gar2 is a nucleolar protein from Schizosaccharomyces ... - Europe PMC

1912-1918 Nucleic Acids Research, 1995, Vol. 23, No. 11

Q--Z) 1995 Oxford University Press

gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation Marie-Pierre Gulli, Jean-Philippe Girard+, Dan Zabetakis1, Bruno Lapeyre§, Teri Melesel and Michble Caizergues-Ferrer* Laboratoire de Biologie Moleculaire Eucaryote du CNRS, 118 Route de Narbonne, 31062 Toulouse, France and 1Columbia University, Department of Biological Sciences, New York, NY, USA Received February 23, 1995; Revised and Accepted Aprl 27, 1995

ABSTRACT Several nucleolar proteins, such as nucleolin, NOP1/ fibrillarin, SSB1, NSR1 and GARI share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevislae. In addition, like these proteins, gar2 has a nucleolar localisatlon. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevsiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSRI. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA. INTRODUCTION In eukaryotes, most steps of ribosome biogenesis occur within a specialized nuclear domain, the nucleolus. The RNA polymerase I transcibes a primary single transcript, the pre-rRNA. In addition to 18S, 5.8S and 25S rRNAs sequences which are maintained in the mature rRNA forms, the pre-rRNA contains spacer regions not found in cytoplasmic ribosomes. Evidence exists that the growing pre-rRNA chain interacts with ribosomal proteins (1), non-ribosomal proteins and small nucleolar RNAs (snoRNAs) (2), leading to the formation of pre-ribosomal particles. In these RNP complexes, the pre-rRNA undergoes base modification, *

EMBL accession no. Z48166

methylation and a series of endo- and exonucleolytic cleavages (pre-rRNA processing) which remove the transcribed spacers. Still, little is known about the mechanisms involved in the post-transcriptional steps of ribosome production. Due to their specific nucleolar location, snoRNAs have long been thought to be required for ribosome biogenesis. Indeed roles in pre-rRNA processing have been clearly demonstrated for U3, U14, snR30, snR10 and MRP RNA in Saccharomyces cerevisiae (3-8) and for U3, U8 and U22 in higher eukaryotes (9-13). According to the generally accepted model, snoRNAs function via direct interactions with the pre-rRNA. Consistent with this idea, base pairing interactions with pre-rRNA has been established in the cases of yeast and mammalian U3 (14-17), snR30 (5) and mammalian U17/E1, E2 and E3 (18). In addition, a subset of snoRNAs from higher eukaryotes, including U16, U18, U20 and U21, contains phylogenetically conserved extended regions of perfect complementarity to rRNA sequences (reviewed in 19 and 20). This suggests that these snoRNAs might interact with various rRNA domains in the nucleolus. Factors able to modulate the structure of the pre-rRNA and the interactions between the pre-rRNA and snoRNAs or between pre-rRNA and proteins must clearly play an important role in ribosome synthesis. Indeed, two putative RNA helicases necessary for assembly of the 60S ribosomal subunit have been described (21,22). Futhermore, the GAR domain found in several nucleolar proteins such as nucleolin (23,24), fibrillarin/NOPI (25-28), SSB1 (29), NSR1 (30) and GARI (31) required for ribosome synthesis, has been reported to have the capacity to modify rRNA structure in vitro (32). The widespread occurrence of the GAR domain among nucleolar proteins characterized so far enabled us to undertake the isolation of novel nucleolar proteins from Schizosaccharomyces pombe using a cDNA fragment encoding the GAR domain of Xenopus fibrillarin as a probe. In this paper we report the characterization of a novel nucleolar protein of fission yeast called gar2. Although the overall structural organization of gar2 resembles of that of nucleolin, gar2 has only two RNA binding domains instead of the four found in nucleolin. The gar2+ gene

To whom correspondence should be addressed

Present addresses: +The Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA and §Division of Biochemistry and Molecular Biology, 401 Barker Hall, University of California at Berkeley, Berkeley, CA 94720, USA

Nucleic Acids Research, 1995, Vol. 23, No. 11 1913 disruption affects normal cell growth at 300C. Additionally, a gar2- mutant accumulates 35S pre-rRNA and has a decrease in the steady-state levels of 18S rRNA. Expression of gar2 in a S.cerevisiae nsrl- mutant restores the wild-type phenotype, suggesting that S.pombe gar2 is a functional homolog of S.cerevisiae NSR1.

