Specific Role for Yeast Homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis
2005; Elsevier BV; Volume: 280; Issue: 46 Linguagem: Inglês
10.1074/jbc.m506916200
ISSN1083-351X
AutoresIsabelle Léger‐Silvestre, Jacqueline M. Caffrey, Rosie Dawaliby, Diana A. Alvarez-Arias, Nicole Gas, Salvatore Bertolone, Pierre‐Emmanuel Gleizes, Steven R. Ellis,
Tópico(s)RNA and protein synthesis mechanisms
ResumoApproximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3′-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis. Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3′-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis. Diamond Blackfan anemia (DBA) 5The abbreviations used are: DBA, Diamond Blackfan anemia; FISH, fluorescence in situ hybridization; rRNA, ribosomal RNA; ITS1, internal transcribed spacer 1; TAP, tandem affinity purification; DAPI, 4′, 6-diamidino-2-phenylindole. is a severe hypoplastic anemia that generally presents early in infancy (1.Freedman M.H. Baillieres Clin. Hematol. 2000; 13: 391-406Crossref PubMed Scopus (23) Google Scholar, 2.Willig T.-N. Gazda H. Sieff C.A. Hematology. 2000; 7: 85-94Google Scholar). Other clinical features of DBA are heterogeneous with some patients presenting craniofacial abnormalities, growth failure, predisposition to cancer, and other congenital abnormalities. Most cases of DBA arise spontaneously, with only a small proportion exhibiting familial transmission typically showing autosomal dominant inheritance. Approximately 25% of the DBA cases have been linked to mutations in the gene encoding ribosomal protein S19 (RPS19), and in these cases haploinsufficiency for this ribosomal protein gives rise to the disease (3.Draptchinskaia N. Gustavsson P. Andersson B. Pettersson M. Willig T.N. Dianzani I. Ball S. Tchernia G. Klar J. Matsson H. Tentler D. Mohandas N. Carlsson B. Dahl N. Nat. Genet. 1999; 21: 169-175Crossref PubMed Scopus (668) Google Scholar, 4.Willig T.N. Draptchinskaia N. Dianzani I. Ball S. Niemeyer C. Ramenghi U. Orfali K. Gustavsson P. Garelli E. Brusco A. Tiemann C. Perignon J.L. Bouchier C. Cicchiello L. Mohandas N. Tchernia G. Blood. 1999; 94: 4294-4306PubMed Google Scholar). The remaining cases are of unknown etiology. The Rps19 protein is a component of the small ribosomal subunit, and as such, defects in some aspect of ribosome structure, function, or synthesis may be an underlying cause of DBA. The human Rps19 protein belongs to a family of ribosomal proteins restricted to eukaryotes and archea. Rps19 does not have a homolog in the eubacterial ribosome where the properties of individual ribosomal proteins have been most extensively studied. As such, little is known regarding the function of members of the eukaryotic Rps19 family. In prokaryotes, ribosomal proteins play critical roles in ribosomal assembly through their interactions with each other and rRNA (5.Nomura M. Hill W.E. Dahlberg A. Garrett R.A. Moore P.B. Schlessinger D. Warner J.R. The Ribosome: Structure, Function, and Evolution. American Society for Microbiology, Washington, D. C.1990: 3-55Google Scholar). These interactions promote both steps in rRNA processing important for subunit maturation and the formation of active sites in mature subunits necessary for ribosome function. The precise functions of the ribosomal proteins in the production of the subunits in eukaryotes are poorly characterized, which calls for a more detailed investigation of the role played by Rps19 in ribosome synthesis and function. The yeast Saccharomyces cerevisiae has proven to be an outstanding system to investigate factors involved in eukaryotic ribosome synthesis, including ribosomal proteins (6.Baudin-Baillieu A. Tollervey D. Cullin C. Lacroute F. Mol. Cell. Biol. 1997; 17: 5023-5032Crossref PubMed Scopus (59) Google Scholar, 7.Ford C.L. Randall-Whitis L. Ellis S.R. Cancer Res. 1999; 59: 704-710PubMed Google Scholar, 8.Tabb-Massey A. Caffrey J.M. Logsden