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Originally published In Press as doi:10.1074/jbc.M506916200 on September 12, 2005

J. Biol. Chem., Vol. 280, Issue 46, 38177-38185, November 18, 2005
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Specific Role for Yeast Homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis*

Isabelle Léger-Silvestre{ddagger}, Jacqueline Marie Caffrey§1, Rosy Dawaliby{ddagger}1, Diana Alehandrovna Alvarez-Arias¶, Nicole Gas{ddagger}2, Salvatore J. Bertolone||, Pierre-Emmanuel Gleizes{ddagger}23, and Steven Robert Ellis§4

From the {ddagger}Laboratoire de Biologie Moléculaire des Eucaryotes (UMR5099) and Institut d'Exploration Fonctionnelle des Génomes (IFR109), CNRS and Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France and the Departments of §Biochemistry and Molecular Biology, Chemistry, and ||Pediatrics, University of Louisville, Louisville, Kentucky 40292

Received for publication, June 24, 2005 , and in revised form, August 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Diamond Blackfan anemia (DBA)5 is a severe hypoplastic anemia that generally presents early in infancy (1, 2). 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, 4). 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). 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 (6–10). 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 (11–13). Ribosomal RNAs are initially synthesized as a polycistronic transcript containing 18 S, 5.8 S, and 25 S rRNAs (14). 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). 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).

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). 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.



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  		FIGURE 1.
Alignment of yeast and human Rps19 proteins. Species designations are indicated as superscripts to the right of each sequence (Sc, S. cerevisiae; Hs, Homo sapiens). The numbers to the left indicate the position of the first residue listed in the amino acid sequence of each protein. The yeast Rps19B protein differs from the Rps19A protein shown by an alanine for proline substitution at residue 2. The line between the yeast and human sequences shows identical residues. Amino acid substitutions in DBA patients are underlined and shown below the human sequence (4, 29). The sequences in bold represent the hotspot for DBA mutations. Some mutations in the hot spot region are found in more than one patient.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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). 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). This plasmid was transformed into a diploid yeast strain heterozygous for RPS19A and RPS19B disruptions generated by crossing strains Y06271 ({Delta}rps19A/RPS19B) and Y11142 [GenBank] (RPS19A/{Delta}rps19B) 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). 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), 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 [{gamma}-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 6x SSC and analyzed by phosphorimaging.

Pulse-chase Analysis—Pulse-chase labeling of pre-rRNA was carried out as described previously (7). 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). Immunolocalization of TAP proteins was performed as described before (10). 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). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 {Delta}rps19B strain had reduced levels of free 40 S subunits and fewer polysomes relative to wild-type. The overall rate of ribosome synthesis in the {Delta}rps19B 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 {Delta}rps19B 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, 22).



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  		FIGURE 2.
Yeast Rps19 proteins are required for cell growth and division. A, outgrowth of spores derived from a diploid heterozygous at the RPS19A/rps19A::kanMX and RPS19B/rps19B::kanMX loci. The heterozygous diploid was sporulated, and tetrads were dissected. Haploid progeny were grown on YPD plates at 30 °C. RPS19A/B genotypes are listed below each tetrad. The genotype for double mutants was inferred. B, depletion of Rps19 by regulated expression from the GAL1 promoter inhibits growth. The strain labeled GAL-RPS19 has the chromosomal copies of RPS19A and RPS19B deleted and plasmid-borne RPS19A under the control of the GAL1 promoter as the only potential source of functional Rps19 protein. The WT strain has wild-type chromosomal copies of RPS19A and RPS19B in addition to the plasmid containing GAL1-RPS19A. Cells were maintained on galactose-containing media and then shifted to glucose-containing media to repress expression from the GAL1 promoter. Cell growth was monitored by optical density measurements at 600 nm.

 
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.



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  		FIGURE 3.
Strains containing disrupted alleles of RPS19 are deficient in 40 S ribosomal subunits. Cell extracts were prepared as described by Baim et al. (18) and loaded onto 7–47% sucrose gradients. Gradients were centrifuged at 18,000 rpm for 16 h. Gradients were broken down using an ISCO 185 gradient fractionator and absorbance at 254nm was measured using a UA-6 absorbance detector. Strain designations are listed above each panel. The wild-type and {Delta}rps19B strains were grown in YPD media and harvested in mid-exponential phase. The GAL-RPS19 strain was grown in synthetic complete media containing 2% galactose and shifted to media containing 2% glucose. Cells were harvested both prior to the nutritional shift, GAL-RPS19(Gal), and after 3 h in glucose, GAL-RPS19(Glc).

