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Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein

2001; Springer Nature; Volume: 20; Issue: 15 Linguagem: Inglês

10.1093/emboj/20.15.4204

1460-2075

Emmanuel Vanrobays, Pierre‐Emmanuel Gleizes, Cécile Bousquet‐Antonelli, Jacqueline Noaillac‐Depeyre, Michèle Caizergues‐Ferrer, Jean‐Paul Gélugne,

RNA Research and Splicing

Article1 August 2001free access Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein Emmanuel Vanrobays Emmanuel Vanrobays LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Pierre-Emmanuel Gleizes Pierre-Emmanuel Gleizes LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Cécile Bousquet-Antonelli Cécile Bousquet-Antonelli Present address: Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, EH9 3JR UK Search for more papers by this author Jacqueline Noaillac-Depeyre Jacqueline Noaillac-Depeyre LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Michèle Caizergues-Ferrer Michèle Caizergues-Ferrer LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Jean-Paul Gélugne Corresponding Author Jean-Paul Gélugne LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Emmanuel Vanrobays Emmanuel Vanrobays LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Pierre-Emmanuel Gleizes Pierre-Emmanuel Gleizes LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Cécile Bousquet-Antonelli Cécile Bousquet-Antonelli Present address: Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, EH9 3JR UK Search for more papers by this author Jacqueline Noaillac-Depeyre Jacqueline Noaillac-Depeyre LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Michèle Caizergues-Ferrer Michèle Caizergues-Ferrer LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Jean-Paul Gélugne Corresponding Author Jean-Paul Gélugne LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France Search for more papers by this author Author Information Emmanuel Vanrobays1, Pierre-Emmanuel Gleizes1, Cécile Bousquet-Antonelli2, Jacqueline Noaillac-Depeyre1, Michèle Caizergues-Ferrer1 and Jean-Paul Gélugne 1 1LBME du CNRS, 118 route de Narbonne, 31062 Toulouse, cedex 04, France 2Present address: Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, EH9 3JR UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:4204-4213https://doi.org/10.1093/emboj/20.15.4204 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Numerous non-ribosomal trans-acting factors involved in pre-ribosomal RNA processing have been characterized, but none of them is specifically required for the last cytoplasmic steps of 18S rRNA maturation. Here we demonstrate that Rio1p/Rrp10p is such a factor. Previous studies showed that the RIO1 gene is essential for cell viability and conserved from archaebacteria to man. We isolated a RIO1 mutant in a screen for mutations synthetically lethal with a mutant allele of GAR1, an essential gene required for 18S rRNA production and rRNA pseudouridylation. We show that RIO1 encodes a cytoplasmic non-ribosomal protein, and that depletion of Rio1p blocks 18S rRNA production leading to 20S pre-rRNA accumulation. In situ hybridization reveals that, in Rio1p depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. This strongly suggests that Rio1p is involved in the cytoplasmic cleavage of 20S pre-rRNA at site D, producing mature 18S rRNA. Thus, Rio1p has been renamed Rrp10p (ribosomal RNA processing #10). Rio1p/Rrp10p is the first non-ribosomal factor characterized specifically required for 20S pre-rRNA processing. Introduction In eukaryotic cells, most steps of ribosomal subunit biogenesis take place in a specialized nuclear structure, the nucleolus, which can be seen primarily, but not exclusively, as the ribosome factory. In the yeast Saccharomyces cerevisiae, transcription by RNA polymerase I of the repeated rDNA genes produces a primary transcript that not only contains the mature ribosomal RNAs (rRNAs), but also internal and external spacer sequences. The primary transcript is chemically modified and undergoes endo- and exonucleolytic