©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Major Protein of Messenger Ribonucleoprotein Particles in Somatic Cells Is a Member of the Y-box Binding Transcription Factor Family (*)

(Received for publication, July 22, 1994; and in revised form, November 17, 1994)

Valentina M. Evdokimova (1) Chia-Lin Wei (2) Albert S. Sitikov (1) Peter N. Simonenko (1) Oleg A. Lazarev (1) Konstantin S. Vasilenko (1) Valentin A. Ustinov (1) John W. B. Hershey (2)(§) Lev P. Ovchinnikov (1)(§)

From the  (1)Institute of Protein Research, Russian Academy of Sciences, Puschino, Moscow Region, Russian Federation and (2)Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A cDNA encoding the major core protein, p50, of cytoplasmic messenger ribonucleoprotein particles (mRNPs) of somatic cells was cloned from a rabbit reticulocyte cDNA library. From the derived 324-amino acid sequence, p50 is identified as a member of the Y-box binding transcription factor family. The protein was earlier described as a repressor of globin mRNA translation. These findings suggest that p50 may affect protein biosynthesis at two levels: mRNA transcription in the nucleus and mRNA translation in the cytoplasm. Together with recently published results showing that masked mRNA in germ cells also is associated with proteins of the Y-box binding protein family, the present finding indicates that these proteins are universal core proteins responsible for the formation of cytoplasmic mRNPs in eukaryotes. Highly purified p50 forms large 18 S homomultimeric complexes with a molecular mass of about 800 kilodaltons and melts RNA secondary structure. This suggests that p50 may affect translation by changing the overall structure of the mRNA.


INTRODUCTION

Messenger RNA in the cytoplasm of eukaryotic cells is associated with specific proteins that form ribonucleoprotein particles (mRNPs or informosomes)(^1)(1, 2, 3) . mRNPs isolated from different cells and tissues contain two major proteins with molecular masses of 70 and 50 kDa(4) . The 70-kDa mRNP protein associates with the poly(A) tail of mRNA(5) . This poly(A) binding protein (PABP) is the most widely studied mRNP protein to date. The protein is highly conserved in evolution and is present in most, if not all, eukaryotes (6, 7, 8) . Genetic and biochemical studies show a crucial role of the PABP in cell viability (9) and demonstrate its participation in the initiation phase of translation(10, 11) . In contrast to PABP, the 50-kDa protein (p50) has not been extensively characterized. This protein is the most abundant protein in free mRNP particles of rabbit reticulocytes. It is also present in polyribosomal mRNPs but in lesser amounts(12, 13) . p50 is the most basic protein among all mRNP proteins, with a pI of about 9.5(13) , and binds the most tightly to RNA(12) . Various polyribonucleotides have the following relative affinities to p50: poly(G) > poly(U) > globin mRNA > poly(A) > poly(C)(13) . Furthermore, p50 is phosphorylated both in vivo and in vitro(13) .

We have shown previously that p50 is responsible for the repressed, nonactive state of globin mRNA within free mRNP particles when such particles are added to the wheat germ cell-free translation system (14) . Furthermore, p50 strongly inhibits translation of exogenous globin mRNA in wheat germ and rabbit reticulocyte lysates(12, 15) . Here we describe the formation of large multimeric complexes of p50 and the effect of p50 on overall mRNA secondary structure. In many respects this protein resembles the core hnRNP proteins. Surprisingly, the amino acid sequence of p50 shows no homology with the sequences of hnRNP core proteins. Instead p50 has striking sequence identity with the Y-box binding transcription factors and binds to DNA containing the CCAAT sequence.


MATERIALS AND METHODS

Preparation of Cell Extracts

Reticulocytes were obtained from rabbits injected with a phenylhydrazine solution as described (16) , separated from the plasma by centrifugation, and washed three times with 140 mM NaCl, 5 mM KCl, and 1.5 mM MgCl(2). Reticulocytes were lysed in two volumes of 5 mM MgCl(2), and the lysate was centrifuged at 12,000 rpm for 15 min in a JA-14 rotor in a J21 centrifuge (Beckman). Rabbit muscle and rat liver were homogenized in a blender with 10 volumes of 10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM MgCl(2), and 0.5% Nonidet P-40 and clarified as above.

Reticulocyte ribosome-free extracts and polyribosomes were obtained as described elsewhere(17) . Postmitochondrial supernatants were layered onto 10 ml of 30% sucrose in 10 mM Tris-HCl, pH 7.6, 10 mM KCl, 1.5 mM MgCl(2) and centrifuged at 35,000 rpm for 3 h in the 35 rotor of the L5-50 centrifuge (Beckman). The upper two-thirds of the supernatant was used as the ribosome-free extract. The polyribosomal pellet was suspended in 10 mM Tris-HCl, pH 7.6, 10 mM NaCl. All preparations were stored at -70 °C.