 
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). 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), 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, 10). 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 strongly suggest that microautophagy of the nucleus is induced by reduced levels of Rps19, a mechanism previously proposed to be involved in down-regulation of ribosome biogenesis (23).



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  		FIGURE 4.
Analysis of ribosomal RNA processing in Rps19-depleted cells. A, pulse-chase analysis of rRNA synthesis. Wild-type and GAL1-RPS19 cells were grown on galactose and then shifted to glucose-containing media. Seven hours after the nutritional shift, cells were harvested, concentrated, and pulse-labeled with [3H]methionine as described under "Experimental Procedures." After pulse labeling, cells were chased for 0, 2, 5, and 10 min in media containing unlabeled methionine. Total RNA was isolated from cells at each time point, fractionated on formaldehyde agarose gels, and transferred to Zetaprobe membrane. The membrane was exposed to Kodak BioMax MS film at –70 °C with a Kodax LE-intensifying screen. B, Northern analysis was performed on total RNAs isolated from GAL-RPS19 cells grown on galactose (Gal) or shifted for 4 and 8 h to glucose-containing media (Glc). RNAs were fractionated on formaldehyde-agarose gels and transferred to nylon membranes. Membranes were blotted with oligonucleotidic probes complementary to mature rRNAs or to transcribed spacers listed under "Experimental Procedures."

 
Rps19 Is Necessary for Recruitment of Pre-ribosomal Factors—To get insight into the stage at which the assembly of the 40 S particle is blocked upon depletion of Rps19, we examined association of several non-ribosomal factors with pre-rRNAs in the GAL-RPS19 strain. We used TAP-tagged genes encoding Noc4, Enp1, Tsr1, and Rio2 as a means of studying the association of these proteins with pre-ribosomal particles. In wild-type cells, Noc4 is found associated with 90 S particles, whereas Tsr1 and Rio2 are present in pre-40 S particles. Enp1 is found associated with both types of pre-ribosomes. The tagged proteins were precipitated with IgG-Sepharose beads, and the co-purifying pre-rRNAs were analyzed by Northern blots with the D-A2 probe (Fig. 7A). Detection of the 25 S rRNA, which does not associate with these tagged proteins, showed that less than 0.1% of this RNA (relative to input) co-precipitated non-specifically (not shown).



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  		FIGURE 5.
Pre-rRNA processing in S. cerevisiae. A, major pre-rRNA processing pathway. Letters designate the endonucleolytic and exonucleolytic cleavage points. Circles indicate the cleavages required to proceed to the next step. B, alternative pathway in Rps19-depleted cells. Cleavage at point A3 substitutes deficient processing at point A2 to yield the 21 S and 27S-A3 pre-rRNAs. The latter is then normally converted to the mature 5.8 S and 25 S species, whereas processing of the former is blocked.

 
In wild-type cells, Noc4 co-precipitated with RNA species found in the 90 S particles (35 S and 32 S), but not significantly with 20 S pre-rRNA. As shown previously, the precursor containing 20 S rRNA was instead isolated in significant amounts in co-precipitates with Enp1, Tsr1, and Rio2. Upon depletion of Rps19, this pattern was dramatically altered. The amount of pre-rRNA co-precipitated with Enp1, Tsr1, and Rio2 was drastically reduced, indicating that these proteins no longer associate with pre-ribosomes in the absence of Rps19. Interestingly, the amount of 21 S pre-rRNA co-precipitating with Noc4 was significantly higher under these conditions, which suggests that some of the proteins associated with the 90 S precursors remain in the abnormal pre-40 S particles, which accumulate in the absence of Rps19.