reactions (see Figure 1) to lead ultimately to the mature rRNAs of 25S and 5.8S found in the large ribosomal subunit of 60S, and to the 18S rRNA found in the small ribosomal subunit of 40S. Concomitantly with the maturation process, numerous ribosomal and non-ribosomal proteins associate with the pre-rRNA intermediates to generate the pre-60S and 43S pre-ribosomal subunits, precursors to the 60S and 40S ribosomal subunits, respectively. These pre-ribosomal subunits are eventually exported to the cytoplasm, where the final assembly and maturation steps occur (for recent and thorough reviews see Kressler et al., 1999; Venema and Tollervey, 1999). Figure 1.Pre-rRNA processing in S.cerevisiae. (A) Structure of the 35S pre-rRNA. In 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 A0–E, and oligonucleotide probes used in northern blot hybridizations by numbers 1–6. (B) 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, generating the 27SA3 pre-rRNA rapidly digested by the 5′–3′ exonucleases Rat1p and Xrn1p to site B1S yielding the 27SBS pre-rRNA. This constitutes the major pathway. Approximately 15% of the 27SA2 molecules are processed at site B1L leading to the 27SBL intermediate. Processing at site B2, the 3′ end of the 25S rRNA, occurs concomitantly with 27SB 5′-end formation. The 27SBS and 27SBL pre-rRNAs both 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. The final maturation of the 20S pre-rRNA by an endonucleolytic cleavage at site D occurs in 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. Download figure Download PowerPoint Processing intermediates formed during 35S pre-rRNA maturation were characterized early in the course of rRNA processing studies. The identification and functional characterization of the trans-acting factors involved in this process constituted one of the 'key questions' in the early nineties (Woolford and Warner, 1991). During the past decade, thanks to the development of new biochemical, genetic and in silico technologies, dozens of these components have been identified in yeast. These are small nucleolar components of ribonucleoprotein particles (snoRNPs) or non-ribosomal nucleolar proteins: putative helicases, ribonucleases and other factors, the actual functions of which have not been defined yet (for reviews see Kressler et al., 1999; Venema and Tollervey, 1999). Although the processing cleavage sites along the 35S precursor have been precisely determined, the endoribonucleases acting on these sites have been identified for very few of them, namely the snoRNP MRP at site A3 and Rnt1p within the 3′ external transcribed spacer (3′ETS) (for review see Venema and Tollervey 1999). Early cleavage steps of the 35S pre-rRNA at sites A0, A1 and A2 yield the 20S and 27SA pre-rRNAs, precursors to the rRNAs of the small ribosomal subunit and the large ribosomal subunit, respectively. In S.cerevisiae, mature 18S rRNA is then formed through an endoribonucleolytic cleavage of the 20S pre-rRNA at site D (Figure 1), which occurs in the cytoplasm (Udem and Warner, 1973; Trapman et al., 1975; Stevens et al., 1991). Impairment of this processing step results in 20S pre-rRNA accumulation and concomitant 18S rRNA depletion. This phenotype could stem from defects in 43S pre-subunit assembly and/or nucleocytoplasmic transport as well, as demonstrated for Ran regulators or nucleoporin mutants (Moy and Silver, 1999). While numerous factors involved in the early cleavages of the 35S pre-rRNA have been identified, very few factors specifically required for the processing of 20S pre-rRNA into 18S rRNA have been described. Deletion of DRS2 (Ripmaster et al., 1993) or of RPS31/UBI3, which encodes a ribosomal protein from the small subunit (Finley et al., 1989), results in decreased efficiency of the conversion of 20S pre-rRNA to 18S rRNA, which is still produced, but at slower rates. On the contrary, deletion of both RPS0A and RPS0B, two redundant genes encoding the rpS0 ribosomal proteins, leads to an arrest of 18S rRNA production correlated with 20S pre-rRNA