Isolation of mRNPs

Free and polyribosomal mRNPs were isolated from reticulocyte ribosome-free extracts or from polyribosomes, respectively, by chromatography on oligo(dT)-cellulose (type 7, P-L Biochemicals, Inc.) as previously described (18) with minor modifications. Binding of free mRNPs with resin was done in 10 mM Tris-HCl, pH 7.6, 500 mM NaCl, 2 mM EDTA, and 0.5 mg/ml heparin at 4 °C. Polyribosomal mRNPs after dissociation of ribosomes with 33 mM EDTA were adsorbed to the column in the same buffer as above but with 250 mM NaCl, and then the column was washed with the same buffer solution with 500 mM NaCl. mRNPs were eluted with 10 mM Tris-HCl, pH 7.6, 2 mM EDTA at 37 °C, pelleted by centrifugation at 100,000 rpm for 2 h in the type TLA-100.3 rotor (Beckman) at 4 °C and dissolved in 10 mM Tris-HCl, pH 7.6, 50 mM NaCl.

Postnuclear extracts of rabbit muscle and rat liver were applied to oligo(dT)-cellulose columns in 10 mM Tris-HCl, pH 8.0, 250 mM NaCl, 2 mM EDTA, and 0.5 mg/ml heparin at 4 °C. mRNPs were eluted and pelleted by centrifugation as described above.

Isolation of p50 from Free mRNPs of Rabbit Reticulocytes

mRNP proteins were dissociated from mRNA with 3 M LiCl, and the mRNA was precipitated overnight at -20 °C. The RNA precipitate was removed by centrifugation at 12,000 rpm in a Microfuge for 15 min at 4 °C. Proteins from the supernatant fraction were precipitated with 70% saturated (NH(4))(2)SO(4), pH 7.6, at room temperature for 15 min and collected by centrifugation at 12,000 rpm for 15 min at 4 °C. Precipitates were dissolved in 10 mM HEPES-KOH, pH 7.6, 100 mM NaCl and dialyzed against the same buffer solution overnight. About 500 µg of mRNP protein were applied to a Superose 6 HR/10/30 column (Pharmacia Biotech Inc.) equilibrated with 10 mM HEPES-KOH, pH 7.6, 100 mM NaCl. Chromatography was done at a flow rate of 0.4 ml/min with the fast protein liquid chromatography system. The fraction volume was 1.5 ml.

p50 for sequencing and immunization was isolated from free mRNPs of rabbit reticulocytes by preparative 10% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R-250, and the p50 protein band was excised. p50 was electroeluted from the gel by using an LKB apparatus in a buffer containing 100 mM NH(4)HCO(3), 0.1% SDS at 25 V for 15 h at room temperature.

Peptide Isolation and Sequence Analysis

p50 was exhaustively digested with V8 protease (Sigma; 0.1 mg/ml) for 5 h at 37 °C in 20 mM Tris-HCl, pH 6.8, and 0.1% SDS, or with trypsin (Worthington, 0.1 mg/ml) for 5 h at 37 °C in 10 mM Tris-HCl, pH 7.3. Peptides were separated by HPLC by using an RP-318 column (Pharmacia) and further purified by rechromatography on the same column. Peptides I, II, III, and V (200 pmol) were sequenced in the Protein Structure Laboratory at the University of California, Davis, with an Applied BioSystems 470A protein sequencer. Peptide IV was sequenced in the Department of Medical Biochemistry, Sylvius Laboratories, The Netherlands.

Cloning of p50 cDNA

cDNA encoding p50 was synthesized from rabbit reticulocyte poly(A) mRNA with Superscript reverse transcriptase (Life Technologies, Inc.). Degenerate primer 1 (5`-CCCAAGCTTGCTC(T/C)GC(T/C)TG(A/G)TT(A/G)TC-3`; a HindIII site is underlined) designed to correspond to peptide V amino acids 315-319 (see Fig. 1) was heated at 70 °C with the mRNA, then extended at 42 °C for 1 h in a 35-µl reaction containing 4 mM MgCl(2), 1 mM deoxyribonucleoside triphosphates, 25 units of RNasin, 4 mM dithiothreitol, and 100 units of enzyme. The cDNA was purified by phenol extraction and ethanol precipitation, then amplified by the polymerase chain reaction (PCR). Primers 1 and 2 (5`-CCCCTGCAGCCGA(A/G)AC(T/G)CA(A/G)CA(A/G)CC-3`; a PstI site is underlined; it corresponds to amino acids 4-8 (Fig. 1) and peptide I) were used at 1 µM to amplify the cDNA in 0.1 the reaction above for 30 cycles (1 min at 94 °C, 1 min at 55 °C, 1.5 min at 72 °C). The amplification mixture (0.1 volume) was subjected to a second PCR with primers 2 and 3 (5`-CCCGGATCC(G/T)GC(G/T)CC (T/C)TG(A/G)TT(A/G)TC-3`; it corresponds to amino acids 315-319 and peptide III) as above except 1 min was used at 72 °C. The second PCR generated a single major band of 563 bp which was gel-purified and sequenced.


Figure 1: Amino acid sequence comparison of p50 and eukaryotic Y-box proteins. The p50 sequence was determined as described under ``Materials and Methods''; the Y-box binding protein sequences are from Wolfe et al.(23) . The p50 protein sequence is compared to the other proteins, where identical amino acid residues are marked with dashes and nonidentical residues are defined. Dots mean the absence of the corresponding residues. Peptide sequences reported in Table 1are shown by above-line dashes and are labeled. Residue numbers of the last p50 residue in each line are noted on the right. The DNA sequence of the cloned cDNA has been deposited in GenBank (accession no. U16821).