P. Taylor S. Trent J.O. Ellis S.R. Nucleic Acids Res. 2003; 31: 6798-6805Crossref PubMed Scopus (31) Google Scholar, 9.Jakovljevic J. de Mayolo P.A. Miles T.D. Nguyen T.M. Leger-Silvestre I. Gas N. Woolford J.L. Mol. Cell. 2004; 14: 331-342Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10.Leger-Silvestre I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). To date, ribosome synthesis in yeast has been broken down to a number of well characterized intermediates, and numerous factors required for discrete steps in the pathway have been identified (11.Fatica A. Tollervey D. Curr. Opin. Cell Biol. 2002; 14: 313-318Crossref PubMed Scopus (425) Google Scholar, 12.Fromont-Racine M. Senger B. Saveanu C. Fasiolo F. Gene. 2003; 313: 17-42Crossref PubMed Scopus (480) Google Scholar, 13.Tschochner H. Hurt E. Trends Cell Biol. 2003; 13: 255-263Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Ribosomal RNAs are initially synthesized as a polycistronic transcript containing 18 S, 5.8 S, and 25 S rRNAs (14.Venema J. Tollervey D. Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (652) Google Scholar). The mature rRNAs are derived from the primary transcript by a series of processing steps most of which occur within the nucleolus. Pre-rRNA processing occurs concomitantly with the assembly of ribosomal proteins onto the rRNA transcripts. The earliest pre-rRNAs are found in a large ribonucleoproteic precursor of 90 S, which splits into pre-40 S and pre-60 S particles after endonucleolytic cleavage in the internal transcribed spacer 1 (ITS1) at site A2 (see Fig. 5). Through proteomic analysis, non-ribosomal factors specifically associated with these pre-ribosomal particles at various maturation stages have been identified (15.Milkereit P. Kuhn T. Gas N. Tschochner H. Nucleic Acids Res. 2003; 31: 799-804Crossref PubMed Scopus (47) Google Scholar). Pre-40 S particles have been found to contain a set of specific nonribosomal proteins that associate in the nucleus and accompany the particles into the cytoplasm where they are required for the final maturation of the small subunit (16.Schafer T. Strauss D. Petfalski E. Tollervey D. Hurt E. EMBO J. 2003; 22: 1370-1380Crossref PubMed Scopus (248) Google Scholar). In yeast, Rps19 is encoded by duplicated genes, RPS19A and RPS19B, which differ from one another by a single amino acid. The yeast Rps19 proteins have over 50% sequence identity with the human protein (Fig. 1). The sequence similarity spans the bulk of the yeast and human proteins and includes blocks of near complete identity. One of these blocks, residues 52–63 in humans, is a hot spot for amino acid substitutions in patients with DBA (4.Willig T.N. Draptchinskaia N. Dianzani I. Ball S. Niemeyer C. Ramenghi U. Orfali K. Gustavsson P. Garelli E. Brusco A. Tiemann C. Perignon J.L. Bouchier C. Cicchiello L. Mohandas N. Tchernia G. Blood. 1999; 94: 4294-4306PubMed Google Scholar). Other amino acid substitutions found in DBA patients that occur outside of this region also fall in residues that are either identical or have conservative substitutions between yeast and humans. Given this high degree of conservation, studies on the function of the yeast Rps19 protein should provide insight into the function of its human homolog and processes affected in Diamond Blackfan anemia. Here we show that Rps19 is an essential protein that is required for the synthesis of the small ribosomal subunit. This phenotype in Rps19-depleted cells is linked to a clear pre-rRNA processing defect at site A2 and to the nuclear retention of 40 S subunit precursors, which fail to recruit certain non-ribosomal factors involved in subunit maturation. In addition, a similar defect in pre-rRNA processing is induced by the expression of yeast Rps19 mutants carrying missense mutations found in DBA patients. These results indicate that Rps19 is a key protein in ribosome biogenesis and establish a link