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  		FIGURE 6.
Depletion of Rps19 results in nuclear retention of pre-40 S particles and induces microautophagy of the nucleus. A, detection of pre-18 S rRNA by FISH with a probe hybridizing within the D-A2 domain of ITS1. Wild-type and GAL-RPS19 cells cultured in galactose (Gal) containing medium were put in the presence of glucose (Glc) for 4 h before fixation. Arrowheads point to the nucleoplasm revealed by DAPI staining. The cytoplasm is indicated by an asterisk. Images of wild-type and Gal-RPS19 cells were created with the same exposure time. B, examples of cells deleted of either RPS19A or RPS19B treated as in A. C, electron micrographs of wild-type and GAL-RPS19 cells after 4 h of culture in glucose-containing medium. Insets illustrate parallel situations as seen by FISH with the D-A2 probe. Cy, cytoplasm; CW, cell wall; Np, nucleoplasm; Nu, nucleolus; Va, vacuole.

 
We next localized the TAP-tagged proteins by immunofluorescence. As seen in Fig. 7B, Noc4-TAP, which is a nucleolar protein in wild-type cells, was also localized in part to the nucleoplasm in GAL-RPS19-depleted cells. This pattern was very similar to that of the pre-40 S particles (Fig. 6A), consistent with the association of Noc4 with the 21 S pre-rRNA detected by TAP purification. In contrast, the late associating Rio2 remained mostly cytoplasmic as in wild-type cells. Enp1 and Tsr1 localization switched from partially cytoplasmic in wild-type cells to fully nuclear in Rps19-depleted cells. The distribution of these two proteins within the nucleus was homogeneous, and thus differed from the ITS1 FISH signal, which remains predominantly nucleolar under these conditions. We therefore infer that, although not binding to pre-40 S particles, these proteins are nevertheless imported in the nucleus.

These data show that Rps19 is necessary for recruitment of certain non-ribosomal factors normally found associated with pre-40 S particles. Absence of these factors may preclude processing at site A2 and further maturation of the precursors to the 18 S rRNA.

Missense Mutations Identified in DBA Patients Influence Growth and rRNA Maturation When Incorporated into the Yeast RPS19A Gene— The results described above indicate that null alleles of RPS19A and RPS19B influence rRNA processing by interfering with cleavage at the A2 site within ITS1. Most of the mutations identified to date in the RPS19 gene of DBA patients would be similar to these null alleles either through large deletions, splicing defects, or frameshift mutations that would interfere with the production of functional Rps19 protein. In addition, several patients have missense mutations in the RPS19 gene, which are presumably responsible for the disease (Fig. 1). We report here a novel missense mutation identified in a DBA patient, which results in a proline for leucine substitution at position 64 in the human protein. This change occurs in the Rps19 consensus sequence adjacent to the hot spot for missense mutations in DBA patients. This polymorphism has not been reported in the RPS19 sequence of over 50 normal control individuals and as such, may represent a pathogenic lesion.6 However, whether this mutation or other reported missense mutations associated with DBA affect Rps19 function has not been determined. Many of these missense mutations fall in regions of Rps19 highly conserved between yeast and humans. Thus, we analyzed the effects of several of these missense mutations on growth and pre-rRNA processing in yeast.

The GAL-RPS19 strain was transformed with a second plasmid carrying mutant alleles of RPS19A under control of its own promoter. When this strain is grown on glucose-containing media, the mutant alleles of RPS19A are the primary source of functional Rps19 protein available to support growth. As shown in Fig. 8A, the I65P substitution, similar to the L64P mutation found in the DBA patient reported here, completely abolishes the ability of the mutant Rps19A protein to support growth on glucose indicating that this missense mutation has a severe effect on Rps19 function. Two other mutations studied, R63Q (position 62 in the human sequence) and I15F, also have demonstrable effects on the ability of the yeast Rps19A protein to support growth. In contrast, two other missense mutations, A62S (codon 61 in humans) and G121S (codon 120 in humans), do not influence the ability of the yeast protein to support growth. Interestingly, A62S is no longer considered a pathogenic alteration in DBA patients, and as such, would not be considered likely to have an affect on Rps19 function.7