The 563-bp PCR fragment was labeled by the random priming method (Amersham Corp.) and used as a probe to screen a rabbit reticulocyte cDNA library (19) in a ZAPII vector, kindly provided by J.-J. Chen (Massachusetts Institute of Technology). Phage (5 times 10^4 plaque-forming units) were plated on a lawn of Escherichia coli XL-1 blue per 150-mm NZCYM agar plate, and plaque DNA was lifted onto nylon filters and screened with heat-denatured P-labeled probe (5 times 10^5 cpm/ml) in 5 times SSC (1 times = 150 mM NaCl, 15 mM sodium citrate), 5 times Denhardt's reagent (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.5% SDS, and 50% formamide at 42 °C for 16 h. The filters were washed at 55 °C in 0.2 times SSC, 0.1% SDS and exposed to Kodak X-omat AR film at -80 °C for 16 h. Twenty-three putative positive plaques were picked and rescreened at lower density (200-500 plaque-forming units/80-mm plate) until four positive plaques were confirmed. Inserts were excised by helper phage from the purified ZAP phages and converted into pBluescript SK according to the manufacturer's instructions (Stratagene). The inserts and subfragments generated with BamHI, EcoRI, and HindIII were subcloned into M13 mp18 and mp19 replicative form DNA and sequenced by the dideoxynucleotide chain-termination method. The plasmid carrying the 1.5-kb insert is called pBSK-p50.

Expression of p50 cDNA in E. coli

The p50 cDNA was expressed from a plasmid called pET-3-1-p50 which was constructed as follows. DNA encoding the N-terminal 60% of p50 was amplified by PCR by using pBSK-p50 DNA as template and an upstream primer (5`-CCCTGCAGTCACCGCACATATGAGCAGCGA-3`, which encodes the first 4 amino acids (see Fig. 1) and contains PstI and NdeI sites (underlined)) and a downstream primer (5`-CCTTCGCCTGCGGTAGGGCCGGA-3`, which corresponds to amino acids 185-192 and overlaps a unique EcoNI site (underlined)). Primers (1 µM) and template DNA were subjected to 35 cycles (1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C) as described above to generate a 0.6-kb DNA. The PCR fragment was digested with PstI and EcoNI and substituted for the PstI-EcoNI fragment in pBSK-p50 to generate pBSK-p50-2. pBSK-p50-2 was digested with NdeI and XhoI and cloned into the same sites of pET-3-1 (20) to generate pET-3-1-p50. Thus, the entire coding region of p50 lies just downstream from the T7 promoter and Shine/Dalgarno sequence, suitable for expression in bacteria. The expression plasmid was transformed into E. coli strain BL21 (DE3), and cells were grown to OD = 0.5 and induced with IPTG for 2 h. Cells were collected and lysed by boiling in SDS-gel sample buffer for analysis by PAGE.

In Vitro Transcription and Labeling of alpha-Globin mRNA

Rabbit alpha-globin mRNA was transcribed by SP6 RNA polymerase from alpha-globin cDNA cloned in the pHST111 vector cleaved by BamHI. The RNA transcript contains the complete alpha-globin coding sequence, the native 5`-UTR lacking the first 22 nucleotides and a poly(A) tail of 35 nucleotides. Transcription was done according to standard procedures (21) with some modifications. The reaction mixture in a total volume of 100 µl contained 1 µg of plasmid DNA, 20 units of RNasin (Amersham), 250 units of SP6 RNA polymerase, 5 mM of each NTP, 2 mM spermidine, 80 mM HEPES-KOH, pH 7.6, 18 mM MgCl(2), and 20 mM dithiothreitol. The reaction was carried out for 2 h at 37 °C, and the mixture was deproteinized with phenol:chloroform and then with chloroform. RNA was precipitated by bringing to 3 M LiCl for 30 min at 4 °C, washed two times with 70% ethanol, and dissolved in water. RNA was precipitated again with ethanol in the presence of 3 M ammonium acetate and reprecipitated twice with 4 M ammonium acetate to remove nonincorporated nucleoside triphosphates.

alpha-Globin mRNA was 3`-labeled by [5`-P]pCp as described elsewhere (22) with minor modifications. The reaction (10 µl) contained 2 µg of alpha-globin mRNA, 10 µM [5`-P]pCp (1000 Ci/mmol; Radioisotope, Tashkent), 1 mM ATP, 50 mM HEPES-KOH, pH 7.6, 15 mM MgCl(2), 1.3 mM dithiothreitol, 16% (v/v) dimethyl sulfoxide, 2.5 µg bovine serum albumin, and 40 units of T4 RNA ligase (Fermentas, Vilnius, Lithuania) and was incubated at 4 °C for 12 h. Labeled RNA was deproteinized with phenol:chloroform and precipitated with ethanol.

