Purification and Nucleic-Acid-Binding Properties of a Saccharomyces Cerevisiae Protein Involved in the Control of Ploidy

Eur. J. Biochem. 249, 309-317 (1997) 0 FEBS 1997

Purification and nucleic-acid-binding properties of a Saccharomyces cerevisiae protein involved in the control of ploidy Viktoria WEBER', Andreas WERNITZNIG', Gudrun HAGER', Masahiko HARATA', Peter FRANK' and Ulrike WINTERSBERGER'

' Department of Molecular Genetics, Institute of Tumor Biology and Cancer Research, University of Vienna, Vienna, Austria * Laboratory of Molecular Biology, Tohoku University, Sendai, Japan

(Received 21 May/l July 1997) - EJB 97 0715/2

Scpl6Op (Succhuromyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum. The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells [Wintersberger, U., Kiihne, C. & Karwan, A. (1995) Yeast 11, 929-9441. Scpl6Op contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins. In this report, we demonstrate that the Scpl6Op sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Cueno- rhubditis elegans as well as with the vigilins, vertebrate RNA-binding proteins with a cellular location similar to that of Scpl6Op. Moreover, we found that Scpl6Op contains a potential nuclear-export signal (NES) near its N-terminus and a potential nuclear-localization signal (NLS) between KH domains 3 and 4. To determine whether the protein is able to bind to RNA, we purified Scpl6Op from yeast cell extract by DNA-cellulose and anti-Scpl6Op affinity chromatography. In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single-stranded and double-stranded DNA. Subcellular fractionation studies revealed that the major part of Scpl6Op is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes. Together, our findings suggest that Scpl6Op is the yeast homologue of the vigilins, and point to a role for Scpl6Op in nuclear RNA export o r in RNA transport within the cytoplasm.

Keywords: KH domain ; nucleic-acid-binding protein ; RNA transport.

A multitude of RNA-binding proteins play key roles in the regulation of gene expression. Characterization of these proteins has led to the identification of several RNA-binding motifs [ 1, 21. One of these motifs, the KH domain (for K protein homology), was first identified as a repeated sequence in human hnRNP K protein [3]. According to secondary-structure predic- tion and NMR spectroscopy, the KH domain folds into a globu- lar structure consisting of an antiparallel three-stranded P-sheet connected by two helical regions and an appended C-terminal helix [4]. Based on this secondary structure, it has been proposed that positively charged residues in the loop between the first two helices form a potential surface for contact with RNA [5]. Since the first description of the KH motif in 1993 [3], a number of proteins containing this domain have been identified in a variety of organisms suggesting that the KH module is an evolutionary ancient structure with important cellular functions. Examples of

Correspondence to V. Weber, Department of Molecular Genetics, Institute of Tumor Biology and Cancer Research, University of Vienna, Borschkegasse Sa, A-1090 Wien

Phone: +43 40154 244. Far: +43 1 406 07 90. E-mail: [email protected] Abbreviations. FMRP, fragile X mental-retardation protein ; KH do-

main, a peptide motif originally identified in hnRNPK; NES, nuclear- export signal; NLS, nuclear-localisation signal; PABP, poly(A)-binding protein; PhMeSO,F, phenylmethylsulfonyl fluoride; Scpl6Op, Saccharo- myces cerevisiae protein involved in the control of ploidy.

Enzyme. T4 polynucleotide 5'-hydroxyl-kinase (EC 2.7.1.78).

KH containing proteins include polynucleotide phosphorylase and the antiterminator NusA from Escherichia coli [6, 71, Merlp from Succharomyces cerevisiae [8], the ribosomal protein S3 found in all taxonomic kingdoms [9, 101, as well as many vertebrate proteins such as murine hnRNP X [ll], GTPase-activat- ing-protein (GAP)-associated tyrosine phosphoprotein p62 [ 121, human poly(rC)-binding and a-complex proteins [13, 141, and the gene products of the fragile X mental retardation gene FMRl [15, 161 and its homologues, FXRl and FXR2 [17]. A protein consisting nearly exclusively of KH domains, vigilin, was found in chicken and human cells [I 8-20]. A direct in vivo interaction between the KH motif of ribosomal protein S3 and rRNA was demonstrated by cross-linking studies in 30s and 50s ribosomal subunits of E. coli and Bacillus stearothermophilus [9].

We have recently reported the cloning of SCP160 (S. cerevisiae gene involved in the control of ploidy). The gene, which is located on chromosome X, codes for a polypeptide with a molecular mass of around 160 kDa, Scpl6Op. Indirect immunofluorescence experiments with anti-Scpl60p serum revealed association of the protein with the nuclear envelope and the endoplasmic reticulum; additionally, a weaker, diffuse signal was observed in the cytoplasm [21]. Deletion of parts of the SCP160 gene results in cells of decreased viability, abnormal morphol- ogy and increased DNA content. Crosses of SCP160 deletion mutants with each other or with unrelated SCP160 competent strains lead to irregular segregation of genetic markers, suggesting that Scpl6Op is necessary for the faithful partitioning of chromosomes during cell division. So far, however, it is not clear

310 Weber et al. (Eur: J. Biockem. 249)

by which mechanism Scpl6Op might influence the process of chromosome segregation.