MATERIALS AND METHODS Plasmids pBL1242. A 1.8 kb PCR product corresponding to the gar2+ cDNA was digested with BamHI and cloned into the BamHI site of the vector pAR3040, for expression of recombinant protein in Escherichia coli under the T7 promoter (33). pBL1252. A 2.1kb insert corresponding to the Hindlll genomic fragment of gar2+ was cloned into the BamHI-Sall site of the pBM272 vector (30). pBL1263. Contains a HindlIl fragment of 11 kb that corresponds to the entire rDNA repeated unit (34). This plasmid was either double digested with KpnI and NdeI (the resulting 1022 bp fragment was used as a 5' ETS probe) or digested with XbaI (the resulting 1460 bp fragment was used as a 18S probe). pWL1O. Contains a 1.6 kb BamHI-SalI fragment that corresponds to the entire coding region of NSR1 inserted into the BamHI-SalI site of pBM272 (30). All DNA manipulations and bacterial transformations were done according to published procedures (35).

Yeast strains

domain of Xenopus fibrillarin (26,38,39). A S.pombe subgenomic library was then constructed with size selected (2.1 kb) HindIll genomic fragments, following a strategy previously described (31,38,39). The isolated clone was characterized by restriction mapping and DNA sequencing using a sequenase kit (USB Corporation). The EMBL accession number is Z48166. A gar2+ cDNA was cloned using a PCR-based strategy previously described (38,39). Briefly, 1 ,ug of S.pombe total RNA was mixed with 1 ,ug of a BamHI-oligo(dT) oligonucleotide [CCCGGATCC(T)17] and denatured for 10' at 70°C before returning to ice. Reverse transcription was then performed using a BRL kit. A PCR amplification of the cDNA thus obtained with the BamHI-oligo(dT) primer and a second BamHI-G2P6 primer (5'-CCCGGATCCATGGCAAAAAAGGATAAAACC-3') complementary to the 5' end of the gar2+ ORF allowed us to obtain a 1.8 kb PCR product corresponding to the gar2 + cDNA. Bacterial expression of the gar2 protein and obtention of polyclonal serum The plasmid pBL1242 was introduced into Ecoli strain BL21 (DE3) LysS. Recombinant gar2 expression was induced with isopropyl P-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM (33). Induced E.coli protein extract was analyzed by 8% SDS-PAGE. Recombinant gar2 protein was electroeluted from 8% polyacrylamide gel and used to raise antibodies in rabbits (40). Antibodies against gar2 were purified from immune serum using recombinant protein as an affinity ligand. For the affinity column, 1 mg of gar2 was coupled with CNBr activated Sepharose (Pharmacia). In this case, the gar2 protein was purified from an E.coli extract by three successive chromatographies, Heparin Sepharose, Q Sepharose and Mono Q Sepharose.

Sp972. h-, this haploid wild-type strain was first described by U. Leupold. SplS. h+, ade6M210, leul-32, ura4DJ8 (gift from P. Fantes). SpJ6. h, ade6M216, leul-32, ura4DJ8 (generous gift from P. Fantes). SpJ8. h+/h-, ade6M210/ade6M216, 1eu132/1eu132, ura4D18/ura4DJ8 (obtained by conjugation of SplS and Spl6). SpJ9. h+/h, ade6M210/ade6M216, leul-32/leul-32, ura4D18/ ura4D18, gar2+/gar2::ura4+ (obtained by transformation of Spl8). Sp22. h-, ade6M216, leul-32, ura4DJ8 (gar2+). Sp24. h+, ade6M216, leul-32, ura4D18, gar2::ura4+(both latter strains were obtained after sporulation of the Spl9 diploid strain). WLY353. MATa, ade2-1, canl-100, ura3-1, leu2-3,112, trpl-1, his3-11,15, nsrl::HIS3 (30). S.pombe cells were cultured either in rich medium YES: 0.5% yeast extract, 3% glucose plus supplements or in EMM minimal medium (36). S.cerevisiae media used were YPG: 1% yeast extract (Difco Laboratories), 2% Bacto peptone (Difco) and 2% galactose, or SG: 0.67% yeast nitrogen base without amino acids (Difco) and 2% galactose. EMM and SD media were both supplemented as required. S.pombe and S.cerevisiae were transformed by standard techniques (36,37).

Iumunofluorescence microscopy The procedure followed was based on that of Hagan and Hyams (41). Cells were fixed in PEMS (100 mM PIPES pH 6.9, 1 mM EGTA, 1 mM MgSO4, 1 M Sorbitol) containing 3.7% formaldehyde for 1.5 h at room temperature. They were then washed three times in PEMS. For digestion of the cell wall, cells were resuspended at a density of 107 cells/ml in PEMS containing 0.3 mg/ml Lysing enzyme (Sigma) and 0.15 mg/ml Zymolyase 20T (ICN Biomedicals) and incubated 30 min at room temperature. After three washes in PEMS, cells were applied to poly-L-lysine coated slides, air dried and permeabilized by three quick washes in acetone at room temperature. Afterwards, cells were incubated at room temperature with primary anti-gar2 antibody at 1/4 dilution in PBS plus 0.05% Tween 20 for 1 h. Following three washes in the same buffer, cells were incubated with secondary antibody (anti-rabbit Ig-Texas Red linked from donkey; Amersham) in PBS plus 0.05% Tween 20, for 1 h at room temperature. After three more washes in PBS plus 0.05% Tween 20 and one in PBS, a drop of mounting medium containing 1 mg/ml p-phenylene diamine and 1 ,ug/ml 4',6-diamidino-2-phenylindole (DAPI) was applied to the air-dried cells. Slides were viewed with a Zeiss photomicroscope.

