From the Institute of Protein Research, Russian
Academy of Sciences, Pushchino, Moscow Region 142292, Russian
Federation and the § Department of Biological Chemistry,
School of Medicine, University of California,
Davis, California 95616
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ABSTRACT |
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The major core protein of cytoplasmic messenger
ribonucleoprotein particles (p50) has been shown previously to inhibit
protein synthesis in vitro and in vivo.
Furthermore, p50 is highly homologous to the Y-box-binding
transcription factor family of proteins, binds DNA containing the Y-box
motif, and thus may have a dual function in cells as a regulator of
both transcription and translation. Here we show that binding or
removal of p50 from rabbit reticulocyte lysate by monospecific
antibodies to p50 strongly inhibits translation of endogenous and
exogenous globin mRNAs as well as prokaryotic -galactosidase
mRNA in a rabbit reticulocyte cell-free system. Thus, depending on
the conditions, p50 not only may act as a translational repressor, but
may also be required for protein synthesis. Translation inhibition with
anti-p50 antibodies is not a result of mRNA degradation or its
functional inactivation. The inhibition does not change the ribosome
transit time, and therefore, it does not affect elongation/termination of polypeptide chains. The inhibition with anti-p50 antibodies is
followed by a decay of polysomes and accumulation of the 48 S
preinitiation complex. These results suggest that p50 participates in
initiation of protein biosynthesis. Although uninvolved in the
formation of the 48 S preinitiation complex, p50 is necessary either
for attachment of the 60 S ribosomal subunit or for previous 5
-untranslated region scanning by the 43 S preinitiation complex.
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INTRODUCTION |
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Regulation of gene expression in eukaryotes often involves modulating the rate of initiation of protein synthesis (for reviews, see Refs. 1-3). Translation is controlled by proteins that are associated with mRNA and/or ribosomes and that either promote or repress the protein synthesis rate. One type of mechanism involves modulation of initiation factor activities (4) that usually affect global translation, whereas the second type involves specific protein repressors that bind to one or a small set of mRNAs (5), leading to specific control. The third type of mechanism may involve proteins associated with many or even all mRNA species, such as proteins present in messenger ribonucleoprotein particles (mRNPs)1 (6, 7). These proteins also may contribute to global control of protein synthesis.
mRNPs isolated from the cytoplasm of different cells and tissues contain two major proteins with molecular masses of 70 and 50 kDa (8). The 50-kDa protein (p50) is the most abundant and the most strongly bound protein within free nontranslatable mRNPs (9) and therefore can be regarded as the core mRNP protein of these particles in mammalian somatic cells. It is also present in mRNPs derived from polysomes, but in lower amounts (9, 10). Regardless of the fact that p50 displayed little or no specificity for RNA in in vitro binding experiments (10, 11), in cell extracts, p50 was found only in association with mRNA (12). This protein is located mostly in the cytoplasm, and its amount is ~0.1% of the total protein, which corresponds to ~5-10 molecules of p50/molecule of mRNA (12). Among all mRNP proteins, p50 is the most basic, with a pI of ~9.5 (10). In the absence of RNA, p50 forms large multimeric complexes with a molecular mass of ~800 kDa (11). When binding to mRNA, p50 melts up to 60% of the RNA secondary structure (11). We have shown earlier that p50 is responsible for the repressed nonactive state of globin mRNA within free mRNPs (13) and that it strongly inhibits translation of exogenous mRNA in cell-free translation systems (9, 14) as well as in vivo translation of mRNA expressed from a reporter gene (12).
According to its amino acid sequence and its affinity for DNA, p50 was identified as a member of the most evolutionarily conserved family of Y-box-binding proteins from bacteria to man (11). Some proteins of this family are known as transcription factors affecting transcription of genes containing Y-box sequence elements in their promoters (15-17). The prokaryotic Y-box-binding protein is also known as a major cold shock protein stimulating gene expression under cold shock conditions (18). Two homologous core mRNP proteins (p54/p56) from Xenopus oocytes have also identified as members of the Y-box-binding protein family and are closely related, if not identical, to FRG Y2, the germ cell-specific form of the transcription factor (19). They were reported to be responsible for the masked state of mRNAs in Xenopus oocytes (20-24). One can therefore conclude that the somatic mRNP protein p50 and the germ cell mRNP proteins p54/p56 have a similar function in the cytoplasm, namely that of preventing mRNA translation. Thus, the Y-box-binding proteins can influence protein synthesis both by affecting transcription of Y-box-containing genes and by inhibiting translation of a wide variety of mRNAs. By interacting with DNA in the nucleus and with mRNA in the cytoplasm, these proteins may establish a balance between gene activity and mRNA content in cells.