accumulation (Ford et al., 1999). Here we report the characterization of a new protein specifically required to process the 20S pre-rRNA to 18S rRNA. This factor, encoded by the essential RIO1/RRP10 gene (Angermayr and Bandlow, 1997), has homologues in all eukaryotes and archaebacterial organisms sequenced so far. We demonstrate that RIO1/RRP10 encodes a cytoplasmic non-ribosomal protein. Its inactivation results in drastic inhibition of cleavage at site D, leading to a strong accumulation of the 20S pre-rRNA in the cytoplasm and to the inhibition of 18S rRNA production. Rio1p/Rrp10p is the first non-ribosomal protein specifically required for 20S pre-rRNA processing in the cytoplasm to be characterized. We suggest that Rio1p/Rrp10p could catalyse, or be part of a complex catalysing, the endonucleolytic cleavage of 20S pre-rRNA at site D. Results A synthetic lethal screen with gar1-10 mutant identifies RIO1 Gar1p, one of the core proteins of H/ACA snoRNPs (Lubben et al., 1995; Ganot et al., 1997; Henras et al., 1998), is an essential protein required for the pseudouridylation of rRNAs and for 18S rRNA synthesis (Girard et al., 1992; Bousquet-Antonelli et al., 1997). In order to characterize factors involved in these processes, we carried out synthetic lethal (sl) screens with mutant alleles of GAR1. Strain YO89 is one of the three non-sectoring, 5′-fluoroorotic acid (5-FOA)-sensitive clones, isolated in a screen undertaken with the gar1-10 temperature-sensitive allele (see Materials and methods and Venema et al., 1997). This clone regained a sectoring phenotype and segregated FOA-resistant (FOAr) cells upon transformation with pJPG67 carrying the GAR1+ allele, but not upon transformation with a vector carrying the gar1-10 allele, demonstrating that it carries mutation(s) synthetically lethal with gar1-10. Strain YO89 was crossed with a MATa derivative of the parental strain YO126, the diploid clones obtained could segregate FOAr cells, and upon tetrad dissection, a 2:2 segregation of the sl character was observed, demonstrating that it is caused by a single recessive mutation. To clone a wild-type allele of this sl mutation, strain YO89-13, a derivative of YO89 in which the LEU2::gar1-Δ disruption has been replaced by a TRP1::gar1-Δ allele, was transformed with a yeast multi-copy genomic library carried by the 2μ LEU2 vector pFL46S (Bonneaud et al., 1991). Eight leu+ sectoring colonies that could grow on 5-FOA plates were obtained (loss of pJPG203: ARS, CEN, ADE3, GAR1). Among these, seven had received from the library a GAR1-containing plasmid since they could segregate lysine-requiring cells (loss of pJPG225: ARS CEN, LYS2, gar1-10). The library plasmid was recovered from the remaining transformant and shown to complement the sl phenotype upon retransformation into strain YO89-13. This plasmid, pEV6, was partially sequenced and shown to carry a fragment from chromosome XV (from nucleotide 548 151 to nucleotide 552 896) containing three complete open reading frames (ORFs): (i) GCY1, which encodes an aldo/keto reductase of unknown substrate specificity (Angermayr and Bandlow, 1997); (ii) an essential gene RIO1 (Angermayr and Bandlow, 1997); and (iii) an uncharacterized ORF, YOR121C (included in GCY1 ORF). When a plasmid carrying the subcloned RIO1 wild-type allele (pEV21) was introduced in the sl strain YO89, red/white sectoring and growth on 5-FOA medium were restored, whereas this was not the case for plasmid pEV22 carrying the rio1-1 mutant allele derived from YO89 genomic DNA (see Materials and methods). Furthermore, this same plasmid pEV22 allows growth in glucose-containing medium of strain YO296, which carries a RIO1+ allele under the control of the GAL10 promoter, demonstrating that the mutation responsible for the colethality with gar1-10 indeed lies within the RIO1 gene. This gene has previously been identified in S.cerevisiae, and was shown to be essential for cell viability (Angermayr and Bandlow, 1997). Occurrence of genes homologous to RIO1 in archaebacteria, Emericella