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  		FIGURE 7.
Behavior of pre-ribosomal factors in Rps19-depleted cells. A, detection of pre-18 S rRNAs co-purifying with TAP-tagged Noc4, Enp1, Tsr1, and Rio2 in Rps19-depleted cells. GAL-RPS19 cells expressing one of the TAP-tagged proteins were grown for 4 h in YP-glucose medium, and total extracts were submitted to immunoprecipitation with IgG-Sepharose. Co-precipitating RNAs were revealed after Northern blot with a probe hybridizing between point D and A2 in ITS1. All samples, were treated in the same conditions; one representative input lane (0.1% of the input) is displayed. The levels of the TAP-tagged proteins were checked in inputs by Western blotting with an anti-protein A antibody. Semi-quantification of the fraction of precipitated RNA was performed by PhosphorImager analysis. B, immunolocalization of TAP-tagged Noc4, Enp1, Tsr1, and Rio2 in WT and GAL-RPS19 strains cultured 4 h in the presence of glucose. Immunofluorescence was carried out with anti-protein A antibodies. Arrowheads indicate the nucleoplasm as visualized by DNA staining with DAPI. The cytoplasm is designated by asterisks.

 
We next determined the influence of these mutations on ribosome synthesis by isolating RNA from each of these strains 22 h after shifting from galactose- to glucose-containing media. Northern blot analysis (Fig. 8B) revealed that strains where growth is compromised by the presence of a DBA-related missense mutation in RPS19A also show increased 21 S rRNA relative to strains where growth is not affected. Moreover, the pre-40 S particles produced in these mutants were retained in the nucleus and accumulated in the nucleolus, as seen by in situ hybridization (Fig. 8C). Thus, there is a close correspondence between the ability of DBA-related missense mutations to affect growth in yeast and their ability to abrogate the function of Rps19 in ribosome biogenesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The gene encoding ribosomal protein S19 is the only gene known to be involved in the pathogenesis of Diamond Blackfan anemia. Approximately 25% of DBA patients have mutations in RPS19. The remaining cases of DBA are of unknown etiology. Studies in the yeast S. cerevisiae have shown that some ribosomal proteins are required for the efficient maturation of ribosomal subunits (6–8, 10). These studies have linked individual ribosomal proteins to specific steps in rRNA processing during subunit maturation. We therefore turned to yeast to gain a better understanding of the function of the Rps19 family of proteins. Disruption of either of the yeast RPS19 genes caused a reduction in growth rate and a deficiency of 40 S ribosomal subunits. Disruption of both RPS19 genes is lethal. This observation is consistent with recent data showing that a homozygous RPS19 deletion in mice is lethal prior to the blastocyst stage indicating that the mammalian Rps19 protein also plays a critical role in cell growth and division (24).



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  		FIGURE 8.
Missense 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.

 
Our data reveal a critical role of Rps19 in the production of the small ribosomal subunit. The most striking defect induced by depletion of Rps19 is a strong inhibition of cleavage at site A2 within ITS1 and of the subsequent maturation of the 3'-end of the 18 S rRNA. The endonucleolytic cleavage at A2 takes place within the nucleolus and results in release of pre-40 S and pre-60 S particles from the 90 S ribosomal precursor. Absence of Rps19 preferentially affects the production of 40 S ribosomal subunits, as cleavage at site A3 still occurs, giving rise to a normal intermediate in the pathway leading to 60 S subunits. The 21 S pre-rRNA is not a dead-end product of the 40 S production pathway, and it may be converted into mature 18 S rRNA, as seen with some mutations in the RRP5 gene (25, 26). However, the 21 S rRNA-containing pre-40 S particles, which accumulate in Rps19-depleted cells are retained in the nucleus and are inefficiently if at all, processed to mature 40 S subunits.

In addition to the structural components of the ribosome, numerous other non-ribosomal factors must assemble with the pre-ribosomes for various steps in pre-rRNA processing and transport. Our data show that the aberrant pre-40 S particles that accumulate in Rps19-depleted cells lack the non-ribosomal factors Enp1, Tsr1, and Rio2, which indicate a major failure in the assembly of the pre-40 S particles. In wild-type cells, Tsr1 and Rio2 are found associated with pre-40 S subunits after the 90 S pre-ribosome is split by cleavage at A2. They are not required for A2 cleavage, but are instead needed for cleavage at site D, which yields mature 18 S rRNA in the cytoplasm (10, 27). The failure of aberrant pre-40 S subunits found in cells depleted of Rps19 to recruit Rio2 could be a consequence of the retention of these particles within the nucleus/nucleolus, as Rio2 is predominantly cytoplasmic. Tsr1, on the other hand, is present in the nucleus and the nucleolus suggesting that the aberrant pre-40 S subunits may be unable to recruit Tsr1 even within the nucleolus. In wild-type cells, Enp1 is a component of early 90 S and late 40 S pre-ribosomes (16). Thermosensitive mutants of ENP1 show a strong accumulation of 21 S rRNA at non-permissive temperatures similar to the phenotype observed here for RPS19 mutants (28). Thus Rps19 and Enp1 could cooperate within the 90 S pre-ribosomes to facilitate cleavage at A2. In addition, the presence of Noc4, a component of the small subunit processome, in the aberrant pre-40 S particles that accumulate in Rps19-depleted cells indicates that dissociation of factors specific to the 90 S particles may also be defective. Together, these data indicate that Rps19 acts as an essential protein for some of the early remodeling steps in the maturation of pre-ribosomes to 40 S ribosomal subunits.