In a detailed study of the effect of p50 on exogenous mRNA translation in reticulocyte lysates, we showed that p50 inhibits mRNA translation when added at a high p50/mRNA ratio. However, with high inhibitory amounts of purified mRNA, addition of low amounts of p50 increases the efficiency of mRNA translation (9). This intriguing result suggests that p50 may also play a positive role in translation. Here we ask if depletion of p50 affects the rate of protein synthesis in reticulocyte lysates. Reduced p50 activity was accomplished by adding p50-specific antibodies. In this instance, translation is inhibited at the level of initiation, thereby providing evidence for a positive role of p50 in promoting protein synthesis.
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EXPERIMENTAL PROCEDURES |
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Preparation of p50--
p50 from free mRNPs of rabbit
reticulocytes was obtained as described earlier (11), except that the
step of protein precipitation with
(NH4)2SO4 was omitted. Recombinant
p50 was expressed in Escherichia coli BL21(DE3) cells, and
the protein was purified by chromatography on heparin-Sepharose 4B and
Superose 12 HR 10/30 columns (Pharmacia Biotechnology, Inc.) as
described (25). The p50 preparations were dialyzed against buffer
containing 10 mM Hepes-KOH, pH 7.8, and 250 mM
KCl and stored at 70 °C in aliquots. Protein concentration was
determined by staining with a Micro-BCA kit (Pierce).
Generation of Polyclonal Antibodies against p50 and Western Blot Analysis-- Polyclonal antibodies were produced either in mice against p50 from rabbit reticulocytes (11) or in rabbits against recombinant p50 synthesized in E. coli (12). Anti-p50 IgG and preimmune IgG were purified on a protein A-Sepharose column (Sigma) (26), dialyzed, and stored in buffer containing 10 mM Hepes-KOH, pH 7.6, and 100 mM KOAc; therefore, the same buffer was added to parallel control incubations throughout the experiments described. For Western blot analysis, proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked overnight at 20 °C with 1% bovine serum albumin, 1% polyvinylpyrrolidone, and 0.05% Tween 20 in 10 mM Tris-HCl, pH 7.6, and 150 mM NaCl and probed with anti-p50 antibodies at 1:500 dilution. Immunocomplexes were detected with alkaline phosphatase-coupled secondary antibodies (Cappel) according to the manufacturer's recommendation.
RNA Isolation--
Globin mRNA was obtained from polysomes
of rabbit reticulocytes by phenol/chloroform extraction followed by
oligo(dT)-cellulose chromatography as described (27). E. coli -galactosidase mRNA was obtained by in
vitro transcription with SP6 polymerase from pJCS-
-galactosidase linearized with HindIII according to
standard procedures (28).
In Vitro Translation Assays-- Translation reactions were performed in rabbit reticulocyte lysates prepared as described (29). Mixtures (30 µl) contained 15 µl of reticulocyte lysate, 10 mM Hepes-KOH, pH 7.6, 100 mM KOAc, 1 mM Mg(OAc)2, 8 mM creatine phosphate, 0.5 mM spermidine, 0.2 mM GTP, 0.8 mM ATP, 1 mM dithiothreitol, 25 µM each amino acid except for the labeled ones, and 6 µCi of [14C]Leu (>300 mCi/mmol; Radioisotop, Obninsk, Russian Federation) or 1 µCi of [35S]Met (>700 Ci/mmol; Radioisotop). When micrococcal nuclease-treated lysates were used, the mixture was supplemented with mRNA as indicated in the figure legends. Translations were carried out at 30 °C for 60 min unless stated otherwise; 5-µl aliquots were treated with deacylation solution (0.3 M NaOH and 150 mM H2O2) and measured for trichloroacetic acid-precipitable radioactivity. When [35S]Met was used, the samples were also subjected to 10-22% SDS-PAGE and autoradiographed.