Here, we demonstrate that the Scpl6Op sequence, which contains 14 KH domains, is colinear with chicken and human vigilin and with the gene product of a sequence from the nematode Caenorhabditis elegans termed C08H9.2 (GenBank accession no. 254342). We present an analysis of the nucleic-acid- binding properties of the purified protein, and, in view of the data that will be presented here, we propose that Scpl6Op might function in RNA export from the nucleus or in RNA transport within the cytoplasm.

As discussed in [21], the HX sequence from S. cerevisiae ([22] ; GenBank accession no. X04679), which has repeatedly been cited as a KH protein, represents the C-terminal half of Scpl6Op (GenBank accession no. X65645 ; SwissProt P06105).

MATERIALS AND METHODS

Yeast strains and growth media. Strain MZ3 (MATa; p p 4 - 3 trpl leu2-A1 ura3-Al) was derived from 20B-12 [21]. Strain AK603 was obtained by transformation of MZ3 with the high copy number 2 p plasmid pFL1/6-1, which was constructed by insertion of the 7.7-kb BamHI-BumHI yeast chromosomal fragment 1211 containing the SCP160 open reading frame under its own promoter into the pFL1 BamHI cloning site. YPD growth medium (1% yeast extract, 2% peptone, 2% dextrose) was used as standard medium for the culture of yeast cells. Syn- thetic complete (Sc) medium was prepared as described by Sher- man [23].

Protein determination, gel electrophoresis and protein blotting. Protein concentration was determined by the method of Bradford [ 241. Discontinuous SDS/PAGE was performed in a Mi:<hty small SE 250 cell or in a SE 600 vertical slab cell, rcspzctively (both from Hoefer), with gels containing 10% acrylamide. Protein bands were visualized with Coomassie blue (32.50 or by silver staining [25]. Proteins were blotted to nitrocellulose or poly(viny1idene difluoride) sheets using a semi-dry blotter (NovaBlot, Pharmacia) and stained with Ponceau S to assess the amounts of proteins transferred. Blots were developed with the ProtoBlot alkaline phosphatase detection system from Promega. Yeast cell extracts for western blotting were prepared as described earlier [26].

Subcellular fractionation. Subcellular fractionation was performed using a procedure adapted from Tibbetts et al. (271. Briefly, log-phase cells were harvested by centrifugation, washed once with lysis buffer (25 mM Tris/phosphate, pH 6.7), frozen in liquid nitrogen, and kept at -70°C for at least 1 h. An equal volume of lysis buffer containing 1 pl/ml 2-mercapto- ethanol and protease inhibitors [ I mM each of phenylmethylsulfonyl fluoride (PhMeS0,F) and sodium sulfite, pH 8.0; 0.1 mM sodium tetrathionate, 1 pM each of Nu-p-tosyl-L-lysine-chloromethane, N-tosyl-L-phenylalanine-chloromethane, pepstatin A, and antipain] as well as chilled glass beads were added to the frozen cell pellet. Cells were broken by vortexing 8-10 times for 30 s and the lysate was kept on ice between the vortexing steps. Lysates were centrifuged for 4 min at 400 g to remove glass beads, undisrupted cells, and cell wall fragments. Aliquots of the clarified lysate were centrifuged at 30000 g and 50000 g, respectively, in a Beckman TLA100.2 rotor yielding two pellet (P30, P50) and two supernatant (S30, S50) fractions. An aliquot of P50 was washed once with lysis buffer and resuspended in 200 pl lysis buffer containing 0.5 M NaC1. After incubation on ice for 20 min with occasional shaking, the mixture was centrifuged for 15 min at 50000 g, producing an additional pellet and supernatant. Pellets and supernatants were precipitated with

trichloroacetic acid and analyzed for the presence of Scpl6Op by immunoblotting.

To examine a possible association of Scpl6Op with ribosomes, a clarified yeast lysate in low-salt buffer (50 mM trietha- nolamine, pH 7.5, 50 mM KCl, 6 mM magnesium acetate, 1 mM dithiothreitol, 0.001 % PhMeS0,F) was prepared as described above. The lysate was centrifuged through a sucrose cushion ( S O % , masdvol. in law-salt buffer) for 10 min at 100000 g in a Beckman TLAlOO rotor. The membrane pellet was treated with low-salt buffer containing 10 mM EDTA for 20 min on ice to dissociate membrane-bound ribosomes, and re-centrifuged as above. Aliquots (10 pg of protein each) of pellet and supernatant withdrawn before and after the treatment with EDTA were analyzed for the presence of Scpl6Op by immunoblotting. In parallel, the blots were developed with antibodies directed against poly(A)-binding protein (PABP) and the ribosomal protein S13, respectively.

