn, N. Gas, and H. Tschochner. Nucleic Acids Res., in press.
    28. 28
  28. Moy, T. I., and P. A. Silver. 1999. Nuclear export of the small ribosomal subunit requires the ran-GTPase cycle and certain nucleoporins. Genes Dev. 13:2118-2133.[Abstract/Free Full Text]
    29. 29
  29. Pfaff, M., and F. A. Anderer. 1988. Casein kinase II accumulation in the nucleolus and its role in nucleolar phosphorylation. Biochim. Biophys. Acta 969:100-109.[Medline]
    30. 30
  30. Rigaut, G., A. Shevchenko, B. Rutz, M. Wilm, M. Mann, and B. Seraphin. 1999. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17:1030-1032.[CrossRef][Medline]
    31. 31
  31. Stade, K., C. S. Ford, C. Guthrie, and K. Weis. 1997. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90:1041-1050.[CrossRef][Medline]
    32. 32
  32. Tollervey, D. 1987. A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA. EMBO J. 6:4169-4175.[Medline]
    33. 33
  33. Tollervey, D., H. Lehtonen, M. Carmo-Fonseca, and E. C. Hurt. 1991. The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J. 10:573-583.[Medline]
    34. 34
  34. Tone, Y., N. Tanahashi, K. Tanaka, M. Fujimuro, H. Yokosawa, and A. Toh-e. 2000. Nob1p, a new essential protein, associates with the 26S proteasome of growing Saccharomyces cerevisiae cells. Gene 243:37-45.[CrossRef][Medline]
    35. 35
  35. Vanrobays, E., P. E. Gleizes, C. Bousquet-Antonelli, J. Noaillac-Depeyre, M. Caizergues-Ferrer, and J. P. Gelugne. 2001. Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein. EMBO J. 20:4204-4213.[CrossRef][Medline]
    36. 36
  36. Warner, J. R. 1999. The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci. 24:437-440.[CrossRef][Medline]
    37. 37
  37. Warner, J. R., J. Vilardell, and J. H. Sohn. 2001. Economics of ribosome biosynthesis, p. 567-574. In J. R. Broach, J. R. Pringle, and E. W. Jones (ed.), The ribosome, vol. LXVI. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    38. 38
  38. Winzeler, E. A., D. D. Shoemaker, A. Astromoff, H. Liang, K. Anderson, B. Andre, R. Bangham, R. Benito, J. D. Boeke, H. Bussey, A. M. Chu, C. Connelly, K. Davis, F. Dietrich, S. W. Dow, M. El Bakkoury, F. Foury, S. H. Friend, E. Gentalen, G. Giaever, J. H. Hegemann, T. Jones, M. Laub, H. Liao, R. W. Davis, et al. 1999. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901-906.[Abstract/Free Full Text]
    39. 39
  39. Wu, L. F., T. R. Hughes, A. P. Davierwala, M. D. Robinson, R. Stoughton, and S. J. Altschuler. 2002. Large-scale prediction of Saccharomyces cerevisiae gene function with overlapping transcriptional clusters. Nat. Genet. 31:255-265.[CrossRef][Medline]
    40. 40
  40. Zhang, J., and T. L. Madden. 1997. PowerBLAST: a new network BLAST application for interactive or automated sequence analysis and annotation. Genome Res. 7:649-656.[Abstract/Free Full Text]

Molecular and Cellular Biology, March 2003, p. 2083-2095, Vol. 23, No. 6
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.6.2083-2095.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Strunk, B. S., Karbstein, K. (2009). Powering through ribosome assembly. RNA 15: 2083-2104 [Abstract] [Full Text]  
  • Lamanna, A. C., Karbstein, K. (2009). Nob1 binds the single-stranded cleavage site D at the 3'-end of 18S rRNA with its PIN domain. Proc. Natl. Acad. Sci. USA 106: 14259-14264 [Abstract] [Full Text]  
  • Zemp, I., Wild, T., O'Donohue, M.-F., Wandrey, F., Widmann, B., Gleizes, P.-E., Kutay, U. (2009). Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2. JCB 185: 1167-1180 [Abstract] [Full Text]  