For ribosome transit time measurements, the volume of the translation mixtures was increased to 150 µl, and translations were carried out as described above. At the indicated intervals, 25-µl aliquots were diluted with 125 µl of ice-cold stop buffer containing 10 mM Tris-HCl, pH 7.6, 100 mM KCl, 5 mM MgCl2, and 0.1 mM cycloheximide (Sigma) and placed immediately on ice. The mixtures were layered onto a 30% glycerol cushion prepared with 10 mM Tris-HCl, pH 7.6, 100 mM KCl, and 5 mM MgCl2 and centrifuged at 100,000 rpm in a TLA-100.3 rotor (Beckman Instruments) for 40 min. This procedure deposits polyribosomes and monomeric ribosomes in the pellet. The ribosomal pellet was dissolved in 1% SDS, and this fraction and the supernatant were assayed for radioactivity as described above.Depletion of p50 Activity in Cell Lysates-- For neutralization of p50 activity, reticulocyte lysates (15 µl) were preincubated with the indicated amounts of antibodies for 10 min at 0 °C. For immunodepletion experiments, nuclease-treated reticulocyte lysates (200 µl) were preincubated with various amounts of antibodies for 10 min at 0 °C with gentle agitation and passed through a 200-µl column of protein A-Sepharose equilibrated with 10 mM Hepes-KOH, pH 7.6, 100 mM KOAc, and 1 mM MgCl2. The first 250 µl of flow-through material from the column was collected and frozen in liquid nitrogen in 15-µl aliquots. Neutralized or immunodepleted reticulocyte lysates were reconstituted by adding the indicated amounts of p50 directly to the lysate and incubating for 3 min at 0 °C. The neutralized, depleted, and reconstituted lysates were then assayed for protein synthesis activity as described above.
Sucrose Gradient Analyses and Dot Blot Assays--
Cell-free
translation mixtures (30 µl) were cooled and immediately layered onto
15-30% (w/v) linear sucrose gradients in buffer containing 10 mM Tris-HCl, pH 7.6, 100 mM NaCl, and 1 mM MgCl2. Centrifugation was carried out at
45,000 rpm in an SW 60 rotor (Beckman Instruments) for the times
indicated in the figure legends. All gradients were monitored for
absorbance at 254 nm during their collection from the bottom. Fractions
of 300 µl were diluted with 700 µl of ice-cold water and treated
with deacylation solution, and [14C]Leu incorporation
into trichloroacetic acid-precipitable material was measured as
described above. For dot hybridization analysis of globin mRNA,
total RNA was extracted from the fractions by an equal volume of
phenol/chloroform, filtered onto a nitrocellulose Hybond-N membrane
(Amersham Corp.), and probed with a full-length -globin cDNA
32P-labeled by the multiprime DNA labeling system (Amersham
Corp.). For quantitation of hybridized RNA, each dot was excised, and radioactivity was measured by Cherenkov counting in a Beckman LS-100C scintillation counter.
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RESULTS |
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Cell-free Translation Systems Deficient in p50 Do Not Synthesize Proteins Efficiently-- To verify the suggestion that p50 may play a positive role in protein biosynthesis, we sought to deplete p50 activity in reticulocyte lysates. For selective reduction of p50 activity in the rabbit reticulocyte cell-free translation system, two different polyclonal anti-p50 antibody preparations were used. The antibodies were raised either in mouse against p50 from free rabbit reticulocyte mRNPs or in rabbit against recombinant p50 expressed in E. coli. Both p50 preparations were purified to homogeneity; no other peptide bands were detected by Coomassie Blue R-250 staining of these proteins resolved by SDS-PAGE (Fig. 1A). The two anti-p50 antibodies were purified on protein A-Sepharose, and their specificities were tested by immunoblotting. The antibodies reacted strongly with purified p50 and recognized only a single antigen in reticulocyte lysates corresponding in electrophoretic mobility to p50 (Fig. 1B). Therefore, the antibodies are monospecific and may be suitable for neutralization of p50 activity due to their specific binding to p50 in the rabbit reticulocyte cell-free system and for p50 immunodepletion by affinity adsorption from the lysate. Both antibody preparations gave identical results, so further reported experimental data are on antibodies against rabbit reticulocyte p50, unless indicated otherwise.