Fractionation of polyribosomes. Ribosomes were fractionated by sucrose-gradient centrifugation according to the method of Deshmukh et al. [28] with minor modifications. Yeast cells were grown to mid-log phase in 50 ml YPD medium. After the addition of 25 mg cycloheximide, the culture was put on ice immediately, cells were harvested by centrifugation and washed twice with a solution containing 10 mM TrisLHC1, pH 7.4, 100 mM NaCI, 30 mM MgCl,, 50 pg cycloheximide/ml, and 200 pg heparin/ml. Washed cells were suspended in 1 ml of the same solution, and after the addition of glass beads, cells were disrupted by vortexing the suspension ten times for 15 s, with a 30-s period of cooling on ice after each vortexing step. An additional 1.5 ml of the same solution was added and the sample was spun at 5000 g for 5 rnin and then at 10000 g for 5 min. An aliquot of the supernatant corresponding to 10 A,,, units (1 A,,, is the amount of material that, where contained in 1 ml of solution gives an absorbance at 260 n m of 1 in 1-cm pathlength cell) was layered over an 11.2-mi linear 7% to 47% sucrose gradient containing 50 mM Tridacetate, pH 7.0, 50 mM NH,Cl, 12 mM MgCl,, and 1 niM dithiothreitol. The gradient was centrifuged at 40000 rpm for 90 min at 13°C in an SW41Ti rotor (Beckman). Fractions of 600 pl were collected from the bottom of the gradient, acetone precipitated and analyzed for the presence of Scpl60p by immunoblotting.

Preparation of the Scpl6Op affinity matrix. Ammonium sulfate was added to 15 ml of rabbit antiserum (40% saturation); after 1 h at 4 ' T the mixture was centrifuged for 20min at 15 000 rpm in a Sorvall SS34 rotor. The pellet was dissolved in 0.1 M Tris/phosphate, pH 7.2 and dialyzed against the same buffer. The dialyzate was loaded onto a protein-A-Sepharose column (Pharmacia), and after extensive washing of the column with the loading buffer, bound IgG was eluted with 0.1 M glycine/HCl, pH 3.0. Fractions containing IgG were pooled and neutralized with 2 M Tris. Coupling of 25 mg of purified IgG to 5 ml of BrCN-activated Sepharose (Pharmacia) was carried out according to the instructions of the manufacturer.

Antibodies. For affinity purification of Scp16Op, the rabbit antiserum described in [21], which detects the C-terminal half of Scpl6Op, was used. Additionally, a polyclonal antiserum against the whole polypeptide was raised by immunizing a Balb/C mouse with about 35 pg of affinity-purified Scp16Op which was applied in three portions at intervals of two weeks. Antisera were taken at 35 days and 48 days after the first injec- tion. To assess antibody specificity, blots of total yeast proteins were probed with the antisera, which were diluted 20000-fold (rabbit) and 10000-fold (mouse) in NaCl/Tris/Tween 20 (10 mM Tris/HCI, pH 8.0, 150 mM NaCI, 0.05% Tween 20), respectively.

Weber et al. (Eur J. Biochem. 249) 31 1

Purification of Scpl6Op from yeast extract. To minimize proteolysis, all purification steps were carried out at 4"C, and protease inhibitors (see above) were included in all buffers. The Scpl6Op overproducing strain AK603 was grown to mid-log phase, harvested by centrifugation, washed once with buffer A (20 mM Tris/HCl, pH 7.9, 1 mM EDTA, 10% glycerol) containing 2 M NaCI, and frozen in liquid nitrogen. The cell pellet (60 g wet cells) was resuspended in buffer A containing 2 M NaC1, and after addition of an equal volume of acid-washed glass beads, the suspension was homogenized and separated from the glass beads, undisrupted cells and large cell fragments as described 1261. The cell extract was subsequently centrifuged for 100 min at 50000 rpm in a Beckman 55.2 Ti rotor. To remove nucleic acids, the supernatant was treated with poly(ethy1ene glycol) 6000 (lo%, mass/vol., final concentration) for 30 min on ice followed by centrifugation for 35 min at 22000 rpm (Beckman 55.2 Ti rotor). After dialysis of the supernatant against buffer A containing 0.1 M NaCl and centrifugation of the dialysate for 25 rnin at 24000 rpm (Beckman 55.2 Ti rotor), the clear supernatant was chromatographed on a 300-ml DNA- cellulose column as described earlier [29]. The protein peak was pooled, dialyzed against 0.1 M Tris/HCl, pH 7.2, containing 0.15 M MgCl,, and applied to the anti-Scpl60p affinity column (see above) at a flow rate of 8 mlh. The column was washed extensively with the same buffer to remove unbound proteins followed by a wash with 5 volumes of 0.1 M TridHC1, pH 7.2. Elution of Scpl6Op was performed with 0.1 M glycineRIC1, pH 3.0. Fractions were immediately neutralized with 2 M Tris and analyzed by SDS/PAGE. The yield of one preparation was about 200 pg of electrophoretically pure protein.