Double-stranded Oligonucleotides

The following oligodeoxyribonucleotides and their complementary strands were synthesized on a Gene Assembler (Pharmacia): CCAAT-containing 26-mer, 5`-TACTT CCACCAATCGGCATGCACGGT-3`; and control oligomer, 5`-AATGCCCGCCGCCGCCGCCGCCGCCCAAAACTG-3`. Synthesized single strands were purified by 15% PAGE in 0.5 times TBE (1 times TBE = 89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.5). The respective complementary strands were combined, denatured for 2 min at 80 °C and annealed for 30 min at room temperature. The double-stranded oligos were 5`-end-labeled by using [-P]ATP (>5000 Ci/mmol; Radioisotope, Tashkent) and T4 polynucleotide kinase (IBI) and purified from unincorporated radioactivity by two precipitations with 2.5 M NH(4)Ac, pH 4.5, and 2 volumes of ethanol. PAGE analysis revealed no single-stranded DNA contamination of the labeled double-stranded DNA.

The p50-alpha-Globin mRNA Interaction by Footprinting

[5`-P]pCp-labeled alpha-globin RNA (20 ng; 50,000 cpm) was mixed with 0.5 µg of p50 and 5 µg of total tRNA from E. coli to decrease nonspecific p50-RNA interactions in a total volume of 10 µl in binding buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 3 mM MgCl(2), 50 mM NaCl, and 5% glycerol). The mixture was incubated for 15 min at 30 °C. RNase T1 (10 or 10 units) was added to the samples and incubation was continued for 10 min. The reaction mixtures were loaded directly onto 6% polyacrylamide gels with 6 M urea and subjected to electrophoresis at 120 V for 6 h. The dried gels were exposed to x-ray beta-Max film (Amersham) at -70 °C.

Preparation of Antibodies and Immunoblotting Procedure

p50 isolated by preparative SDS-PAGE was used to immunize BALB/c mice. Mice were injected with p50 three times intraperitoneally at 1-month intervals. The first 50 µg were injected in Freund's complete adjuvant; for the second and third immunizations, 25 µg were injected in incomplete Freund's adjuvant. Two days before the last immunization the mice were inoculated intraperitoneally with Krebs II ascites cells, and 1 week later ascites fluid and blood samples were taken, mixed, and incubated for 1 h at 37 °C and then for 12 h at 4 °C. The coagulate was removed by centrifugation, and the antibodies from the supernatant were precipitated with 25% saturated (NH(4))(2)SO(4), pH 7.6, and dissolved in the initial volume of 10 mM HEPES-KOH, pH 7.6, 50 mM NaCl, and 3 mM NaN(3).

For immunoblotting, proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked overnight at 4 °C with 1% BSA, 1% polyvinylpyrrolidone, 0.05% Tween 20 in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl and probed with p50 antibodies at 1:2,000 dilution. Immunocomplexes were detected by using the ECL Western blotting analysis system (Amersham) or alkaline-phosphatase-conjugated antibodies (Cappel) with chromogenic reagents nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate according to the manufacturers' recommendations.


RESULTS

To better study the structure and function of p50, we set out to clone and sequence its cDNA. p50 protein was purified from isolated free mRNP particles derived from rabbit reticulocytes as described under ``Materials and Methods.'' The pure protein was completely digested by V8 protease or trypsin, and p50 peptides were separated by HPLC and purified by rechromatography on the same column as described under ``Materials and Methods.'' Isolated peptides were sequenced by the Edman degradation method. The amino acid sequences of five peptides are reported in Table 1. Three peptides (III, IV, and V) gave unique sequences; one preparation obviously represented a mixture of two peptides (I and II). A comparison of these sequences with the GenBank data base shows their complete or nearly complete identity with sequences contained in proteins known as Y-box binding proteins(23) . The five peptides (labeled and indicated by dashed overlines above the p50 sequence in Fig. 1) are homologous to regions from the N terminus to the C terminus of the mammalian proteins YB-1, EF1(A), MSY1, and MUSYB and the Xenopus protein FRG Y1. The relatedness of p50 to Y-box binding proteins is described in detail below.

cDNAs encoding p50 were cloned from a rabbit reticulocyte cDNA expression library by using PCR and hybridization strategies as described in detail under ``Materials and Methods.'' Two degenerate primers were synthesized based on peptides I and V from the N- and C-terminal regions (as deduced from the homology to known Y-box binding proteins). The downstream primer was used to synthesize cDNA from rabbit reticulocyte poly(A) mRNA, and both were used to amplify the cDNA. A portion of the reaction was amplified again with the upstream primer and a nested primer based on peptide III to generate a 563-bp fragment. The fragment was sequenced and was found to encode a protein homologous to YB-1 and other Y-box binding proteins. The 563-bp fragment was radiolabeled and used to probe a rabbit cDNA library in ZAPII. From 23 putative positive plaques from 1 times 10^6 plaques screened, four phages were selected and purified which contained insert sizes of 2.6, 1.5, 1.5, and 1.3 kb. From the DNA sequences, the two 1.5-kb inserts are identical and contain a 972-bp open reading frame encoding a 324-amino acid protein and 122 and 409 bp of 5`- and 3`-untranslated regions. The 1.3-kb clone lacks cDNA encoding the first 4 amino acid residues, whereas the 2.6-kb clone appears to contain a fusion of other DNAs and was not analyzed further. The 1.5-kb cDNA insert in bacteriophage was converted into the recombinant Bluescript SK plasmid called pBSK-p50. The DNA sequence of the 1.5-kb insert has been deposited in GenBank (accession no. U16821).