nidulans (formerly Aspergillus nidulans), Caenorhabditis elegans and Homo sapiens has been reported (Anaya et al., 1998). Our search in databases identified homologues of Rio1p in all eukaryotic and archaebacterial sequenced genomes (for sequence alignments see Supplementary figure 1 in the Supplementary data, available at The EMBO Journal Online). The predicted polypeptide has an unusual composition, containing 18% glutamic and aspartic acids and 11.5% serine and threonine. The only mutation found in the rio1-1 allele that causes lethality in gar1-10 strains is a missense mutation leading to the change of the universally conserved threonine 62 in the S.cerevisiae protein to isoleucine. Since a mutant allele of RIO1 was retrieved as synthetically lethal with a mutant allele of the GAR1 gene, the product of which is clearly required for pre-rRNA processing and post-transcriptional modification (Girard et al., 1992; Bousquet-Antonelli et al., 1997), we investigated the possible involvement of Rio1p in the production of mature rRNAs. As shown below, RIO1 appears to be involved in the processing of pre-rRNA and consequently it has been renamed RRP10 (ribosomal rNA processing #10). Depletion of Rrp10p affects 18S rRNA production Since RIO1/RRP10 has been shown to be an essential gene (Angermayr and Bandlow, 1997), a conditional RIO1/RRP10 allele was constructed by replacing its promoter by the inducible GAL10 promoter. The 5′ end of this construct was also tagged with two protein A epitopes (see Supplementary data for the description of this construction). Transcription driven by the GAL10 promoter allows high expression of the gene in culture media that contain galactose (YPG, YNB Gal), but is repressed in culture media containing glucose (YPD, YNB Glu), leading to the depletion of ProtA-Rrp10p (see Supplementary figure 2). We made use of this conditional RRP10 allele to determine whether Rrp10p is necessary for pre-rRNA processing. We first assessed the steady-state levels of mature rRNAs by ethidium bromide staining and northern blotting of RNAs prepared from cells grown in permissive YPG medium or shifted to non-permissive YPD medium for 2–24 h. As shown in Figure 2A, depletion of Rrp10p has no effect on 25S rRNA accumulation but drastically affects the level of 18S rRNA. Furthermore, after 16 h culture in YPD medium, RNA molecules that migrate similarly to the 20S pre-rRNA accumulate at sufficient level to be detected by ethidium bromide staining (Figure 2A). Figure 2.Depletion of Rrp10p specifically affects the steady-state levels of mature 18S rRNA species and results in 20S pre-rRNA accumulation. Northern blot analysis of pre-rRNA processing: YO296 (GAL::PROTA-RRP10) cells were grown in YPG medium (Gal), or in YPD medium for up to 24 h (Glu). At the indicated time points, total RNA was extracted and separated in 1% agarose-formaldehyde gels to analyse 35S, 32S, 27SA2, 25S, 20S and 18S species, and in 6% polyacrylamide gels for 7S(L), 7S(S) and 5.8S species analysis. Equal amounts of total RNA (5 μg) were loaded in every lane. (A) Ethidium bromide staining of the gel. Northern blots of the gel were hybridized to: (B) probe 2 complementary to ITS1 upstream of site A2; (C) probe 1 complementary to 18S rRNA, a 7 h exposure (upper panel in C) and a 90 min exposure (lower panel in C) of the same blot are shown; (D) probe 4 complementary to 5.8S, probe 5 complementary to ITS2 and probe 6 complementary to 25S rRNA. Pre-rRNA and rRNA species are schematically represented on the right side; full rectangles represent the mature rRNAs and thin lines the transcribed spacers. Download figure Download PowerPoint To determine whether this RNA species is related to the 20S pre-rRNA, precursor to the 18S rRNA, northern hybridizations using probe 1 complementary to the 18S rRNA, and probe 2 complementary to an ITS1 sequence between sites D and A2 (see Materials and methods and Figure 1), were performed. Results of these hybridizations are shown in Figure 2B and C. The RNA species that accumulates in