Isolation of the gene encoding the S.pombe gar2 protein A 2.1 kb HindHu fragment containing the gar2+ gene was first detected by Southern blot analysis of S.pombe genomic DNA, at low stringency with a cDNA probe corresponding to the GAR

Disruption of the gar2+ gene and tetrad analysis The chromosomal copy of the gar2+ gene was replaced with a partial gar2 deletion by the one-step gene disruption method (42). The 0.49 kb SacI-KpnI fragment of the gar2 + gene (aa 163-325)

1914 Nucleic Acids Research, 1995, Vol. 23, No. 11 was cut and replaced by the 1.8 kb SacI-KpnI fragment containing the S.pombe ura4+ gene from the plasmid pBKS+ura4 + (gift from G. Basi). A linear 3.7 kb fragment containing the ura4+ gene flanked by the sequences of the gar2 + gene (0.672 kb at 5' end and 0.860 kb at 3' end) was isolated and used to transform the diploid

ura4- strain Spl8 obtained from SpiS and Spl6. ura4+ transformants were checked by Southern analysis to confirm that the interrupted gene was integrated into the homologous gar2+ locus. Transformants which contained one wild-type copy and one disrupted copy of the gar2 +gene (gar2+1gar2:: ura4+) were sporulated, the tetrads dissected and analyzed according to (43). The steady-state levels of rRNAs were analyzed by Northern blot (35).

Ribosomal profiles Analysis of ribosomes was done essentially as described (44). S.pombe strains were grown in YES medium, while S.cerevisiae strains were grown overnight in SG medium to select for the plasmid, and then for several generations in YPG medium. Cells were collected and washed with buffer A (10 mM Tris-Cl pH 7.4, 100 mM NaCl, 30 mM MgCl2 and 50 ig of cycloheximide/ml). Cells were lysed with glass beads and the supernatants collected. Ribosomes were analyzed by centrifugation through a sucrose gradient (50 mM Tris-acetate pH 7, 50 mM NH4Cl, 12 mM MgCl2, 1 mM dithiothreitol and 7, 17, 27, 37 and 47% sucrose). Gradient were centrifuged at 89 000 g for 5.5 h in a SW27 rotor (Beckman). Samples were analyzed with an ISCO model 640 gradient fractionator and the effluent was monitored as OD254.

RESULTS Isolation and characterization of the gar2+ gene The similarities between the GAR domains of the two nucleolar proteins nucleolin and fibrillarin are sufficient to obtain, in a low stringency hybridization experiment, a cross reaction between the DNA sequences encoding these domains (26). In order to characterize new genes encoding proteins containing GAR domains in S.pombe, we first performed Southern analysis using total genomic DNA from S.pombe digested either by EcoRl or by HindIu and a fragment of the Xenopus laevis fibrillarin cDNA as a probe (38,39). Four strong hybridization signals were detected, cwrresponding to HinduI fragments of 5.2 kb, 3.5, 2.2 and 2.1 kb (38). In order to clone the corresponding fragments, we prepared and screened different subgenomic libraries, each containing the sized fragments 5.2, 3.5, 2.2 and 2.1 kb. The isolated clones have been analyzed and sequenced. The 5.2 kb fragment has not been further characterized, the 3.5 kb fragment contains the S.pombe gene for fibrillarin (39), the 2.2 kb fragment contains the S.pombe gari + gene (38), the equivalent of a S.cerevisiae gene that we have characterized (31) and the 2.1 kb fragment corresponds to a new S.pombe gene. This 2.1 kb HindIl fragment has been mapped and sequenced on both strands. Sequence analysis reveals the presence of a 1500 bp open reading frame (ORF) that could encode a protein of 500 residues. After data base comparison, it appeared to be a new protein of S.pombe containing a GAR domain located at the C-terminus of the protein; therefore, we have called it gar2. Since many genes of S.pombe, as opposed to S.cerevisiae, do contain one or more introns, we have cloned a gar2 + cDNA from S.pombe total RNA using a PCR-based strategy (see Materials

and Methods). Detailed restriction mapping, followed by sequencing using different primers, allowed us to confirm the absence of introns within this gene (data not shown). A Northern blot was performed using total RNA from S.pombe to define the size of the gar2 mRNA. A unique S.pombe mRNA of 1750 nt was detected (39), which is consistent with the size of the gar2+ ORF and the absence of intron within the gene.