  • Kimmelman, A. C., Hezel, A. F., Aguirre, A. J., Zheng, H., Paik, J.-h., Ying, H., Chu, G. C., Zhang, J. X., Sahin, E., Yeo, G., Ponugoti, A., Nabioullin, R., Deroo, S., Yang, S., Wang, X., McGrath, J. P., Protopopova, M., Ivanova, E., Zhang, J., Feng, B., Tsao, M. S., Redston, M., Protopopov, A., Xiao, Y., Futreal, P. A., Hahn, W. C., Klimstra, D. S., Chin, L., DePinho, R. A. (2008). Genomic alterations link Rho family of GTPases to the highly invasive phenotype of pancreas cancer. Proc. Natl. Acad. Sci. USA 105: 19372-19377 [Abstract] [Full Text]  
  • Rosado, I. V., Dez, C., Lebaron, S., Caizergues-Ferrer, M., Henry, Y., de la Cruz, J. (2007). Characterization of Saccharomyces cerevisiae Npa2p (Urb2p) Reveals a Low-Molecular-Mass Complex Containing Dbp6p, Npa1p (Urb1p), Nop8p, and Rsa3p Involved in Early Steps of 60S Ribosomal Subunit Biogenesis. Mol. Cell. Biol. 27: 1207-1221 [Abstract] [Full Text]  
  • Bax, R., Raue, H. A., Vos, J. C. (2006). Slx9p facilitates efficient ITS1 processing of pre-rRNA in Saccharomyces cerevisiae. RNA 12: 2005-2013 [Abstract] [Full Text]  
  • DE BOER, P., VOS, H. R., FABER, A. W., VOS, J. C., RAUE, H. A. (2006). Rrp5p, a trans-acting factor in yeast ribosome biogenesis, is an RNA-binding protein with a pronounced preference for U-rich sequences. RNA 12: 263-271 [Abstract] [Full Text]  
  • Granneman, S., Nandineni, M. R., Baserga, S. J. (2005). The Putative NTPase Fap7 Mediates Cytoplasmic 20S Pre-rRNA Processing through a Direct Interaction with Rps14. Mol. Cell. Biol. 25: 10352-10364 [Abstract] [Full Text]  
  • Leger-Silvestre, I., Caffrey, J. M., Dawaliby, R., Alvarez-Arias, D. A., Gas, N., Bertolone, S. J., Gleizes, P.-E., Ellis, S. R. (2005). Specific Role for Yeast Homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis. J. Biol. Chem. 280: 38177-38185 [Abstract] [Full Text]  
  • LaRonde-LeBlanc, N., Wlodawer, A. (2005). A Family Portrait of the RIO Kinases. J. Biol. Chem. 280: 37297-37300 [Abstract] [Full Text]  
  • Lebaron, S., Froment, C., Fromont-Racine, M., Rain, J.-C., Monsarrat, B., Caizergues-Ferrer, M., Henry, Y. (2005). The Splicing ATPase Prp43p Is a Component of Multiple Preribosomal Particles. Mol. Cell. Biol. 25: 9269-9282 [Abstract] [Full Text]  
  • Borovjagin, A. V., Gerbi, S. A. (2005). An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA. Nucleic Acids Res 33: 4995-5005 [Abstract] [Full Text]  
  • Loar, J. W., Seiser, R. M., Sundberg, A. E., Sagerson, H. J., Ilias, N., Zobel-Thropp, P., Craig, E. A., Lycan, D. E. (2004). Genetic and Biochemical Interactions Among Yar1, Ltv1 and RpS3 Define Novel Links Between Environmental Stress and Ribosome Biogenesis in Saccharomyces cerevisiae. Genetics 168: 1877-1889 [Abstract] [Full Text]  
  • Dez, C., Froment, C., Noaillac-Depeyre, J., Monsarrat, B., Caizergues-Ferrer, M., Henry, Y. (2004). Npa1p, a Component of Very Early Pre-60S Ribosomal Particles, Associates with a Subset of Small Nucleolar RNPs Required for Peptidyl Transferase Center Modification. Mol. Cell. Biol. 24: 6324-6337 [Abstract] [Full Text]  
  • ROSADO, I. V., DE LA CRUZ, J. (2004). Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae. RNA 10: 1073-1083 [Abstract] [Full Text]  
  • VANROBAYS, E., GELUGNE, J.-P., CAIZERGUES-FERRER, M., LAFONTAINE, D. L.J. (2004). Dim2p, a KH-domain protein required for small ribosomal subunit synthesis. RNA 10: 645-656 [Abstract] [Full Text]  
  • Geerlings, T. H., Faber, A. W., Bister, M. D., Vos, J. C., Raue, H. A. (2003). Rio2p, an Evolutionarily Conserved, Low Abundant Protein Kinase Essential for Processing of 20 S Pre-rRNA in Saccharomyces cerevisiae. J. Biol. Chem. 278: 22537-22545 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vanrobays, E.
Right arrow Articles by Caizergues-Ferrer, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vanrobays, E.
Right arrow Articles by Caizergues-Ferrer, M.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»