Northwestern blot analysis. Polynucleotides were 5'-end labelled with [y-"P]ATP (Amersham) using T4 polynucleotide kinase (New England Biolabs). Purified Scpl6Op (200 ng/lane in the experiment of Fig. 3, and 10, 30, and 100 ng per lane in the experiment of Fig. 4) was subjected to SDS/PAGE on a 7.5 9% gel and electrophoretically transferred to a nitrocellulose membrane in a buffer of 12.5 mM Tris and 95 mM glycine. Blots were treated with binding buffer [ l o mM Tris/HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.2% BSA, 0.2% Ficoll 400, 0.2% polyvinylpyrrolidone] for 1 h at room temperature. Subse- quently, individual lanes were probed with labelled polynucleotides (probe concentration, 0.1 pg/ml) in binding buffer. To avoid unspecific binding of the probe to the membrane, a 20- fold excess of cold yeast tRNA (which shows no significant interaction with Scpl6Op as determined in a separate experiment) was added to the binding buffer. For the competition experiment, the binding buffer was supplemented with 0.5, 2, 8 or 32 pg/ml of unlabelled poly(rC), crude yeast rRNA, sheared dsDNA from E. coli, sheared and heat denatured (single- stranded) E. coli DNA, and yeast tRNA, respectively. Excess "P-labelled nucleic acids were removed by washing the blots for three 15-min periods in binding buffer. The blots were air dried and analyzed for bound radioactivity with the BAS2000 system (Fuji).

Electrophoretic mobility-shift assay. Ribosomal RNA from bovine liver was obtained from Sigma. Prior to the experiment, the RNA was heated to 65°C and slowly cooled to room temperature to allow refolding. A reaction mixture of 14 p1 consisted of 0.2-1 pg of purified Scpl6Op, 1 pg RNA and 50 U RNasin (Promega) in diethylpyrocarbonate-treated water. After incubation on ice for 20 min, 6 pl of loading buffer (450 mM Tris/ borate, pH 8.0, 10 mM EDTA, 30% glycerol, 0.025% bromo- phenol blue) was added, and the samples were subjected to electrophoresis in a native 0.8% agarose gel in Tris-borateEDTA electrophoresis buffer (TBE; 90 mM Tris/borate, pH 8.0, 2 mM EDTA). To detect the position of RNA, gels were stained with

ethidium bromide. After equilibration for 10 min in cathode buffer for western blotting [4& mM Tris HC1, 39 mM glycine, 20% (by vol.) methanol, 1.3 mM SDS], gels were blotted to nitrocellulose membranes (Schleicher & Schull) in the same way as SDS/PAGE gels. The position of Scpl6Op was visualized using the mouse anti-Scpl60p serum at a dilution of 1 : 10000 in NaCl/Tris/Tween 20.

RESULTS

Scpl6Op contains 14 KH domains and is the yeast homologue of the vertebrate vigilins. With the exception of its N-terminal part (amino acids 1 - 107), Scpl6Op consists almost entirely of KH motifs. With respect to number and arrangement of its KH domains, the protein closely corresponds to the vertebrate vigilins, although its primary structure shows only about 20% identity with the vigilins based on the alignment presented in Fig. 1. Domains 2, 8-12 and 14 of Scpl6Op contain the conserved GXXG loop [7] and thus represent classical KH motifs. Do- mains 1, 3-7 and 13, however, are degenerate, as they have insertions or deletions in their GXXG motif; nevertheless, they can be identified as KH domains based on their expected secondary structure [5]. The central positions of the GXXG (or GKXG) loop, a predicted site of RNA interaction [5], are occu- pied by semiconserved positively charged amino acids. It is no- table that only domains 5 , 8, and 10 of the Scpl6Op sequence possess a perfect loop, whereas chicken and human vigilin contain 11 and 12 out of 15 such motifs, respectively. Fig. 1 shows a complete alignment of the Scpl60 polypeptide sequence with chicken vigilin (Vig-C), human vigilin (Vig-H), and with the putative gene product of an open reading frame from C. elegans termed C08H9.2 (GenBank accession no. 254342). The four proteins are colinear, and each of the degenerate KH domains of Scpl6Op matches the position of a KH domain in the vigilins and in C08H9.2. Due to an insert of 61 amino acids [ 5 ] , KH domain 13 of Scpl6Op extends over domains 13 and 14 of the vigilins and of C08H9.2. The two vigilin sequences exhibit 87 % identity to each other, i.e., they differ in only 163 out of 1270 (1268) residues. The letters X, C, and H above the alignment in Fig. 1 refer to the comparison of Scpl6Op to the vigilins, with X indicating residues which are different in all three proteins, and C or H indicating positions where either chicken (C) or human (H) vigilin is identical to Scpl6Op. In 28% of the cases (22 C and 23 H) where the vigilins differ from each other, one of them possesses a residue identical to Scpl6Op. This percentage rises to 33% if one ignores the 26 X residues that fall into one of the two regions where a longer gap has to be inserted for Scp16Op in the alignment (KH domain 12 and C-terminus, see Fig. 1). Thus, every third residue which is different between the two vigilins is conserved between one vigilin and Scpl6Op. A similar result is obtained for the comparison of C08H9.2 and the vigilins (in 34% of the cases where the vigilins differ from each other, one of them is identical to C08H9.2). These findings strongly suggest that CO8H9.2 and the vigilins represent the nematode and vertebrate homologues of Scpl60p.