gar2 is structurally related to NSR1 and nucleolin While the characterization of gar2 was being carrried out, the sequence of a protein of S.cerevisiae, called NSR1, was reported (30). The alignment of the amino-acid sequences of the two proteins reveals that gar2 and NSR1 exhibit 53% identity (Fig. lA). In addition, the structure of these two proteins is strongly reminiscent of that of nucleolin from vertebrates. Like nucleolin and NSR1, gar2 has four different domains (Fig. iB). The first domain of 80 residues is reminiscent of the N-terminal domain of nucleolin, containing numerous doublets of lysine surrounded by hydrophobic residues and a potential cdc2 phosphorylation site (S50PKK). In nucleolin, eight threonine residues in the N-terminal domain are phosphorylated in vitro and in vivo by the cdc2 kinase (45,46). The major difference between nucleolin or gar2 and NSR1, is that the latter has only a very short N-terminal basic domain and does not contain a cdc2 phosphorylation site. The second domain in gar2 is formed by long stretches of serines and acidic residues. Similar serine/acidic rich domains are found in NSR1 while the four acidic domains of nucleolin are different in the sense that they contain more acidic than serine residues. In gar2, NSR1 and nucleolin some serine residues in the acidic domain are CKII phosphorylation sites (47; Gulli, Faubladier and Caizergues-Ferrer, unpublished data). The third domain is the putative RNA binding domain, formed by the repetition of a long conserved domain of -80 residues, which is present in a large family of nuclear RNA binding proteins and is referred to as the RRM (for RNA recognition motif; 48,49). gar2 and NSR1 contain two RRMs and nucleolin contains four RRMs. The fourth domain is the GAR domain. The typical motif FGGRGG, found in multiple copies in the nucleolar GAR proteins (31) is repeated twice in the gar2 protein. This motif has been described as a target for post-translational modifications such as methyl incorporation in the form of N9,N9-dimethylarginine in various nuclear RNA binding proteins, e.g. hnRNP A and B proteins, fibrillarin and nucleolin (50 and references therein).

gar2 is a nucleolar protein of S.pombe The expression of the gar2+ gene in S.pombe, was analyzed by Western blots. Rabbit antibodies against gar2 have been produced and affinity purified (see Materials and Methods). Western blot analysis of crude protein extracts from S.pombe with these antibodies revealed a single protein that migrates on SDS-PAGE as a 72 kDa polypeptide (result not shown). This apparent molecular mass of the gar2 protein is larger than the predicted 52 kDa calculated from the sequence. Such an abnormal mobility during electrophoresis is probably due to the presence in gar2 of long stretches of serine/acidic residues, as previously shown for other nucleolar proteins containing acidic stretches (51). The affinity-purified antibodies were used to localize gar2 by immunofluorescence. 4',6-diamidino-2-phenylindole (DAPI) stains both mitochondrial and nuclear DNA in fixed S.pombe cells. When double labeling of the same cells was performed with

Nucleic Acids Research, 1995, Vol. 23, No. 11 1915

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Figure 1. gar2 is structurally related to NSRl and nucleolin. (A) Comparison of the sequences of Spombe gar2 and S.cerevisiae NSRl. The sequence of the gar2 protein deduced from the nucleotide sequence of the gene has been compared to the NSRI sequence (33). Identical residues (:), similar residues (.) and gaps (-) are indicated. (B) Comparison of the primary structures of S.pombe gar2, S.cerevisiae NSRl, and vertebrate nucleolin. The three proteins are very similar in their C-terminal part but differ in the length of their N-terminal part. The codes used to indicate each domain are represented on the left.

DAPI (Fig. 2A and C) and by indirect immunostaining with anti-fibrillarin (Fig. 2B), or with affinity purified anti-gar2 antibodies (Fig. 2D), the immunolabeling is found in the non-chromatin hemisphere of the nucleus demarcated by the DAPI staining and corresponds precisely to the nucleolus (52). Thus, gar2 is a novel nucleolar protein in S.pombe.

gar2 is required for normal cell growth To investigate the role played by gar2 in S.pombe, a disruption of the gar2+ gene by the one-step gene replacement method (42) was performed. A diploid ura4- strain was transformed with a construct in which the ura4+ gene replaced the 0.49 kb central part of the gar2+ coding sequence (i.e. the last two acidic serine rich domains and the first RNA recognition motif). By Southern blot analysis, stable ura4+ transformants were checked for the replacement of the wild-type gar2+ by the disrupted allele (Fig. 3A and B). Diploid strains exhibiting the appropriate hybridization pattern were then sporulated and the tetrads dissected. In the 18 tetrads analyzed, four viable spores were recovered, two of which grew abnormally slow in rich medium (Fig. 3C). The slow-growing colonies were always ura4+. These results indicate that gar2 is required for normal cell growth. gar2+ disruption reduces 18S rRNA and 40S ribosomal subunit levels Due to the homology ofthe gar2 protein with NSR1 and nucleolin and to its nucleolar localization, we wondered whether this slow-growth phenotype could be related to any defect in ribosome biogenesis. Thus, we compared the major rRNAs in the wild-type strain Sp22 with those in the gar2::ura4 + strain, Sp24. Total RNA was isolated from both strains, separated by agarose-formaldehyde gel electrophoresis and either stained with