between partial or total loss of function of the DBA-linked protein Rps19 and a strong defect in pre-rRNA processing and subunit maturation. Yeast and Bacterial Strains—The yeast strains used in this study were generated by the Saccharomyces genome deletion project consortium and obtained from Research Genetics or Euroscarf. Media used in cultivating yeast were YPD (1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose) and synthetic (0.67% (w/v) yeast nitrogen base without amino acids and 2% (w/v) glucose). Where appropriate, nutrients were added to synthetic media in amounts specified by Sherman (17.Sherman F. Methods Enzymol. 1991; 194: 1-21PubMed Google Scholar). In some experiments glucose was replaced as a carbon source by 0.2% (w/v) sucrose and 2% (w/v) galactose. Diploids were sporulated on solid sporulation media (1% (w/v) potassium acetate, 0.1% (w/v) yeast extract, 0.05% (w/v) glucose, and 2% (w/v) agar). Additional nutrients were added to sporulation media in amounts corresponding to 25% of that used in synthetic media. A haploid yeast strain (GAL-RPS19), with chromosomal alleles of RPS19A and RPS19B disrupted and growth supported by a plasmid-borne allele of RPS19A under control of the GAL1 promoter, was created to analyze the effects of depletion of both Rps19 proteins. The RPS19A gene was amplified from genomic DNA by PCR and subcloned into pFL38-GAL (10.Leger-Silvestre I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). This plasmid was transformed into a diploid yeast strain heterozygous for RPS19A and RPS19B disruptions generated by crossing strains Y06271 (Δrps19A/RPS19B) and Y11142 (RPS19A/Δrps19B) obtained from Euroscarf. Resulting transformants were sporulated, and tetrads were dissected on rich media with 2% (w/v) galactose as carbon sources. Haploid progeny were initially tested for growth on 2% (w/v) glucose to identify strains whose growth was galactose-dependent. The status of the chromosomal alleles of RPS19A and RPS19B in these strains was assessed using oligonucleotides flanking the kanMX disruption cassette in each gene. Two haploid strains were identified that had growth dependent on the plasmid-borne allele of RPS19A under control of the GAL1 promoter. The Escherichia coli strain used in this study was XL1-Blue (Stratagene, La Jolla, CA). RPS19 Mutations—The UAS and entire coding sequence of RPS19A were amplified by PCR using Pfu Ultra Hotstart polymerase (Stratagene, La Jolla, CA). The PCR product was cloned into pRS315. Missense mutations in RPS19 reported for DBA patients were introduced at corresponding positions in the RPS19A gene using the Genetailor site-directed mutagenesis kit (Invitrogen). Mutations introduced into RPS19A were confirmed by sequencing, which also ruled out the introduction of spurious mutations during the mutagenesis procedure. Plasmids carrying either wild-type or mutant alleles of RPS19A were transformed into the GAL-RPS19 strain growth on 2% galactose. The functions of RPS19A alleles on the pRS315 plasmid were analyzed after shifting growth to media containing 2% glucose. Polysome Analysis—Polysomes were prepared and fractionated on 7–47% sucrose gradients as described by Baim et al. (18.Baim S.B. Pietras D.F. Eustice D.C. Sherman F. Mol. Cell. Biol. 1985; 5: 1839-1846Crossref PubMed Scopus (100) Google Scholar). Centrifugation was generally carried out at 18,000 rpm for 16 h in an SW28.1 rotor (Beckman Instruments, Fullerton, CA). Sucrose gradients were fractionated, and absorbance at 254 nm was monitored using an ISCO model 185 gradient fractionator and a UA-6 absorbance detector. Data were digitized using UN-SCAN-IT software (Silk Scientific, Orem, UT) or Adobe Photoshop. Northern Analysis—Total RNA was prepared from yeast by hot phenol extraction (19.Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1152) Google Scholar), separated on a 1% agarose gel, and passively transferred to a nylon membrane (Amersham Pharmacia Biotech). In Fig. 4, the membrane was hybridized overnight at 50 °C with the following oligonucleotides: probe 5′-A0 (5′-GCAGATCTGACGATCACC-3′), probe A0–A1 (5′-GATCGTTCTCCCTTACCCAC-3′), probe 18 S (5′-CATGGCTTAATCTTTGAGAC-3′), probe D-A2 (5′-TTAAGCGCAGGCCCGGCTGG-3′), probe A2–A3 (5′-GATTGCTCGAATGCCCAAAG-3′), probe E-C2 (5′-GGCCAGCAATTTCAAGTTA-3′) and probe 25 S (5′-CCATCTCCGGATAAACC-3′). In Fig. 8, membranes were hybridized overnight at 37 °C with probe A2–A3 (5′-ATGAAAACTCCACAGTG-3′) and probe 18S (5′-TGATCCTTCCGCAGGTTCACCTACGGAAAC-3′). The probes (10 ng) were labeled with [γ-32P]ATP using T4 polynucleotide kinase (Promega). After hybridization, the blots were washed twice in 0.1% SSC, 0.1% SDS, or 3 times in 6× SSC and analyzed by phosphorimaging.FIGURE 8Missense mutations found in DBA patients influence rRNA processing and affect the ability of RPS19A to support growth in yeast. The GAL-RPS19 strain was transformed with a second plasmid (pRS315) harboring alleles of RPS19A under the control of its own promoter. A, growth assay. Cultures were grown overnight in synthetic media lacking uracil and leucine with galactose as carbon source, diluted to 0.1 A600, subjected to 10-fold serial dilutions, and spotted on rich media containing galactose (Gal) or glucose (Glc). RPS19A alleles carried on the pRS315 plasmid are listed above each dilution series. B, Northern analysis. Strains were grown overnight in synthetic media lacking uracil and leucine with galactose as carbon source, transferred to glucose-containing media, and grown for 22 h before cells were harvested and total RNA prepared. Filters were hybridized with probe A2–A3 and a probe to mature 18 S rRNA. Values below each lane represent the ratio of 21 S pre-rRNA to 18 S rRNA derived from PhosphorImager analysis. The ratio in cells with RPS19A was arbitrarily set at 1. Other values were normalized against the RPS19A value. A.U., arbitrary units. C, pre-18 S rRNA FISH with a probe complementary to the D-A2 segment of ITS1. Strains were grown for 4 h in glucose-containing media. Arrows point to the nucleoplasm as visualized by DNA staining with DAPI. Asterisks indicate the cytoplasm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pulse-chase Analysis—Pulse-chase labeling of pre-rRNA was carried out as described previously (7.Ford C.L. Randall-Whitis L. Ellis S.R. Cancer Res. 1999; 59: 704-710PubMed Google Scholar). The GAL-RPS19 strain was used in the pulse-chase experiments. Cells were initially grown in synthetic media lacking uracil containing 0.2% (w/v) sucrose and 2% (w/v) galactose followed by depletion of Rps19 by shifting to synthetic media containing 2% (w/v) glucose. After 7 h in glucose-containing media, cells were recovered from 40 ml of each culture (A600 0.2–0.3) by centrifugation. Cells from both cultures were each suspended in 1 ml of glucose-containing synthetic media lacking uracil and methionine. Each culture was pulse-labeled for 2 min at 30 °C with 250 μCi of [methyl-3H]methionine. Labeled cells in 250-μl aliquots were diluted into synthetic media containing 1 mg/ml methionine and either placed on ice (0 chase period) or incubated for 2, 5, or 10 min at 30 °C. After the chase period cells were quickly chilled on ice prior to the isolation of total RNA. RNA was fractionated on 1.5% agarose-formaldehyde gels and transferred to Zeta-probe membrane. Membranes were baked for 2 h at 80 °C and exposed to BioMax MS film at –70 °C using a BioMax LE intensifying screen (Eastman Kodak Co., Rochester, NY). Film was exposed from 2 weeks to 1 month. The long development time resulted from the fact that the strains in the deletion collection are methionine auxotrophs. Inclusion of methionine in the culture medium during and after the shift to glucose reduces the efficiency of radiolabeling with [methyl-3H]methionine during the pulse period. Co-immunoprecipitation and Immunolocalization