A short stretch of amino acids (Leu52-Leu61) of the Scpl60p sequence exhibits similarity to the leucine-rich nuclear export signals from human immunodeficiency virus (HIV)-l Rev [30] and FMRP [31], with the reservation that the last two leucines of the Scpl6Op sequence are separated by two residues instead of only one as in the consensus sequence for nuclear export proposed by Bogerd et al. [32]. No such signal exists at the corresponding positions of the vigilins or of C08H9.2. The vigilins possess a short basic nuclear-localization signal (NLS) between KH domains 3 and 4 [33]. Although this

312 Weber et al. (Eur: J . Biochem. 249)

Fig. 1. Multiple sequence alignment of Scpl6Op, chicken vigilin (Vig-C), human vigilin (Vig-H) and COSH9.2 from C. eleguns. The alignment was generated in two steps, using the ClustalW program [41] and visual inspection of the sequences. Identities (*) and similarities (.) between the four proteins are marked below the alignment. KH domains of Scpl6Op are numbered and coloured, those of the vigilins and C08H9.2 are shaded; bold letters indicate the conserved GXXG loops. For each KH domain the sequence-conserved parts are shown in dark yellow for the classical KH domains (2, 8-12, 14) and in light yellow for the degenerate ones, i.e. for those with an altered or missing GXXG motif (1, 3-7, 13); the a-helical C-terminal parts are shown in blue. Domain 13 of Scpl6Op has an insert of 61 amino acids [5]. Letters X, C and H above the aligned sequences mark residues not conserved between chicken and human vigilin, with X indicating residues different also in Scpl6Op; C marks identity between chicken vigilin and Scpl6Op, and H identity between human vigilin and Scpl6Op. The potential nuclear-export and nuclear-localization signals of Scpl6Op as well as the vigilin NLS are boxed. For further details, see text. EMBLlGenBank accession numbers of the proteins used for the alignment are as follows: Scpl60p, X65645; C. elegans protein, 254342; chicken vigilin, X65292; human vigilin, M64098.

motif is not fully conserved in Scpl6Op, the residues at the corresponding position of the Scpl6Op sequence (Lys321 to Lys333) show all characteristics of a bipartite nuclear localization signal, which typically comprises two basic amino acids, followed by a spacer of about ten residues (seven in the case of Scpl6Op) and a basic cluster in which three out of five amino acids must be basic.

Purification of Scpl60 protein. Scpl6Op was purified from an overproducing yeast strain. As described in detail in Materials and Methods, the purification protocol included chromatography on DNA-cellulose, to which Scpl6Op bound quantitatively under low-salt conditions (0.1 M NaC1). In addition to enriching Scpl60p, this step served to separate proteases from the Scpl6Op containing fractions. In this way we were able to largely avoid proteolytic degradation of both Scpl6Op and the affinity matrix (a column containing anti-Scpl60p antibodies linked to BrCN-activated Sepharose) used in the next purifica-

tion step. As shown in Fig. 2A, the purification procedure resulted in a fraction containing mainly one polypeptide which migrates as a band of approximately 160kDa by SDSffAGE. This band coincides with the Scpl60p-band as detected by immunostaining with anti-Scpl60p serum (Fig. 2B).

The electrophoretic mobility of Scpl6Op is slower than expected from its relative molecular mass of 134750, as calculated from the amino acid sequence [21]. Considering post-transla- tional modification as an explanation for the difference between calculated and observed molecular mass, we examined a possible glycosylation of Scpl60p, which contains ten potential sites for N-linked glycosylation. However, as described in detail in [34], this kind of modification could be excluded: Scpl6Op did not bind to Concanavalin-A-Sepharose, and treatment with peptide-N-glycosidase F did not result in higher electrophoretic mobility of the protein. In a control experiment the carbohydrate part of ovalbumin was completely removed even after treatment with only 10% of the enzyme dose used for Scpl6Op. A slower

A

Weber et al. (Eur: J . Biockem. 249)

160

313

205 - 116 - 97 - 66 - 45 -

1 2 3

Fig. 2. Purification of Scp160 protein from S. cerevisiue. (A) Aliquots of cell extract from strain AK603 (3 pg, lane I), pooled DNA cellulose peak (4 pg, lane 2) and eluate from the affinity column (1 pg, lane 3) were resolved on a 10% SDS/polyacrylamide gel, and proteins were visualized by silver staining. Molecular-mass markers are indicated on the left. (B) Demonstration of antibody specificity by immunoblotting : together with the fractions indicated in (A), proteins of 10 pg of cell extract were separated, the lane was cut off and probed with the mouse anti-Scpl60p serum.

Fig. 3. Northwestern blot of ribohomopolymers bound to Scp16Op. Blots of affinity-purified Scpl6Op (200 ngilane) were prepared as described in Materials and Methods. They were incubated with '*P-labelled poly(rA) (lane I), poly(rU) (lane 2), poly(rG) (lane 3), and poly(rC) (lane 4). Radioactivity bound to the blots was detected by autoradiography.