The 972-bp open reading frame codes for a protein whose sequence is shown in Fig. 1. All of the peptide sequences shown in Table 1match the cDNA-derived sequence except for the Leu residue which is Lys in the peptide sequence (Fig. 1). To demonstrate that the cDNA clone encodes p50, the 972-bp open reading frame was expressed in E. coli as described under ``Materials and Methods.'' A 50-kDa protein was specifically overexpressed in cells transformed with pET-3-1-p50 (Fig. 2). This protein precisely comigrates with purified p50 on SDS-PAGE and reacts with anti-p50 antibodies (Fig. 2). The results indicate that the p50 cDNA is full-length.


Figure 2: Expression of p50 in E. coli. p50 protein was expressed in E. coli from pET-3-1-p50 as described under ``Materials and Methods.'' The cell lysate was subjected to SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue G-250 (Panel B) and immunoblotted with anti-p50 antibodies and alkaline phosphatase-conjugated secondary antibodies as described under ``Materials and Methods'' (Panel A). Lanes 1 and 2, lysate protein from E. coli transformed with the control vector pET-3-1, without and with induction for 2 h with IPTG, respectively; lanes 3 and 4, lysate protein from E. coli transformed with pET-3-1-p50, without and with induction for 2 h with IPTG, respectively; lane 5, purified p50 from rabbit reticulocytes. Migration positions of molecular mass markers are shown in kilodaltons to the left of Panel B.



A computer-assisted comparison of the entire p50 amino acid sequence with sequences in the GenBank shows that p50 is homologous to a family of proteins known as Y-box binding transcription factors (Fig. 1). The Y-box is a 14-bp DNA element that contains the sequence CCAAT in reverse. The structurally related Y-box binding proteins regulate the transcriptional activity of promoters and are found in essentially all cells, from bacteria to vertebrates. The p50 protein clearly is a member of the Y-box binding protein family, exhibiting about 97% sequence identity with the human proteins EF1(A) and YB-1 and 86% identity with FRG Y1. FRG Y2 and mRNP3, other members of this class of proteins, are identified as germ line-specific mRNP proteins in Xenopus oocytes. Although these mRNP proteins closely resemble p50 and the other Y-box binding transcription factors in the so-called cold shock domain (p50 residues 52-133), they share little or no homology to p50 in the C-terminal half of their structures.

Since many Y-box binding proteins bind both to RNA and to DNA containing the CCAAT sequence, we tested purified p50 for such activities. In nitrocellulose filtration assays with labeled DNA, p50 binds to a double-stranded 26-mer containing the CCAAT sequence both at low and at high salt concentration, whereas a random double-stranded oligodeoxyribonucleotide interacts with p50 only under low salt conditions (Fig. 3A). In competition experiments, a single-stranded 26-mer DNA complementary to the CCAAT-containing oligodeoxy-ribonucleotide is a poor competitor with the double-stranded DNA for p50 binding (Fig. 3B). Thus p50 isolated from mRNP particles possesses the basic property of Y-box binding proteins, namely binding to double-stranded DNA containing the CCAAT sequence. In the competition experiments we also show that mRNA strongly binds with p50 and dissociates complexes of p50 with DNA containing CCAAT, whereas tRNA and ribosomal RNA are very weak competitors (Fig. 3C). Thus, p50 protein possesses the ability to bind both to CCAAT-containing DNA and to still unknown regions within mRNA.


Figure 3: Binding of p50 with various DNAs and RNAs. Panel A, purified p50 (50 ng) was incubated on ice for 20 min with 10 ng P-labeled CCAAT-containing double-stranded 26-mer DNA (upper row) or nonspecific double-stranded oligomer DNA (lower row) (see ``Materials and Methods'') in 10 µl of buffer containing 10 mM Tris-HCl, pH 7.6, 5 mM MgCl(2), 2 mM EDTA, 1 µg of poly(dI-dC), and NaCl at the indicated concentrations. The p50-nucleic acid complexes were filtered through nitrocellulose filters (0.45-µm pore diameter; Whatman) and autoradiographed. Panels B and C, purified p50 binding to P-labeled CCAAT-containing double-stranded 26-mer DNA was determined as described in Panel A except that reactions contained 300 mM NaCl and included 100 ng of unlabeled competitor nucleic acids as indicated.