of TAP Proteins— Affinity purification of TAP-tagged Noc4p, Enp1p, Tsr1p, Ltv1p, Rio2p, and analysis of co-precipitating RNAs were performed as described previously (20.Dez C. Noaillac-Depeyre J. Caizergues-Ferrer M. Henry Y. Mol. Cell. Biol. 2002; 22: 7053-7065Crossref PubMed Scopus (58) Google Scholar). Immunolocalization of TAP proteins was performed as described before (10.Leger-Silvestre I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). Briefly, spheroplasts were incubated for 2 h at room temperature with rabbit anti-protein A antibodies (Sigma) in phosphate-buffered saline/bovine serum albumin (1:50,000 dilution). Fluorescence detection was achieved with Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) antibodies (Molecular Probes), and DNA was counterstained with DAPI. Images were captured with a Coolsnap ES camera (Roper Scientifics) mounted on a DMRB microscope (Leica) using the Metavue software (Universal Imaging). Fluorescence in Situ Hybridization and Electron Microscopy—Detection of pre-rRNA by FISH was performed as described by Gleizes et al. (21.Gleizes P.E. Noaillac-Depeyre J. Leger-Silvestre I. Teulieres F. Dauxois J.Y. Pommet D. Azum-Gelade M.C. Gas N. J. Cell Biol. 2001; 155: 923-936Crossref PubMed Scopus (46) Google Scholar). The sequence of the ITS1 oligonucleotidic probe is TT*GCACAGAAATCTCT*CACCGTTTGGAAT*AGCAAGAAAGAAACT*TACAAGCT*T (ITS1 probe), where T* represents amino-modified deoxythymidine conjugated to Cy3. Cells were prepared for electron microscopy by high pressure freezing (EM Pact, Leica). The samples were then transferred to –90 °C for cryosubstitution in acetone containing 0.2% uranyl acetate, 0.1% glutaraldehyde, 0.2% osmium tetroxide for 72 h. This fixative was then replaced by pure acetone, and temperature was raised to –45 °C for embedding in Lowicryl HM20 resin. The resin was polymerized under UV light for 2 days at –45 °C and 1 day at +20 °C. Rps19 Is Essential for Cell Viability—To determine the extent to which yeast cell growth and division is dependent on the RPS19 genes, strains deleted for one or the other RPS19 gene (obtained from the Yeast Deletion Collection) were crossed to create a diploid strain heterozygous for both RPS19 deletions. After sporulation, the resulting tetrads were dissected, and the haploid meiotic progeny from individual tetrads were examined for growth on rich media at 30 °C. The results from this analysis indicate that disruption of either RPS19 gene alone reduces growth rate, whereas spores fail to germinate when both genes are deleted (Fig. 2A). To examine whether Rps19 is required for vegetative growth, a strain (GAL-RPS19) was created where the chromosomal copies of RPS19A and RPS19B are disrupted and a plasmid-borne copy of RPS19A is expressed under control of the GAL1 promoter. These data reveal that growth ceases when cells are shifted to glucose and RPS19 expression is shut off, indicating that the yeast Rps19 proteins are essential for cell growth and division (Fig. 2B). Shortage of Rps19 Preferentially Affects the Production of 40 S Subunits—To address whether deleting either RPS19 gene influences the translational machinery we prepared cell extracts from wild-type and RPS19 deletion strains and passed these extracts through sucrose gradients to generate polysome profiles. This method provides a steady-state measure of free ribosomal subunits and ribosomes engaged in protein synthesis. Fig. 3 shows that extracts from the Δrps19B strain had reduced levels of free 40 S subunits and fewer polysomes relative to wild-type. The overall rate of ribosome synthesis in the Δrps19B strain is also decreased, which is likely secondary to the 40 S subunit deficiency and is correlated with reduced growth rate. Experiments with the GAL-RPS19 strain showed that the level of free 40 S subunits was reduced and free 60 S subunits increased 3 h after a shift to glucose-containing media (Fig. 