0 50 100

Amount of ScplGOp per lane (ng)

t p o l y ( r A ) t p o l y ( r U ) -A-poly(rG) I -+poly(rC) +poly(dC)

Fig. 4. Binding of labelled homopolynucleotides to increasing amounts of Scpl60 protein. Northwestern blots were carried out as described in Materials and Methods and shown in Fig. 3. Data are presented as relative amounts of bound radioactivity in comparison to the value obtained for binding of poly(rA) to 10 ng of Scpl6Op, which was arbitrarily fixed as one.

0 10 20 30 Competitor concentration (pgfml)

electrophoretic mobility than expected from the molecular mass has been observed for a variety of acidic proteins [35, 361. This could also explain the migration behaviour of Scp16Op, as its predicted isoelectric point is 5.74, which is consistent with its migration by chromatofocusing (data not shown).

As the rabbit anti-Scpl60p serum originally used [21] was directed only against the C-terminal half of Scpl6Op, we additionally raised a polyclonal mouse antiserum against the purified protein. When tested for specificity on blots of yeast cell extract, this antiserum recognized a single band at 160 kDa (Fig. 2B). Minor bands of higher mobility that were occasionally observed in addition to the 160-kDa band were most probably due to proteolysis.

Nucleic-acid-binding activity of Scpl6Op. Considering the sequence similarity of Scpl6Op to known RNA-binding proteins, we performed northwestern blotting experiments to determine whether Scpl6Op is able to associate with RNA in vitro. As shown in Fig. 3, Scp16Op binds to all four ribohomopolymers, and exhibits the highest affinity for poly(rG). To test whether the difference in binding is more pronounced at lower concentrations of Scpl6Op, the experiment depicted in Fig. 4 was carried out and poly(dC) was included as a probe. Again, poly(rG) bound most efficiently, and remarkably, the difference in binding of poly(rC) and poly(dC) was insignificant. In an attempt to

t p o l y ( r C ) t r R N A -A-dsDNA -A-SsDNA -%+tRNA L__ -__. I Fig. 5. Competition of 3ZP-labelled poly(rC) with different unlabelled nucleic acids for binding to Scpl6Op (100 ng/lane). Inhibition of poly(rC) binding by crude yeast rRNA, dsDNA and heat-denatured DNA from E. coli, and yeast tRNA (competitor concentration, 0, 0.5, 2, 8, and 32 pg/ml, respectively). Increased binding in the presence of low concentrations of tRNA may be due to suppression of unspecific binding of probe to the nitrocellulose membrane.

compare the RNA-binding and DNA-binding activities of Scpl60p more rigorously, we performed a competition experiment with "P-labelled poly(rC) as a probe and unlabelled poly(rC), crude yeast ribosomal RNA, sheared dsDNA from E. coli, sheared and heat-denatured E. coli DNA, and yeast tRNA as competitors (Fig. 5). Under the in vitro conditions, rRNA competed most efficiently with poly(rC) for binding to Scpl60p. Both double-stranded and single-stranded (heat-denatured) DNA led to a significant reduction of probe binding as well, although the effect was somewhat weaker than that observed for rRNA. Yeast tRNA caused no significant reduction of probe binding except at the highest concentration employed.

To test a naturally occurring RNA in contrast to artificial homopolymers, we performed an electrophoretic mobility-shift assay with purified Scpl6Op and rRNA from bovine liver. Higher mobility of Scpl6Op on a native agarose gel when incubated with RNA before electrophoresis was considered indica-

3 14 Weber et al. (Eur. f. Biochem. 249)

RNA + + + + -

S~pl6Op + + + - +

+ 1 2 3 1 5

Fig.6. Binding of Scpl6Op to ribosomal RNA. Samples of commer- cially obtained rRNA (1 pg each) were incubated with 0.2, 0.4 or 1 pg of purified Scpl6Op, respectively (lanes 1-3) and without Scpl60p as control (lane 4). The reaction mixtures were subjected to electrophoresis under non-denaturing conditions. After determining the position of rRNA, the gel was blotted to nitrocellulose and the migration of Scpl60p alone (lane 5 ) as well iib together with the rRNA (lanes 1-3) was visualized by immunostaining with the mouse anti-Scpl60p serum. The arrow indicates the direction of migration.

lysis buffer 0.5 M NaCl --- p30 s30 P S O SSO PSa SSO

205 - Scpl6Op- &m ““rs

116 - c

Polysomes 1 I

I

TOD Bottom ~~

40s 60s 80s Polysomes I I I -

Fig. 8. Separation of ribosomal particles and polysomes on a 7 % to 47% sucrose gradient. A yeast cell lysate was subjected to sucrose- gradient centrifugation as described in Materials and Methods. The gradient was fractionated and the absorbance at 260 nm was monitored. The positions of 40S, 60.5 and 80s ribosomal particles and polysomes are indicated. Below the absorbance profile, an immunoblot of the gradient fractions is shown. The position of Scpl60p in the gradient was detected using the rabbit anti-Scpl60p antibody.

low sal t buffer low sal t buffer + lOmM EDTA

Fig. 7. Membrane-association of Scpl6Op. Cells from strain MZ3 were lysed as described in Materials and Methods. Aliquots of clarified lysate were centrifuged at 30000 g and 50000 g, respectively, yielding fractions P(ellet)30, P50, S(upernatant)30, and S50. P50 was treated with 0.5 M NaCl and recentrifuged at 50000 g. Aliquots of pellets and supernatants corresponding to 15 pg of protein each were resolved on a 7% SDS/polyacrylamide gel and Scpl60p was detected by immunoblotting.