It is known that Y-box binding proteins possess the ability to form quaternary structures(24, 25, 26) . To determine whether or not the p50 mRNP protein possesses this property, preparations of free mRNPs from rabbit reticulocytes were depleted of mRNA either by ribonuclease treatment or by the LiCl extraction procedure and were gel filtered through a fast protein liquid chromatography Superose 6 column (Pharmacia) (Fig. 4). Free mRNPs and isolated globin mRNA (not shown) elute in the void volume (near fraction 6). A substantial amount of protein elutes from the column ahead of other proteins, in the region corresponding to a molecular mass of about 800 kDa (fractions 7-9). This peak contains only p50, as shown by SDS-PAGE analysis of the Superose 6 column fractions (Fig. 5). Thus p50 derived from free mRNPs after mRNA has been removed is found as a large homo-multimeric aggregate apparently containing more than a dozen p50 molecules. The 800-kDa protein particles formed by p50 aggregation are quite stable and remain intact during rechromatography on Superose 6 under a wide range of ionic strength conditions (50-400 mM NaCl; results not shown). An A/A ratio of 1.1 for this particle excludes the possibility of considerable amounts of RNA in the complex. Upon centrifugation in sucrose density gradients, the 800-kDa particles sediment with a sedimentation coefficient of about 18 S (Fig. 6).


Figure 4: Gel-filtration analyses of free mRNP proteins. Protein was isolated from free mRNP particles either by treatment with 20 µg/ml RNase A for 1 h at 37 °C (Panel A) or with LiCl as described under ``Materials and Methods'' (Panel B). Both preparations were subjected to fractionation by Superose 6 HR/10/30 column chromatography (Pharmacia). Arrows indicate the elution positions of marker proteins: thyroglobulin, 669 kDa; ferritin, 440 kDa; bovine serum albumin, 67 kDa; RNase A, 13.7 kDa.




Figure 5: SDS-PAGE analysis of protein fractions. Proteins from the Superose 6 column fractions shown in Fig. 4were precipitated with 5% trichloroacetic acid at 4 °C, washed with 90% acetone, and separated by SDS-PAGE according to Laemmli(33) , except that linear 10-20% acrylamide gradient slab gels were used. Proteins were stained in the gels with Coomassie Brilliant Blue G-250. Panel A, lane 1, molecular mass markers; lane 2, total proteins from free mRNPs; lanes 3-6, fractions 6-9 from Fig. 4A; lanes 7-10, fractions 12-15 from Fig. 4A. Panel B, lane 1, molecular mass markers; lane 2, total proteins from free mRNPs; lanes 3-10, fractions 7-14 from Fig. 4B.




Figure 6: Sedimentation distribution of the p50 complex in sucrose density gradients. p50 was isolated by gel-filtration of LiCl-extracted protein on a Superose column (see Fig. 4B). The p50 complex was analyzed on 5-20% sucrose density gradients in 10 mM Tris-HCl, pH 7.6 and 100 mM NaCl, without fixation (Panel A) or after fixation with 0.1% glutaraldehyde (Panel B). Centrifugation was performed in a TLS-55 rotor (Beckman) at 55,000 rpm for 2 h at 4 °C. Gradients were scanned for absorbance at 280 nm. Vertical lines indicate positions of 9 S globin mRNA and 18 S and 28 S rRNA.



As p50 recognizes DNA containing the CCAAT sequence but also binds to mRNA, it is of interest to identify the p50 binding site on mRNA. To define binding sites for p50 on a mRNA molecule, we used a footprinting approach (Fig. 7). Rabbit alpha-globin mRNA was synthesized and 3`-labeled in vitro as described under ``Materials and Methods.'' The alpha-globin [P]mRNA was mixed with purified p50 and treated with RNase T1 at two concentrations. Free alpha-globin [P]mRNA treated with RNase T1 under the same conditions served as a control (lanes 3 and 5). To our great surprise, p50 considerably increases mRNA sensitivity to RNase over its entire length (compare lanes 2 and 3, lanes 4 and 5), although p50 itself has no RNase activity (lane 6). We were unable to detect any specific mRNA fragment that is protected by p50. We believe that the increase in mRNA sensitivity to RNase in the presence of p50 is caused by melting the mRNA secondary structure, possibly on the surface of the protein particle.


Figure 7: The effect of p50 on the sensitivity of alpha-globin mRNA to RNase T1. An in vitro alpha-globin transcript was prepared, 3`-end labeled with [P]phosphate, and analyzed for RNase T1 sensitivity in the presence or absence of p50 as described under ``Materials and Methods.'' Electrophoresis of cleaved transcripts was performed in 6% polyacrylamide gels in the presence of 6 M urea and the dried gels were subjected to autoradiography. Lane 1, alpha-globin mRNA without treatment; lanes 3 and 5, alpha-globin mRNA alone treated with RNase T1 at 1 and 0.1 unit/ml, respectively; lanes 2 and 4, the alpha-globin mRNA-p50 complex treated with RNase T1 at 1 and 0.1 unit/ml, respectively; lane 6, the alpha-globin mRNA-p50 complex without RNase treatment. Arrows indicate marker positions: xylene cyanole (XC) and bromphenol blue (B).



To verify this assumption, the effect of p50 on mRNA absorbance at 260 nm and on circular dichroism was studied. The increase in UV absorption with increasing amounts of added p50 (Fig. 8A) indicates that p50 partially disrupts mRNA secondary structure. The hypochromicity effect saturates at a p50 to RNA mass ratio of 1.5 to 1. The change of hypochromicity and amplitude of the CD spectrum in the region of the absorption band at 260 nm (Fig. 8B) shows that the process of melting of RNA secondary structure is noncooperative and incomplete. The CD spectrum of the p50-RNA complex at a p50 to RNA mass ratio of 4 to 1 at 20 °C virtually coincides with the spectrum of free mRNA at 50 °C. The amount of mRNA secondary structure in such complexes can be estimated as 40% of the original, by comparison with the mRNA CD spectrum at 70 °C.