Figure 2. gar2 is localized to the Spombe nucleolus. Nuclei were first stained with DAPI (A and C), the arrows point to the nucleolus which is slightly stained. Indirect immunofluorescence was then performed on the same cells with antibodies affinity purified against Fibrillarin (B) and gar2 (D), respectively. In this case, the immunostaining (white arrow) corresponds precisely to the nucleolus.

ethidium bromide or transferred onto nylon membrane and hybridized with specific DNA probes from the S.pombe 5' ETS or 18S rDNA (obtained from pBL 1263) (Fig. 4A-C). Quantification of the bands were obtained either with an ultrascan (LKB) or a phosphoimager (Fuji). The amount of 25S rRNA was the same per cell in wild-type and mutant strains but the ratio of 18S to 25S in the Sp24 strain was different from that in the wild-type strain. The level of 18S rRNA was reduced by -20-30% and the amount of the pre-rRNA precursor recognized by the 5' ETS probe, which we shall call 35S pre-rRNA, was increased by a factor of two. Thus gar2, like NSR1, NOPI, SOFI and GARI, is required for normal production of 18S rRNA (31,44,53,54). The observation that the gar2 mutant was defective in its ability to produce 18S rRNA prompted us to consider whether this defect was reflected in the distribution of ribosomal subunits. In order to test this we analyzed the levels of free 40S and 60S ribosomal subunits, 80S monoribosomes and polyribosomes in wild-type strains and in gar2:: ura4+ strains. Extracts from these cells were fractionated by sucrose velocity gradient centrifugation as previously described (44). The results are shown in Figure 5. Compared with the gar2+ strain (Sp22) the gar2-strain (Sp24) had no free 40S ribosomal subunit and a large surplus of free 60S ribosomal subunits. This result is similar to that observed for a nsrl strain (44). In the S.pombe wild-type strain, Sp22, the 60S free subunit is not present in as great amounts as the 40S free subunit. We would expect the 60S peak to have a greater absorbance due to its greater RNA content, as is the case for wild-type S.cerevisiae profiles. Nevertheless, the disruption of the gar2 + gene clearly results in a great increase in the free 60S pool. One difference between NSR1 and gar2 disruptions is that in the case of gar2- mutant there seems to be a deficiency of

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Figure 3. Creation of a disrupted allele of the gar2+ gene. (A) The internal SacI-KpnI fragment ofthe gar2+ coding region was replaced by the ura4 + gene as described in Materials and Methods, and the resulting fragment was used to replace the gar2+ gene in a diploid. The hatched segment corresponds to the gar2+ ORF while the black segment represents the S.pombe ura4+ gene. Restriction sites are abbreviated as follows: E, EcoRl; H, HindIl; K, KpnI; S, SalI; Sc, Sacl. (B) Southem blot hybridization. Total Spombe DNA was purified from diploid Spl8 strain wild-type (lane 1) and from diploid disrupted strain Spl9. The DNA was digested with EcoRI, run in a 0.7% agarose gel, transferred onto nylon membrane and probed either with a HindU fragment of ura4+ gene or with a Hindli fragment of the gar2 + gene. (M): x BstEII marker, 5' 32P-labeled with T4 DNA kinase is shown on the left. A 5.5 kb EcoRl fragment is clearly seen in the wild-type (1) and is split into two fainter bands in the mutant, one of which is also detected with the ura4+ probe. (C) Tetrad analysis of the gar2+/gar2:: ura4+ strain. A diploid heterozygous for gar2+/gar2:: ura4+ was sporulated and tetrads were dissected with a micromanipulator. The figure shows the dissection ofthree tetrads. Spores were grown 4 days on YES medium at 30°C.

polysomes, while no change was noted for nsrl-. Due to the presence of a large, perhaps larger, peak of 80S ribosomes in gart strain (Sp24) it is unlikely that the defect in polysomes is the result of problems in translation initiation. Instead, the decrease in amount of polysomes may be related to a decrease in ribosome number caused by the deficiency in the 40S subunit. These results support the hypothesis that gar2 is involved in the production of 40S subunit, and that the growth phenotype observed with the disruption of this gene is due to the reduced ability to synthesize ribosomes.