P S P S

S~pl6Op-

PABP - S13 -

tive for association of Scpl60p with the RNA. As shown in Fig. 6, Scpl6Op migrated significantly faster after incubation with RNA than free Scpl60p, which nearly stayed at the position of the loading slot. We conclude that at least a subset of rRNA is able to associate with Scpl60p.

Subcellular fractionation. In subcellular fractionation experiments, more than 90% of Scpl60p was found in the pellet (P50) of the cytoplasmic lysates centrifuged at 50000 g. (Fig. 7). To obtain information on the nature of the membrane association of Scpl6Op, P50 was treated with salt, and the supernatant and pellet fractions were assayed by immunoblotting. Over 50% of Scpl60p was released from the membrane pellet after incubation with 0.2 M NaCI, and incubation with 0.5 M NaCl resulted in almost complete removal of Scpl60p from the pellet (Fig. 7).

The finding that Scpl60p binds to RNA in vitro together with its affiliation to the endoplasmic reticulum prompted us to ask whether ScplGOp might be associated with ribosomes. To

Fig. 9. Scpl6Op can be partially released from the membrane fraction by treatment with EDTA. A rough microsomal pellet obtained by centrifugation of yeast lysate at 100000g was treated with 10 mM EDTA. Aliquots of pellet and supernatant (10 pg of protein each) were withdrawn before and after the treatment with EDTA and analyzed for the presence of Scpl60p by immunoblotting. In parallel, the blots were developed with antibodies directed against PABP and the ribosomal protein S13, respectively, to monitor the dissociation of ribosomes from the membrane pellet.

answer this question, two experiments were performed: firstly, cytoplasmic lysates were fractionated by sedimentation on linear sucrose gradients, and the A,,, of each fraction was monitored to obtain the profile shown in Fig. 8. The position of Scpl6Op in the gradient was determined by western blotting of individual fractions. Under the conditions employed, Scpl60p did not migrate with ribosomal subunits or polysomes, but was found at the top of the gradient. The fractions containing Scpl6Op also

Weber et al. (Eue J . Biochein. 249) 315

showed the strongest signal when tested with an antiserum against Kadp, which we used to monitor the position of micro- somes in the sucrose gradient (data not shown). Thus, we conclude that like Kar2p Scpl6Op is not associated with cytosolic ribosomes. In a second experiment, we treated rough micro- somes with EDTA at low-salt conditions to examine whether dissociation of the ribosomes would lead to a liberation of Scpl60p from the membrane fraction. The dissociation of ribosomes (and the concomitant release of mRNA) was checked by immunoblotting using antisera against the ribosomal protein S 13 and against the poly(A)-binding protein. About SO% of Scpl6Op was released from the membrane pellet by incubation with 10 mM EDTA (Fig. 9). The amount of PABP released by the EDTA treatment was comparable to that of Scpl6Op, whereas a clearly higher percentage (about 80% as judged by western blotting) of S 13 was liberated.

DISCUSSION

We are studying the yeast protein Scpl6Op, which, though not absolutely essential for cell survival under laboratory conditions, proved to be necessary for maintaining the exact ploidy, an observation leading to our hypothesis that the protein might be involved in chromosome distribution or nuclear division [21]. Scpl6Op contains seven classical and seven less well conserved KH domains, sequence motifs that have recently been found in a number of RNA-binding proteins. It possesses the same domain organization as the vertebrate vigilins, i.e., the KH motifs are arranged with little or no space in between. A closer look at the KH domains of Scp16Op and the vigilins reveals that sequence conservation of their GXXG loops differs significantly : while only 3 of the 14 Scpl6Op KH domains contain a perfect loop with at least one positively charged residue, 11 and 12 such motifs are found in chicken and human vigilin, respectively. However, it cannot be inferred from this finding that Scpl6Op has a lower affinity for RNA than the vigilins, as even for KH domains with perfect loops significant differences in the RNA- binding ability have been reported [38].

Another member of the Scpl60p/vigilin family most probably is the gene product of C08H9.2 from the nematode C. eleguns. Its deduced amino acid sequence contains 15 KH domains and in this respect is more similar to the vigilins than to Scpl6Op. However, whereas the human and the chicken vigilin genes are composed of 27 and 29 exons, respectively (the latter gene comprising some 22 kb, the former being more than twice as large), the nematode gene contains only four relatively small introns (45, 50, 49 and 57 nucleotides, respectively) and thus is more similar to the intron-free yeast SCPI60 gene.