Figure 8: Effect of p50 on UV absorbance and CD spectrum of mRNA. alpha-Globin mRNA (8 µg) generated by in vitro transcription as described under ``Materials and Methods'' was mixed with various amounts of p50 in 30 µl of buffer containing 10 mM Tris-HCl, pH 7.6, and 100 mM NaCl. The mixtures were incubated for 30 min at 30 °C, then for 30 min at 0 °C, and were diluted to 200 µl with the same buffer. Panel A, UV spectra were measured by using a Varian Cary 219 spectrophotometer (cell length 1 mm, 0.2-ml sample). Light scattering and p50 absorbance effects are subtracted. Panel B, CD spectra were obtained on a JASKO J-600 spectropolarimeter equipped with a variable temperature accessory (cell length 1 mm, 0.2-ml sample). Curves 1-3 are spectra of mRNA (8 µg) at 20, 50, and 71 °C, respectively. Curves 4-9 are mRNA spectra at 20 °C in the presence of 0.4, 0.8, 1.2, 6.4, 12, and 36 µg of p50. Curve 10 is the spectrum of p50 without mRNA. Inset, effect of p50 on the RNA CD spectrum at 260 nm.



Free mRNP particles from rabbit reticulocytes contain primarily globin mRNA, and obviously the p50 in these preparations is mainly bound with globin mRNA. The question arises whether this protein is specific to globin mRNA or whether it interacts with some or all other mRNAs. To answer this question we also analyzed mRNP preparations from rat liver and rabbit muscle for the presence of p50 using immunoblot procedures with anti-rabbit-p50 monospecific mouse antibodies. p50 is detected in comparable amounts in the three mRNP preparations studied, assuming that the rabbit and rat proteins react comparably with antibodies (Fig. 9A). We conclude that p50 participates in the formation of mRNP particles with many different mRNAs. The protein is not detected in nuclear extracts (data not shown) and apparently is not a part of hnRNPs.


Figure 9: Detection of p50 in polyribosomal and free mRNPs of different tissues. Panel A, cytoplasmic free mRNP proteins from rabbit reticulocytes (lane 1), rat liver (lane 2), and rabbit muscle (lane 3) were subjected to 10-20% SDS-PAGE. Immunoblots with anti-p50 antibodies and the ECL kit (left three lanes) and Coomassie Blue staining (right four lanes) are shown. Lane 4 shows molecular mass markers. Panel B, immunoblots with anti-p50 antibodies and the ECL kit (left two lanes) and Coomassie Blue staining (right two lanes) of gels with free (lane 1) and polyribosomal (lane 2) mRNP proteins from rabbit reticulocytes.



We have shown earlier by two-dimensional isoelectric focusing/SDS-PAGE that the protein with a mass and pI similar to those of p50 is present in polyribosomal mRNPs(13) . Here it is shown that antibodies prepared against p50 from the free mRNPs cross-react with the p50 from polyribosomal mRNPs (Fig. 9B, lane 2). Thus, it is likely that p50 is a universal protein associated with many mRNAs both in translating polysomes and nontranslating mRNP particles.


DISCUSSION

The amino acid sequence of the major cytoplasmic mRNP protein, p50, reveals strong homology with proteins of the Y-box binding protein family throughout their entire length. Members of this family include transcription factors that bind to double-stranded DNA containing the CCAAT sequence within the Y-box. Examples from mammalian species include YB-1, MSY1, MUSYB, EF1(A), and YB3. The binding of p50 to double-stranded DNA containing the CCAAT sequence but not to DNA lacking CCAAT supports the view that p50 belongs to the Y-box binding protein family. Two homologous Y-box binding proteins also have been identified in Xenopus laevis oocytes: FRG Y1 and FRG Y2. FRG Y1, a transcription factor, resembles p50 and the mammalian Y-box proteins over its entire length, whereas FRG Y2 is homologous in the cold-shock domain (corresponding to residues 52-133 in the p50 sequence) but shares less than 25% sequence identity outside this region. It is noteworthy that FRG Y2, along with the highly related protein, mRNP3, are germ line-specific mRNP proteins.

The p50 cDNA encodes a protein whose calculated mass is 35 kDa, whereas the cDNA expressed in E. coli generates a product whose migration during SDS-PAGE corresponds to 50 kDa and is identical to that of p50 purified from reticulocytes. The discrepancy between the apparent and calculated masses may be explained by aberrant migration of p50 during SDS-PAGE. Such discrepancies have been observe with other members of the Y-box binding proteins and frequently occur with proteins that possess a high percentage of charged amino acid residues. For example, FRG Y2, whose cDNA-calculated mass is 37 kDa, migrates as a 56-kDa protein in SDS-PAGE(26, 27, 28) ; similarly, the mouse Y-box protein, MSY1, migrates much more slowly on SDS-PAGE than predicted from its sequence(29) . Thus it is possible that rabbit p50 corresponds to one or more of the previously characterized mammalian or Xenopus proteins whose cDNAs have been cloned.