S.pombe gar2 is a functional homolog of S.cerevisiae NSR1 The similarity between gar2 and NSR1 led us to test whether gar2 could substitute for NSR1. One phenotype observed for nsrlstrains, as well as gar2 strains, is a deficiency of 40S subunits and a great excess of free 60S subunits. If gar2 is a functional

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Nucleic Acids Research, 1995, Vol. 23, No. 11 1917

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control Figure 6. Ribosomal profiles of strains expressing NSRl and gar2. S.cerevisiae strains lacking NSRI but containing either a control plasmid with no fused protein or plasmid allowing the expression of NSR I or gar2 were grown in YPG media and ribosomes and ribosomal subunits were isolated and separated on a 7-47% sucrose gradient (54). The gradient was analyzed by monitoring the OD254 with a ISCO Model 640 Gradient Fractionator. The OD scale is approximate. (A) pWLIO, expressing NSRl. (B) pBL1252, expressing gar2. (C) Control, expressing no protein from plasmid. The top and bottom of the gradient are marked as are the ribosomes and subunits. Numbered peaks refer to polysomes that contain the indicated number of ribosomes.

homolog, its expression in a nsrl- strain should restore the native ribosomal profile. For this study, the genes for NSR1 and gar2 were expressed using the GAL] galactose-inducible promoter (plasmids pWLIO and pBLI252). As control, a plasmid with no fused gene was used. The S.cerevisiae strain WLY353, which carries a disruption of the NSRJ gene was transformed separately with the three plasmids. Ribosomal profiles are shown in Figure 6. The control strain, with no fused gene shows a typical nsrlphenotype (Fig. 6C). The 40S free ribosomal subunit is essentially absent while the free 60S subunit is greatly elevated. Both NSR1 and gar2 expression are able to restore wild-type level of free ribosomal subunits (Fig. 6A and B). Another phenotype displayed by the nsrl- strain is an increased sensitivity to the drug paromomycin (44). Expression under the GALI promoter of gar2 in the nsrl- strain is able to reduce paromomycin sensitivity (results not shown). Thus, expression of gar2 in the nsrl- strain restores a wild-type phenotype, supporting the idea that gar2 is a functional homolog of NSR1 in the fission yeast S.pombe.

We report the identification and characterization of the gar2+ gene encoding a novel nucleolar protein of S.pombe. gar2 was identified in S.pombe as a member of the family of nucleolar proteins containing a motif rich in glycine and arginine residues called the GAR domain (31). Other members of this family include in vertebrates the well known nucleolin, and in yeast the essential nucleolar proteins fibrillarin/NOPI, GARI and the non-essential nucleolar proteins SSB1 and NSR1. We show in this paper that gar2 is able to restore aberrant ribosomal profile and drug sensitivity of a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. Those and previous results (38,39,55) show clearly that the family of nucleolar GAR proteins has been well conserved during evolution and suggest that the components interacting with these proteins should be well conserved too. In overall structure, gar2 more closely resembles the S.cerevisiae NSR1, and mammalian nucleolin, than SSB1, NOPI or GARL. Besides the GAR domain, gar2 has consensus RNA binding motifs and also an acidic-serine rich N-terminus that forms a number of putative casein kinase II phosphorylation sites. Like nucleolin, gar2 can be phosphorylated in vitro by the cdc2 kinase (45,46; Gulli, Faubladier and Caizergues-Ferrer, unpublished results). In contrast, no consensus phosphorylation site can be found in NSR1. The absence of a cdc2 phosphorylation site in NSR1 may indicate a function for nucleolin and gar2 that does not overlap with NSR1. The availability of several cell cycle conditional mutants in the fission yeast S.pombe will allow the study of the nature of this phosphorylation and its effects on gar2

function(s). In a S.pombe gar2- mutant, processing of the initial pre-rRNA is slowed, steady-state levels of 18S rRNA are reduced by 30% and there is no detectable free pool of 40S ribosomal subunits. On the contrary, the steady-state amount of 25S rRNA is not affected. This phenotype is similar to the one observed in a nsrl- mutant or upon depletion in S.cerevisiae of the nucleolar proteins NOP 1, GARI, SSB1, SOFI (31,53,54,56) or of the snoRNAs U3, U14, snR30 and snRIO (3-6). Hence the proposal that these factors participate in a common processing complex required for 18S rRNA production recently termed 'the processome' (19). The precipitation of snoRNAs with NOPI, GARI, SOF1 and SSB 1 suggests strongly that these proteins interact with the pre-rRNA via snoRNA-pre-rRNA base-pairing. On the contrary, there is no evidence yet that NSR1 or gar2 are directly associated with snoRNAs. The overall structural similarity between gar2, NSR1 and nucleolin and the observation that nucleolin binds rRNA in vitro suggest rather that gar2 and NSR1 might bind rRNA directly. Nucleolin, containing four putative RNA binding domains (RRMs), interacts in vitro with the pre-rRNA at different sites in the 5' external transcribed spacer (5' ETS) as well as within the 18S and 28S rRNA regions (F. Amalric, personal communication). If rRNA sequences are well conserved between different species, the sequences present in the spacers are much more divergent. This divergence could explain why nucleolin failed to complement the nsrl- mutant, even though nucleolin appears to be correctly localized in the nucleolus of S.cerevisiae (57). Moreover, unlike nucleolin, NSRl/gar2 are not a priori expected to bind 25S rRNA since they do not seem to be involved in its production. Therefore, the roles of nucleolin in vertebrates might be played by multiple proteins in yeast. Recently, an