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Inhibition of Protein Synthesis with Anti-p50 IgG Is Not Caused by Acceleration of mRNA Degradation-- Antibody binding to p50 may stimulate mRNA decay in the cell-free translation system, thereby causing translation inhibition. To verify this idea, RNAs were isolated from lysates incubated under cell-free translation conditions with and without anti-p50 IgG and tested for messenger activity. Both mRNA preparations possessed equal messenger activity in the cell-free translation system and produced full-length globin chains as determined by SDS-PAGE (Fig. 4). The messenger activity of these RNAs was the same as that of RNA from a nonincubated lysate (data not shown). Thus, anti-p50 IgG-induced protein synthesis inhibition is not caused by mRNA degradation.
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Specific Anti-p50 Antibodies Inhibit Initiation but Not Elongation/Termination of Globin Synthesis in Reticulocyte Lysates-- Fig. 5 shows the effect of anti-p50 IgG on the kinetics of protein synthesis in rabbit reticulocyte lysates with endogenous (panel A) and exogenous (panel B) globin mRNAs. With either type of mRNA, anti-p50 IgG inhibited strongly at high concentrations, whereas preimmune IgG had little or no effect. The inhibition of endogenous mRNA translation with a high anti-p50 IgG concentration resembled the inhibition caused by edeine, a specific inhibitor of the initiation phase of protein synthesis (30, 31). Thus, the shape of the curves suggests that the antibodies inhibit mainly translation initiation.
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Specific Anti-p50 Antibodies Inhibit Initiation but Not Elongation
of -Galactosidase Synthesis in Reticulocyte Lysates--
p50 is a
universal mRNP protein associated with all or almost all mRNAs of
mammalian somatic cells (11). The question arises as to whether p50
promotes only initiation of eukaryotic mRNAs having an
m7G cap, a characteristic consensus sequence around the
initiator AUG codon and 3
-poly(A) tail, or whether it would exert the
same positive effect on translation of a bacterial mRNA lacking
these features. To answer this question, an uncapped mRNA
transcript encoding E. coli
-galactosidase was translated
in the reticulocyte cell-free system without antibodies or with
anti-p50 IgG added either at zero time or after 10 min of incubation
(when only elongation occurs). The kinetics of
-galactosidase
synthesis was studied by SDS-PAGE analysis of the product obtained
after different incubation times. Synthesis of full-length
-galactosidase was completed in 20-40 min in the system without
antibodies (Fig. 8A) (33). When anti-p50 IgG was added before incubation, no synthesis of full-length or even partial-length protein was observed (Fig. 8B), consistent with inhibition at initiation. With anti-p50
IgG added after 10 min of incubation,
-galactosidase synthesis
occurred and yielded full-length product after 40 min of incubation
(Fig. 8C). The pattern of partial proteins seen does not
differ detectably from that obtained with the control lysate (Fig.
8A). These results show that p50 is strongly required for
initiation of bacterial mRNA translation, and it does not affect
elongation even at antibody concentrations providing the complete
inhibition of initiation. This is in good agreement with our previous
findings on eukaryotic globin mRNA (Figs. 5-7).
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Specific Anti-p50 Antibodies Cause Accumulation of 48 S
Preinitiation Complexes--
To identify the step in the initiation
pathway that is inhibited by anti-p50 IgG, the cell-free translation
systems were incubated without antibodies, with anti-p50 IgG, or with
edeine and then fractionated by centrifugation in sucrose gradients.
The distribution of -globin mRNA in the gradient was determined
by dot hybridization with 32P-labeled
-globin cDNA.
In the control cell-free translation system, mRNA was found mainly
on 80 S ribosomes and to a lower extent in the region of free globin
mRNPs (~20 S) (Fig. 9A).
After incubation in the presence of edeine blocking the joining of the 60 S subunit (30, 31), most of the
-globin mRNA was found in the
region where the small 40 S ribosomal subunit sediments and can be
regarded as the 48 S preinitiation complexes (40 S·Met-tRNAi·eukaryotic initiation factor-2·GTP·
mRNA) (Fig. 9C). In the system incubated in the presence
of anti-p50 IgG, the bulk of
-globin mRNA was also found in 48 S
preinitiation complexes (Fig. 9B). Thus, anti-p50 IgG does
not inhibit binding of 40 S ribosomes to mRNA, but rather inhibits
a subsequent step in the initiation pathway.