Rrp10p depleted cells is detected with probes 1 and 2, and migrates to the same position as the normal 20S pre-rRNA intermediate (Figure 2B and C, lane Gal). Probing the same blot with probe 3 (complementary to ITS1 sequences between sites A2 and A3) did not reveal any pre-rRNA intermediate of this size or close to it (data not shown), indicating that the RNA species accumulating in Rrp10p depleted cells is the 20S pre-rRNA that extends from the 5′ end of 18S to site A2 in ITS1 (Figure 1). These data show that depletion of Rrp10p severely impairs 18S rRNA production and leads to accumulation of its 20S pre-rRNA precursor. Phosphorimager quantifications of the 20S and 18S bands in the northern blot shown in Figure 2C reveal a 40-fold reduction in the amount of 18S rRNA and a 20-fold increase of the 20S pre-rRNA levels, in cells grown for 16 h in YPD medium compared with the ones in cells grown in galactose medium. Considering the observed evolution of the 18S/25S ratio, and that the same amount of total cellular RNA has been loaded in every lane and the corresponding number of cells used to get this amount of RNA (given the dramatic inhibition of 18S rRNA production upon depletion of Rrp10p, more cells, collected from the later time point samples of the culture in YPD medium, were needed to get the same amount of total cellular RNA), the actual drop in 18S rRNA level is even more dramatic than 40-fold. Moreover, the small amount of 18S rRNA found in these cells (Figure 2A) probably stems from incomplete depletion of Rrp10p due to leaks in glucose transcriptional repression of the GAL10 promoter and/or survival of Rrp10p molecules produced before or early after the switch to glucose medium. Consequently we think that upon deletion of Rrp10p, production of 18S rRNA is completely inhibited. It also appears that the 20S pre-rRNA does not accumulate stoichiometrically with respect to 25S rRNA, although arising from the processing of the very same 35S pre-rRNA molecules. This suggests that the 20S pre-rRNA is intrinsically unstable if not converted to 18S rRNA and degraded by the exosome complex (Allmang et al., 2000), or that Rrp10p depletion leads to a less stable form of this precursor, possibly due to some defect in the assembly of the 43S pre-subunits, or both. The high molecular weight blot shown in Figure 2B was also probed with probes 5 and 6, complementary to the ITS2 and the 25S rRNA, respectively (see Figure 1 and Materials and methods). A low molecular weight blot was probed with probes 4 (complementary to the 5.8S rRNA) and 5 (complementary to the ITS2). Apart from what was already observed for 18S and 20S rRNAs, neither changes in the levels of normal pre-rRNA processing intermediates, nor accumulation of aberrant species was detected upon depletion of Rrp10p in these experiments (Figure 2D). The kinetics of pre-rRNA processing and rRNA accumulation were analysed in (methyl-3H) methionine pulse–chase experiments (Figure 3). In a GAL::PROTA-RRP10 strain grown in the presence of galactose, most of the labelled 27S and 20S pre-rRNAs have disappeared after a 10 min chase. In contrast, after 20 min of chase, 20S pre-rRNA is still present and 18S rRNA is only faintly detectable in cells from the same GAL::PROTA-RRP10 strain grown for 14 h in glucose-containing medium. On the contrary, Rrp10p depletion does not affect the time course of the 27S pre-rRNA conversion to 25S rRNA. Moreover, no other pre-rRNA intermediate is observed during the chase experiment, indicating that the processing pathways leading to the 20S pre-rRNA and 25S rRNA are fully functional in Rrp10p depleted cells, while 18S rRNA formation is strongly inhibited in such conditions due to a defect in 20S pre-rRNA processing. Figure 3.Depletion of Rrp10p results in reduced synthesis of 18S rRNA. (A) YO296 (GAL::PROTA-RRP10) strain grown at 30°C on YNB Gal without methionine; (B) YO296 grown at 30°C in YPG medium (Gal) then shifted for 14 h to YPD medium (Glu), and finally grown for 9 h in YNB Glu without methionine. Cells