1918 Nucleic Acids Research, 1995, Vol. 23, No. 11 essential nucleolar protein termed NOP4/NOP77 containing three canonical RRMs and required for 25S production and pre-rRNA methylation was characterized (58,59). We propose that gar2 might interact with the pre-rRNA via its two RRMs in combination with the GAR domain. Futhermore gar2, like NSRl or nucleolin, might directly help recruit or assemble some highly basic ribosomal proteins with the prerRNA via its acidic/serine rich N-terminus (60). To test these assumptions, we are currently investigating by mutational analyses the roles of the gar2 subdomains, searching for its RNA target(s) and protein partners.

ACKNOWLEDGEMENTS We thank Y. Henry, T. Kiss and P. Ferrer for critical reading of the manuscript and other members of our group for fruitful discussions. We are grateful to J. Feliu and M. Faubladier for excellent technical assistance, to Y. de Preval for oligonucleotides synthesis, and P. Fantes and B. Ducommun for providing strains and vectors. We are indebted to F. Amalric for his continuous support. MPG and JPG had a fellowship from the Ministere de l'Enseignement Superieur et de la Recherche (MESR). This research was carried out under contract by the Association pour la Recherche sur le Cancer and by the Universite Paul Sabatier.

REFERENCES 1 Chooi, W. Y., and Leiby, K. R. (1981) Proc. NatI. Acad. Sci. USA 78, 4823-4827. 2 Mougey, E. B., O'Reilly, M., Osheim, Y., Miller Jr, 0. L., Beyer, A., and Sollner-Webb, B. (1993) Genes Dev. 7, 1609-1619. 3 Hughes, J. M. X., and Ares, M. Jr (1991) EMBO J. 10, 4231-4239. 4 Li, H. V., Zagorski, J., and Fournier, M.J. (1990) Mol. Cell. Biol. 10, 1145-1152. 5 Morrissey, J., and Tollervey, D. (1993) Mol. Cell. Biol. 13, 2469-2477. 6 Tollervey, D. (1987) EMBO J. 6, 4169-4175. 7 Schmitt, M. E., and Clayton, D. A. (1993) Mol. Cell. Biol. 13, 7935-7941. 8 Lygerou, Z., Mitchell, P., Petfalski, E., Seraphin, B., and Tollervey, D. (1994) Genes Dev. 8, 1423-1433. 9 Savino, R., and Gerbi, S. (1990) EMBO J. 9, 2299-2308. 10 Kass, S., Tyc, K., Steitz, J.A., and Sollner-Webb, B. (1990) Cell 60, 897-908. 11 Mougey, E. B., Pape, L. K., and Sollner-Webb, B. (1993) Mol. Cell. Biol. 13, 5990-5998. 12 Peculis, B.A., and Steitz, J.A. (1993) Cell 73, 1233-1245. 13 Tycowski, K., Shu, M. D., and Steitz, J. A. (1994) Science 266, 1558-1561. 14 Maser, R. L., and Calvet, J. P. (1989) Proc. Natl. Acad. Sci. USA 86, 6523-6527. 15 Stroke, I. L., and Weiner, A. M. (1989) J. Mol. Biol. 210, 497-512. 16 Tyc, K., and Steitz, J.A. (1992) Nucleic Acids Res. 20, 5375-5382. 17 Beltrame, M., and Tollervey, D. (1992) EMBO J. 11, 1531-1542. 18 Rimoldi, 0. J., Raghu, B., Mihir, K. N., and Eliceiri, G. L. (1993) Mol. Cell. Bio. 13, 4382-4390. 19 Foumier, M. J., and Maxwell, E. S. (1993) Trends Biochem. Sci. 18, 131-135. 20 Sollner-Webb, B. (1993) Cell 75, 403-405. 21 Sachs, A. B., and Davies, R. W. (1990) Science 247, 1077-1079. 22 Ripmaster, T. L., Vaughin, G. P., and Woolford Jr., J. L. (1992) Proc. Natl. Acad. Sci. USA 89, 11131-11135. 23 Lapeyre, B., Bourbon, H. M., and Amalinc, F (1987) Proc. Natl. Acad. Sci. USA 84, 1472-1476.