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DISCUSSION |
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The results reported here indicate that p50, the major protein of cytoplasmic mRNPs, not only functions as a repressor of mRNA translation, but also is required for protein biosynthesis. Two independent preparations of monospecific anti-p50 antibodies raised against either rabbit or recombinant p50 were used to reduce the activity of endogenous p50 in rabbit reticulocyte lysates or to prepare depleted lysates. Immunoneutralization or immunodepletion of p50 by both antibody preparations caused inhibition of protein synthesis. Addition of highly purified rabbit or recombinant p50 proteins to such systems restored the translational activity completely or nearly completely, which confirms the positive role of p50 in mRNA translation.
How can p50 function both to promote and to repress translation? The in vitro assays for p50 activity suggest that the level of p50 determines whether it stimulates or inhibits translation. With the p50/mRNA weight ratio increasing up to 2 (which is characteristic of polysomal mRNPs), p50 stimulates protein biosynthesis, whereas a further increase of the ratio up to 5-6 (close to the ratios characteristic of free mRNPs) causes a gradual inhibition of translation until it ceases completely (9). Thus, the inactive-to-active transition of mRNA may be connected with a decrease in the amount of p50 attached to an mRNA molecule.
The amount of p50 on mRNA can possibly be regulated by phosphorylation. It is known that p50 is a phosphoprotein (10, 34) and may be phosphorylated by a protein kinase present in mRNPs that resembles casein kinase II.2 Furthermore, the Xenopus proteins p54/p56 are phosphoproteins, and their binding to RNA is enhanced upon phosphorylation by casein kinase II (35). Phosphorylation of p54/p56 results in inhibition of protein synthesis, whereas inhibitors of casein kinase II activate translation (24, 35). Work is in progress to elucidate how phosphorylation of p50 may affect its binding to RNA and its activity in stimulating and inhibiting translation in vitro.
Immunoneutralization of p50 does not change the ribosome transit time
for globin synthesis and does not affect elongation of prokaryotic
-galactosidase. This means that p50 is not required for elongation
and termination, and consequently, it is required only for initiation.
The effect of anti-p50 IgG on the kinetics of protein synthesis in the
cell-free translation system and on the decay of polysomes also points
to inhibition of translation initiation.
Analysis of the distribution of globin mRNA in reticulocyte lysates
inhibited by anti-p50 IgG shows that mRNA accumulates in 48 S
preinitiation complexes. This means that p50 is not required for
attachment of the small subunit of the ribosome to mRNA, although it is necessary for subsequent binding of the 60 S ribosomal subunit to
the complex. We suggest that either p50 directly participates in
attachment of the 60 S ribosomal subunit to the 48 S preinitiation complex or that it is involved in the previous step of 5-untranslated region mRNA scanning by the 43 S preinitiation complex.
Several mechanisms of p50 participation in translation initiation can
be proposed. (i) Nonspecific affinity of p50 for RNA and the presence
of many copies of p50 on mRNA can protect mRNA against
nonspecific binding of initiation factors along its entire length, thus
contributing to their specific binding to the 5-untranslated region
(9, 36). (ii) Since p50 possesses RNA-unwinding activity (11), its
direct participation in 5
-untranslated region scanning is quite
possible. (iii) p50 provides the general mRNA structure favorable
for translation initiation. (iv) Finally, we cannot rule out that p50
affects translation initiation by its direct interaction with
translation initiation factors. These suggestions are being verified
currently.
p50 is not the only mRNP protein implicated in the initiation phase of protein synthesis. Another major mRNP protein, p70 (or poly(A)-binding protein), appears to be involved in protein synthesis initiation, too (37-40). Whether poly(A)-binding protein and p50 interact directly on mRNAs has not yet been determined.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Denisenko for valuable
advice and for the -galactosidase gene and to Dr. Ilan for the
-globin gene.
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FOOTNOTES |
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* This work was supported by Grants N 93-04-06548 and N 96-04-48903 from the Russian Basic Research Foundation, Grant MCB-91-23549 from the National Science Foundation, Grant N MUCOOO from the International Science Foundation, and Grant RBI-282 from the United States Civilian Research and Development Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel./Fax: 95-924-0493; E-mail: ovchinn{at}sun.ipr.serpukhov.su.
1 The abbreviations used are: mRNPs, messenger ribonucleoprotein particles; PAGE, polyacrylamide gel electrophoresis; FRG Y2, frog Y-box transcription factor 2.
2 V. A. Ustinov, M. A. Skabkin, V. M. Evdokimova, J. W. B. Hershey, and L. P. Ovchinnikov, manuscript in preparation.
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REFERENCES |
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