It appears that some of the Y-box binding proteins possess dual functions: regulation of transcription by binding to DNA in the nucleus and regulation of translation by binding to mRNA in the cytoplasm. p50 appears capable of both functions, as it was isolated as a component of free cytoplasmic mRNP particles and represses translation in vitro, and furthermore binds to DNA containing CCAAT sequences. The closely related Xenopus oocyte proteins, p54 and p56, also are found in free mRNP particles and the latter is nearly identical to FRG Y2(28, 30) . FRG Y2 binds to CCAAT sequences in DNA and is known to regulate transcription of promoters containing the Y-box(27, 28, 31, 32) . Similarly, the mouse MSY1 protein, like p50, is homologous to FRG Y2 in the cold-shock domain and is an mRNP component in spermatocytes(29) . p50, MSY1, and p54/56 all bind nonspecifically to RNA sequences, and all have been shown to be abundant proteins in mRNP particles in their respective cells or tissues.

Xenopus p54/56 are present in eggs and embryos up to the gastrula stage but not in tissues of the adult organism(28) . Furthermore, they are detected in the cytoplasm but not in the nucleus. These members of the Y-box binding family are homologous to p50 only in the so-called cold-shock domain, but differ greatly in the C-terminal half of the protein. However, proteins immunologically related to p54/p56, but with slightly different electrophoretic mobilities in SDS-PAGE, were found by the same authors in bovine testis and in Xenopus liver. Although many mRNP proteins belonging to the Y-box family have been isolated from germ cells or embryonic tissues, it is possible that such proteins also are present in other cell types. Here we have shown that a cytoplasmic mRNP protein isolated from a somatic cell is a member of the Y-box binding protein family. The view that the Y-box family is present in most or all somatic cells is supported by the fact that antibodies to rabbit p50 cross-react with proteins of similar size in two other mammalian somatic tissues. Furthermore, FRG Y1 mRNA, which encodes a protein highly homologous to p50, is found in frog somatic tissues. We therefore believe that mRNP proteins in embryos and in tissues of adult organisms occur as two different yet very similar forms of Y-box binding proteins. We have isolated the adult form, p50 from rabbits, whereas others have identified embryonic forms of this protein family, namely p54/p56 from Xenopus and MSY1 from mouse. The results described here show for the first time that a Y-box protein (p50) exists within cytoplasmic mRNP particles of somatic mammalian cells.

It has been noted that the Y-box binding transcription factors FRG Y1 and FRG Y2 are capable of multimerization(26) . P54/56 from Xenopus oocytes form a 6 S heterodimer complex and a 15 S complex containing in addition to p54/56 two other proteins, p60 and p100(24) . The Xenopus proteins appear to bind RNA as dimers or possibly higher oligomers(25) . In contrast, p50 forms a very large homomultimeric complex of 18 S (800 kDa). Our results also indicate that the high molecular weight complex is formed in the absence of RNA.

Numerous workers propose that p54/p56 from Xenopus mRNPs is responsible for mRNP masking in oocytes(25, 29, 32) . Earlier we identified rabbit reticulocyte p50 as a repressor of translation, which together with an activator seems to regulate the distribution of mRNA between polyribosomes and free mRNP particles(12, 14, 15) . Taking into account that p50 forms large particles and alters considerable portions of mRNA secondary structure, we propose that p50 affects mRNA translation by changing the overall mRNA structure on its surface, thereby affecting the interactions of the mRNA with translation factors and other regulatory proteins. Thus it may play a fundamental structural role for mRNP particles in the cytoplasm, analogous to that of core hnRNP proteins in the nucleus which possess a similar mass but quite different primary structures. One can surmise that p50 represents the universal structural protein for packaging cytoplasmic mRNAs.


FOOTNOTES

*
This work was supported by grants N 93-04-6548 from the Russian Fundamental Research Foundation, MUC000 from the International Science Foundation, a grant from the Russian State Programme ``Cell-Free Protein Biosynthesis'' (to L. P. O.), United States Public Health Service, National Institutes of Health Grant GM22135 (to J. W. B. H.), and National Science Foundation Grant MCB-91-23549 (to L. P. O. and J. W. B. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16821[GenBank].

§
To whom correspondence should be sent: Dept. Biological Chemistry, UCD School of Medicine, Davis, CA 95616. Tel.: 916-752-3235; Fax: 916-752-3516; jwhershey{at}ucdavis.edu.

(^1)
The abbreviations used are: mRNP, ribonucleoprotein particle; PABP, poly(A) binding protein; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; bp, base pair(s); kb, kilobase pair(s); hnRPN, heterogeneous nuclear ribonucleoprotein particle.


ACKNOWLEDGEMENTS

We are grateful to A. S. Spirin for critical comments on the manuscript and for constructive suggestions. We thank J. Ilan for the alpha-globin gene, A. Oleinikov for the nonspecific oligonucleotide, and R. Amons for sequencing peptide IV.


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