24 Caizergues-Ferrer, M., Mariottini, P., Curie, C., Lapeyre, B., Gas, N., Amalric, F., and Amaldi, F. (1989) Genes Dev. 3, 324-333. 25 Shimmang, T., Tollervey, D., Kern, H., Frank, R., and Hurt, E.C. (1989) EMBO J. 8, 489-497. 26 Lapeyre, B., Mariottini, P., Mathieu, C., Ferrer, P., Amaldi, F., Amalric, F., and Caizergues-Ferrer, M. (1990) Mol. Cell. Biol. 10, 430-434. 27 Henriquez, R., Blobel, G., and Afis, J. P. (1990) J. Bio. Chem. 265,

2209-2215. 28 Aris, J. P., and Blobel G. (1991) Proc. Natl. Acad. Sci. USA 88,931-935. 29 Jong, A. Y. S, Clark M. W., Gilbert, M., Oehm, A., and Campbell, J. L. (1987) Mol. Cell. Biol. 7, 2947-2955. 30 Lee, W.C., Xue, Z., and Melese, T. (1991) J. Cell Biol. 113, 1-12. 31 Girard, J. P., Lehtonen, H., Caizergues-Ferrer, M., Amalric, F., Tollervey, D., and Lapeyre, B. (1992) EMBO J. 11, 673-682. 32 Ghisolfi, L., Joseph, G., Amalric, F., and Erard, M. (1992) J. Biol. Chem. 267, 2955-2959. 33 Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorf J. W. (1990) Methods Enzymol. 185, 60-68. 34 Lapeyre, B., Michot, B., Feliu, J., and Bachellenie, J.P. (1993) Nucleic Acids Res. 21, 3322. 35 Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 36 Moreno, S., Klar, A., and Nurse, P. (1991) Methods Enzymol. 194, 795-823. 37 Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168. 38 Girard, J. P., Caizergues-Ferrer, M., and Lapeyre, B. (1993) Nucleic Acids Res. 21, 2149-2155. 39 Girard, J. P., Feliu, J., Caizergues-Ferrer, M. and Lapeyre, B. (1993) Nucleic Acids Res. 21, 1881-1887. 40 Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 41 Hagan, I. M., and Hyams, J. S. (1988) J. Cell. Sci. 89, 343-357. 42 Rothstein, R.J. (1983) Methods Enzymol. 101, 203-211. 43 Sherman, F., Hicks, J. B., and Fink, G. R. (1986) Methods in Yeasts Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 44 Lee, W. C., Zabetakis, D., and Melese, T. (1992) Mol. Cell. Biol. 12, 3865-3871. 45 Belenguer, P., Caizergues-Ferrer, NI., Labbe, J. C., Doree, M., and Amalric, F. (1990). Mol. Cell. Biol. 10, 3607-3618. 46 Peter, M., Nakagawa, J., Doree, M., Labbe, J. C., and Nigg, E. A. (1990) Cell 60, 791-801. 47 Caizergues-Ferrer, M., Belenguer, P., Lapeyre, B., Amalric, F, Wallace, M. O., and Olson, M. 0. J. (1987) Biochemistry 26, 7876-7883. 48 Mattaj, I. W. (1993) Cell 73, 827-840. 49 Kenan, D. J., Query, C. C., and Keene, J. D. (1991) Trends Biochem. Sci. 16, 214-220. 50 Najbauer, J., Jonhson, B. A., Young, A. L., and Aswad, D. W. (1993) J. Biol. Chem. 268, 10501-10509. 51 Alderuccio, F., Chan, E. K. L., and Tan, E. M. (1991) J. Exp. Med. 173, 941-952. 52 Hirano, T., Konoha, G., Toda, T., and Yanagida, M (1989) J. Cell Biol. 108, 243-253. 53 Tollervey, D., Lehtonen, H., Carmo-Fonseca, M., and Hurt, E. C. (1991) EMBO J. 10, 573-5837. 54 Jansen, R., Tollervey, D., and Hurt, E. C. (1993) EMBO J. 12, 2549-2558. 55 Jansen, R. P., Hurt, E. C., Kern, H., Lehtonen, H., Carmo-Fonseca, M., Lapeyre, B., and Tollervey, D. (1991) J. Cell Biol. 113, 715-729. 56 Clark, M. W., Yip, M. L. R., Campbell, J., and Abelson, J. (1990) J. Cell Biol. 111, 1741-175 1. 57 Xue, Z., Shan, X., Lapeyre, B., and Melese, T. (1993) Eur J. Cell Biol. 62, 13-21. 58 Sun, C., and Woolford, J. L. Jr (1994) EMBO J. 13, 3127-3135. 59 Berges, T., Petfalski, E., Tollervey, D., and Hurt, E. C. (1994) EMBO J. 13,

3136-3148. 60 Xue, Z., and Melese, T (1994) Trends Biol. Cell 4, 414-417.