were labelled for 4 min with (methyl-3H) methionine and chased with a large excess of unlabelled methionine for 1–20 min. Download figure Download PowerPoint In order to determine whether Rrp10p depletion specifically affects the processing of 20S pre-rRNA, levels of small nuclear and nucleolar RNAs were also assessed in cells of strain YO296 shifted to YPD medium. All tested snoRNAs were found to be unaffected (data not shown). Likewise, levels of the RNase MRP RNA and of U1 snRNA were not altered even after 24 h of growth in YPD medium. Northern hybridizations with probes specific for ACT1 and rpS11 ribosomal protein mRNAs did not reveal any defect in pre-mRNA processing or accumulation (data not shown). Altogether these data show that depletion of Rrp10p strongly and specifically affects processing of the 20S pre-rRNA to 18S rRNA. Cells depleted of Rrp10p accumulate 20S pre-rRNA in the cytoplasm In yeast, it has been shown previously that the last steps of 18S rRNA maturation occur in the cytoplasm. After cleavage at site A2, the 20S pre-rRNA is exported from the nucleolus as part of a 43S pre-ribosomal particle and is subsequently processed at site D by an unknown endonuclease (Udem and Warner, 1973; Trapman et al., 1975), leading to 18S rRNA and a D-A2 5′-ITS1 fragment that is degraded by the cytoplasmic 5′–3′ exoribonuclease Xrn1p (Stevens et al., 1991). Thus, accumulation of the 20S pre-rRNA may be either due to nuclear retention of the 43S particles, precursors of the small ribosomal subunit, or to a defect in the cytoplasmic processing of 20S pre-rRNA. To sort out these two possibilities, the localization of the 20S pre-rRNA accumulating in Rrp10p depleted cells was determined by in situ hybridization with a probe complementary to the D-A2 fragment. Ultrastructural localization of the probe was analysed by electron microscopy (Figure 4). In wild-type cells (Figure 4A) and in YO296 cells grown in galactose medium (not shown), the labelling, which corresponds to the 35S, 32S or 20S precursors, is essentially restricted to the nucleolus. In contrast, Rrp10p depleted cells exhibit both a nucleolar and a cytoplasmic labelling (Figure 4C), a phenotype similar to the one observed in a Δxrn1 strain (Figure 4B). Thus, 20S pre-rRNA accumulation in Rrp10p depleted cells does not result from a defect in the nuclear export of the 43S particle, but rather from the impairment of the cytoplasmic endonucleolytic processing of 20S pre-rRNA. Figure 4.Cells depleted of Rrp10p accumulate 20S pre-rRNA in the cytoplasm. Electron microscopic detection of the ITS1: (A) in wild-type yeast cells, the labelling is exclusively detected in the nucleolus (35S, 33S, 32S and 20S pre-rRNAs); (B) in Δxrn1 cells, the labelling is found in the nucleolus and in the cytoplasm (fragment D-A2 from the ITS1); (C) in GAL::PROTA-RRP10 cells grown for 16 h in YPD medium (Glu), the labelling is detected in the nucleolus and in the cytoplasm. np, nucleoplasm; no, nucleolus; ct, cytoplasm. Bar, 200 nm. Download figure Download PowerPoint Rrp10p is a non-ribosomal cytoplasmic protein Subcellular localization of Rrp10p was investigated using a green fluorescent protein (GFP)-Rrp10p fusion protein. Strain YO296 (GAL::PROTA-RRP10) regained the ability to grow on YPD upon transformation with the pEV24 plasmid, which directs expression of the GFP-Rrp10p construct, showing that this fusion is functional. In strain JG540 (wild type) expressing a GFP-RRP10 fusion allele (pEV24), the green fluorescence is detected in the cytoplasm (Figure 5A). In the same strain, a fusion of the nucleolar protein Gar1p with the GFP localizes to the nucleolus, as expected (Girard et al., 1992) (Figure 5B). The cytoplasmic localization of Rrp10p is confirmed by immunodetection of a ProtA-Rrp10p fusion protein. Immunodetection of ProtA-Rrp10p and Gar1p-ProtA fusions by electron microscopy is shown in Figure 5C and D, respectively. Gar1p is found in the nucleolus, but Rrp10p exclusively localizes to the cytoplasm. Since Rrp10p is required for the