It is not merely their identical KH domain organization that argues for Scpl6Op and the vigilins being homologues: both proteins have been localized to the endoplasmic reticulum by immunofluorescence microscopy [21, 371. Recently, vigilin has been reported to be present also in the nucleus and to contain a functional nuclear-localization signal [33]. Although the short basic NLS found in the vigilins is not conserved in Scpl6Op, the corresponding residues of the Scpl6Op sequence (Lys321 and Lys322) might form a bipartite NLS together with the 11 residues following. In addition to this potential NLS, Scpl6Op contains a motif (LPSLKDLPSL61) reminiscent of the leucine- rich nuclear-export signals of HIV-1 Rev and FMRP [30, 311. This motif is located in the N-terminal part of the protein, the only region free of KH domains. That the residues corresponding to the potential NES of Scpl6Op are not conserved in the vigilins does not necessarily argue against Scpl6Op and the vigilins being homologues: as discussed in [33], newly synthesized

vigilin is thought to be imported into the nucleus via its NLS and then re-exported to the rough endoplasmic reticulum despite the lack of a (known) NES.

As the numerous KH domains of Scpl6Op suggest RNA- binding ability, we purified the protein to homogeneity and analyzed its nucleic-acid-binding properties. Northwestern blotting experiments using radiolabelled ribohomopolymers demonstrated that Scpl6Op indeed interacts with RNA in vitro, show- ing a strong preference for poly(rG) under the experimental conditions applied. Additionally, the protein is capable of binding to single-stranded and double-stranded DNA, albeit with lower affinity than to RNA. This seems to be another parallel to the vigilins, as a fusion protein containing the first four KH domains of human vigilin has recently been shown to interact with poly(rG) and, less strongly, with ssDNA and dsDNA 1381. Al- though binding studies with artificial homopolymers do not ex- actly reflect the in uivo conditions, our data, above all the strong preference of Scpl6Op for poly(rG), argue against sequence-in- dependent interaction between Scpl6Op and nucleic acids.

In agreement with the results from immunofluorescence experiments 121 ], subcellular fractionation experiments revealed that most of Scpl6Op is membrane associated. That more than 50% of Scpl6Op was released after treatment of the membrane pellet with 0.2 M NaCl indicates a relatively weak membrane association of Scpl6Op via ionic interactions.

Motivated by our finding of the in vitro interaction between Scpl6Op and ribosomal RNA, we investigated a possible association of the protein with ribosomes. However, as Scpl6Op did not migrate to the positions of ribosomal subunits or polysomes in a sucrose gradient (under conditions comparable to those employed by Siomi et al. [39], who recently demonstrated association of FMRP with 60s ribosomal subunits), we exclude that Scpl6Op is associated with cytosolic ribosomes. This conclusion is corroborated by the finding that in subcellular fractionation experiments Scpl6Op was sedimented almost completely under conditions leaving free ribosomes in the supernatant. To test a potential association of Scpl60p with membrane-bound ribosomes, we investigated whether release of ribosomes from the membrane fraction would coincide with a release of Scpl6Op. The classical procedure for stripping membranes [40] could not be applied as it involves high-salt concentrations. Therefore, we tried to dissociate ribosomes by EDTA treatment under low-salt conditions. About half of Scpl6Op was liberated from the membranes by treatment with EDTA. The same was true for PABP, which associates with ribosomes via mRNA, whereas a con- siderably higher amount of the ribosomal protein S13 was liberated. These findings suggest that a part of Scpl6Op is associated with membrane-bound ribosomes, and that this association occurs via binding to mRNA rather than directly to the ribosomal subunits.

What might the function of Scpl6Op be? Considering its RNA-binding ability, we propose a role either in nuclear export of RNA or in the transport of RNA within the cytoplasm. The presence of both a nuclear-export and a nuclear-localization signal makes it conceivable that Scpl6Op shuttles in and out of the nucleus, functioning as an RNA transporter, as has been proposed for the vigilins [33]. However, as yet we do not see how to explain the relationship between the phenotype of SCPI60 deletion mutants, foremost their increased chromosome number, and the RNA-binding ability of Scpl6Op. We assume that the effect of Scpl6Op exerted on the process of chromosome segregatiodseparation is indirect, e.g. via the transport of se- lected mRNAs. Also, at this stage, and in the light of the association of Scpl6Op with membrane-bound ribosomes described here, a function of the protein in the process of translation, possi-

316 Weber et al. (Eur. J. Biochem. 249)

bly the regulation of translation of specific mRNAs, cannot be excluded.

We are grateful to A. Karwan for constructing strain AK 603, B. Rosenwirth and E. Pursch for preparing the mouse anti-Scpl60p antibody, and A. Krimer for drawing our attention to the KH domains of Scpl60p. G. Neu-Yilik, B. Dobberstein and T. Gibson kindly informed us about their own results before publication. Rabbit anti-Kar2p serum was generously supplied by M. Rose; anti-S13 serum was kindly supplied by J. Stahl. The research was supported by the Fonds zur Fiirde- rung der wisseiischaftlichen Forschung in Osterreich (grant number S 5806-MOB) and the Herzfeldersche Familienstlftung.

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Purification and Nucleic-Acid-Binding Properties of a Saccharomyces Cerevisiae Protein Involved in the Control of Ploidy