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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.
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
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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), were chosen for further functional analyses.
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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).
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RESULTS |
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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).
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::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.
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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.
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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.
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DISCUSSION |
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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).
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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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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