maturation of the 20S pre-rRNA and is located in the cytoplasm, we assessed the possibility that Rrp10p is a ribosomal protein. In this analysis ProtA-Rrp10p could not be detected in either ribosomal subunit, whereas, as expected, the ribosomal proteins rpL3 and rpL32 are present in the 60S subunit, and rpS4 in the 40S subunit (see Supplementary figure 3). Moreover, Rrp10p is not found in the 80S ribosomes fractions of the gradient shown in Figure 6A (see below). These results show that although localized in the cytoplasm and required for cleavage at site D of the 20S pre-rRNA, Rrp10p is not a ribosomal protein. In agreement with these data, Rrp10p was not referenced in the recent compilation of ribosomal proteins (Planta and Mager, 1998). Figure 5.Rrp10p is a cytoplasmic protein. Subcellular localization using GFP-tagged proteins (A and B). (A) GFP-Rrp10p: cells transformed by pEV24 (GFP::RRP10) exhibit a cytoplasmic pattern of fluorescence. (B) Gar1p-GFP: in cells transformed with pZUT3 (GAR1::GFP) a punctate pattern characteristic of a nucleolar staining is observed. Positions of nuclei were determined by DAPI staining (blue). Overlay images are shown by superposition of the blue and green stainings. Immunolocalization by electron microscopy (C and D). (C) ProtA-Rrp10p; (D) Gar1p-ProtA. Tagged proteins were detected by treatment with anti-protein A antibodies followed by incubation with colloidal gold-conjugated protein A. no, nucleolus; np, nucleoplasm; ct, cytoplasm. Bars in C and D, 200 nm. Download figure Download PowerPoint Figure 6.Sedimentation profiles of ProtA-Rrp10p and 20S pre-rRNA in a glycerol gradient. A total extract was loaded on a 10–30% glycerol gradient and subjected to centrifugation. Fractions were collected, and proteins and RNA in each fraction were analysed. (A) Sedimentation profiles of ProtA-Rrp10p, rpS4p, rpL3p and rpL32p. Proteins precipitated with TCA and separated by SDS–PAGE were revealed by western blotting. (B) Sedimentation pattern of 18S and 25S rRNAs and of 20S pre-rRNA. Fractions containing the peaks of 40S, 60S ribosomal subunits and 80S ribosomes are indicated. (C) 20S pre-RNA selectively co-purifies with Rrp10-ProtAp. 1/400 of the clarified cell lysate (T) or the bulk of the immunoprecipitated RNA (IP) was probed with either a mix of probes 1 and 6 in Figure 1 (18S, 25S) or a D-A2 probe prepared by multiprime labelling (20S). The fraction of each rRNA or pre-rRNA recovered in the immunoprecipitate was determined by phosphoimager quantification (IP/T). Download figure Download PowerPoint Rrp10p is associated with higher order structures but not found in translating ribosomes To assess whether Rrp10p is present in the cytoplasm as free molecules or associated with higher order structures, a cellular extract from strain YO296 expressing ProtA-Rrp10p was fractionated by sedimentation through 10–30% glycerol gradients, and fractions from this gradient were analysed for their protein and RNA contents. The relative positions of 40S, 60S ribosomal subunits and 80S monosomes were determined by probing the RNA extracted from the fractions with 18S- and 25S-specific probes. Probing the same blot with a 20S pre-rRNA specific probe (fragment D-A2 in Figure 1) revealed that this precursor of 18S rRNA is exclusively found in particles that exhibit a sedimentation coefficient of ∼40S (Figure 6B). Likewise, immunodetection with PAP antibody showed that Rrp10p sediments in the very same fractions as 20S pre-rRNA (Figure 6A). Less than 5% of the ProtA-Rrp10p fusion protein is found in the first fractions of the gradient corresponding to soluble factors, and none is detected in the 80S fraction (Figure 6A). Thus, although Rrp10p is associated with higher order structures with a sedimentation coefficient of ∼40S, it is found neither in monosomes nor in polysomes, indicating that Rrp10p is not an integral component of translating ribosomes. Since Rrp10p depletion blocks 20S pre-rRNA processing, and Rrp10p is not found in monosomes or polysomes, the observed cose