Rps19 haploinsufficiency is thought to give rise to DBA. In many cases, DBA patients harbor mutations in an RPS19 gene that deletes the gene entirely, interferes with pre-mRNA splicing, or affects translation by introducing frameshifts or premature termination codons (4, 29). Less frequently, DBA patients have been identified that have missense mutations in RPS19. These missense mutations, particularly those that arise spontaneously, are considered pathogenic if they are not found in the general population and/or in unaffected family members. As such, they are classified as pathogenic solely on the basis of sequence analysis. The observation, that many of these missense mutations fall in regions of RPS19 highly conserved between the human and yeast genes, led us to assess the effect of these amino acid substitutions on the function of the yeast protein. Three of the mutations studied, including a novel mutation reported here, have measurable effects on Rps19 function in yeast. These missense mutations yield alterations of pre-rRNA processing equivalent to those observed in cells lacking Rps19, indicating that they result in the loss of function of the protein. Of the two mutations that did not affect the function of the yeast protein, the A62S mutation is no longer considered pathogenic and, as such, would not be expected to affect function. The reason why the G121S does not affect the function of the yeast protein is less clear. It is possible that the glycine to serine codon change at position 120 in the human sequence may be an innocuous polymorphism rather than a pathogenic mutation. Alternatively, the affect of this change on function may be more specific to the human protein and not be manifested by the yeast protein. Because the identification of pathogenic mutations in RPS19 is important for assessing genotype/phenotype correlations in DBA, these data reveal a critical need for the ability to monitor Rps19 function in mammalian systems.

Other diseases have recently been identified that result from defects in genes known to affect ribosome synthesis. The X-linked form of dyskeratosis congenita is caused by mutations in the dyskerin gene, which encodes a pseudouracil synthase involved in ribosomal RNA processing and telomere maintenance (30, 31), whereas the Treacher Collins syndrome gene TCOF1 is involved in ribosomal DNA transcription (32). Each of these diseases, including DBA, has defining characteristics that differ from one another, but all exhibit extreme clinical heterogeneity with some overlap in secondary characteristics between diseases. These data indicate that additional genetic or environmental factors significantly influence the nature and course of each disease. Other diseases that may result from defects in ribosome synthesis that also show considerable clinical heterogeneity include cartilage hair hypoplasia (33) and Shwachman-Diamond syndrome (34, 35). Thus, the failure to synthesize normal amounts of ribosomal subunits is a contributing factor to the clinical profile of a growing number of human diseases.


   FOOTNOTES
 
* This work was supported in part by grants from the Kentucky Lung Cancer Research Program and NHLBI, National Institutes of Health (to S. R. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 Received funds from the CNRS, the French Ministry of Research (Actions Concertées Incitatives Microbiologie) and the Association pour la Recherche contre le Cancer (Paris, France). Back

3 To whom correspondence may be addressed. Tel.: 33-561-335-926; Fax: 33-561-335-886; E-mail: gleizes{at}ibcg.biotoul.fr. 4 To whom correspondence may be addressed. Tel.: 502-852-5222; Fax: 502-852-6222; E-mail: srellis{at}louisville.edu.

5 The 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. Back

6 H. Gazda, personal communication, Dana Farber Cancer Institute. Back

7 S. Ball, personal communication, St. George's Hospital Medical School. Back


   ACKNOWLEDGMENTS
 
We thank Valérie Choesmel, Sébastien Ferreira-Cerca, Cécile Guinefoleau, and Marlène Faubladier for their participation to this study.



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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
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