3, compare GAL-RSP19(Gal) with GAL-RPS19(Glu)). Under these conditions the secondary effects on global ribosome synthesis are not as dramatic as those seen in the Δrps19B strain. Data for the RPS19 deletion strains are similar to results reported for disruption of genes encoding other ribosomal proteins that are essential components of the 40 S ribosomal subunit (8.Tabb-Massey A. Caffrey J.M. Logsden P. Taylor S. Trent J.O. Ellis S.R. Nucleic Acids Res. 2003; 31: 6798-6805Crossref PubMed Scopus (31) Google Scholar, 22.Demianova M. Formosa T.G. Ellis S.R. J. Biol. Chem. 1996; 271: 11383-11391Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Production of 18 S rRNA Is Blocked in the Absence of Rps19—The reduced amount of 40 S subunits in RPS19 deletion strains could be the consequence of a defect in subunit synthesis or enhanced subunit degradation. We examined flux through the yeast rRNA-processing pathway in wild-type and GAL-RPS19 cells by pulse-chase analysis. The strains were grown in glucose containing medium for 7 h to deplete Rps19 in the GAL-RP19 cells, and ribosomal RNA precursors were radiolabeled with a brief pulse of [methyl-3H]methionine. The radiolabeled methionine rapidly equilibrates with S-adenosylmethionine, which is used in the methylation of ribosomal RNA precursors. Methylation occurs early in the synthesis of rRNA, efficiently labeling the 35S primary transcript. Processing was then followed after chase periods in the presence of unlabeled methionine. Fig. 4A shows that during the labeling period wild-type cells rapidly processed the 35 S/32 S precursors to 27 S and 20 S species, which were then matured to 25 S and 18 S rRNAs in the first 2 min of the chase period. The GAL-RPS19 strain, in contrast, showed delayed processing to the 35 S and 32 S precursors during the labeling period. Although there was some delay in the production of 25 S rRNA upon depletion of Rps19, the amount of 25 S rRNA produced by 5 to 10 min of chase approached that observed in wild-type cells. The production of 18 S rRNA, on the other hand, was severely compromised in the absence of Rps19: maturation of the 18 S rRNA stopped after synthesis of a late precursor, which corresponds to 21 S pre-rRNA (see below). Thus, although flux through the entire rRNA processing pathway appears reduced in Rps19-depleted cells mutants relative to wild-type, steps leading to the production of 18 S rRNA are clearly impacted more severely than those leading to 25 S rRNA. Rps19 Is Required for Pre-rRNA Processing at Site A2—To determine more precisely the pre-rRNA processing defect(s) induced by the depletion of Rps19, we next performed Northern analysis on total RNAs isolated from GAL-RPS19 cells using oligonucleotidic probes (Fig. 4B). Cells were grown either on galactose or shifted for 4 and 8 h to glucose-containing medium to shut down Rps19 synthesis. An outline of the yeast rRNA processing pathway is shown in Fig. 5A. While the amount of 25 S rRNA stays roughly constant upon shutting down Rps19 expression the level of 18 S rRNA decreases (Fig. 4B, probes 18S+25S). The decrease in 18 S rRNA is accompanied by an increase in 35 S and 23 S precursors indicating that cells depleted of Rps19 are inefficient at cleaving rRNA precursors at the A0 and A1 sites within ETS1 (Fig. 4B, probes 5′-A0 and A0–A1). Blots with an oligonucleotide that hybridizes to sequences between the D and A2 cleavage sites revealed a severe reduction of the amount of 20 S pre-rRNA, a normal intermediate in the pathway leading to 40 S subunits, and an increase in what appears to be 21 S pre-rRNA, which extends from the 5′-end of mature 18 S rRNA to the A3 cleavage site (Fig. 4B, probe D-A2). An oligonucleotide complementary to the A2–A3 segment confirmed that this band was 21 S pre-rRNA (Fig. 4B, probe A2–A3). This probe also showed that the 23 S rRNA intermediate extends past the A2 cleavage site. Thus, cells depleted of Rps19 are defective in cleavage at the A2 site within ITS1, as illustrated in Fig. 5B. The relative amounts of 23 S and 21 S rRNA precursors accumulating in the Rps19-depleted cells indicate that the primary defect is in ITS1 with secondary effects on cleavage within ETS1, consistent with previous reports on the coupling between cleavage at sites A0, A1, and A2 sites (14.Venema J. Tollervey D. Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (652) Google Scholar). In parallel, the 27 S-A2 precursor was decreased in cells depleted of Rps19 providing further support for the influence of Rps19 on cleavage at the A2 site (Fig. 4B, probe A2–A3). In contrast, the levels of 27 S-A3 and 27 S-B showed only a modest reduction in Rps19-depleted cells (Fig. 4B, probe E-C2) indicating that relatively efficient cleavage of 35 S and 32 S precursors at site A3 can still generate normal intermediates in the pathway leading to mature 25 S rRNA. Very similar pre-rRNA profiles were obtained when only one RPS19 gene was deleted: the major processing defect observed in these strains was accumulation of 21 S pre-rRNA (data not shown). These data indicate that Rps19 is necessary for efficient maturation of the pre-rRNA ETS1 and pinpoint the strict requirement of this ribosomal protein for processing at the A2 site in the ITS1. Nuclear Retention of Pre-40 S Particles—We next examined the intracellular fate of the 21 S pre-rRNA-containing particles accumulating upon depletion of Rps19 and determined whether they can be exported to the cytoplasm or are retained in the nucleus and degraded. Fig. 6A shows the localization by fluorescence in situ hybridization of the pre-40 S particles with a probe complementary to the D-A2 domain of ITS1, in wild-type and mutant cells. As previously observed (10.Leger-Silvestre I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar), wild-type cells display labeling in the nucleolus, where most ribosome biogenesis is performed, and in the cytoplasm where conversion of the 20 S pre-rRNA to 18 S mature RNA occurs. In GAL-RPS19 cells depleted of Rps19, one observes a striking decrease of the cytoplasmic signal, which indicates that the pre-40 S particles are no longer efficiently exported from the nucleus. Precursors to the small subunit accumulate in the nucleus with most of the signal being restricted to the nucleolus, which can be distinguished from the nucleoplasm due to very low staining with DAPI. Labeling with a probe complementary to the A2–A3 segment also yields a nucleoplasmic signal in Rps19-depleted cells and not in wild type, consistent with the nuclear accumulation of pre-40 S particles containing 21 S pre-rRNA (data not shown). Single deletion of RPS19A or RPS19B is sufficient to induce a similar phenotype (Fig. 6B). These data are very reminiscent of our previous observations of cells depleted of Rps18, which is also required for efficient cleavage at the A2 cleavage site within ITS1 (8.Tabb-Massey A. Caffrey J.M. Logsden P. Taylor S. Trent J.O. Ellis S.R. Nucleic Acids Res. 2003; 31: 6798-6805Crossref PubMed Scopus (31) Google Scholar, 10.Leger-Silvestre I. Milkereit P. Ferreira-Cerca S. Saveanu C. Rousselle J.-C. Choesmel V. Guinefoleau C. Gas N. Gleizes P.-E. EMBO J. 2004; 23: 2336-2347Crossref PubMed Scopus (88) Google Scholar). The strong nucleolar signal is compatible with a defect in early pre-rRNA cleavage steps taking place in the nucleolus. Together with the accumulation of pre-rRNA precursors in the nucleus, another conspicuous feature in a majority of Rps19-depleted cells is the fact that the nucleolus (as seen with the D-A2 probe) becomes round and appears to detach from the DNA. We visualized this phenotype at a higher resolution by electron microscopy (Fig. 6C). In several sections, we observed what appeared to be the vacuole engulfing the nucleolus. These images stron
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