The mRNA-binding Protein YB-1 (p50) Prevents Association of the Eukaryotic Initiation Factor eIF4G with mRNA and Inhibits Protein Synthesis at the Initiation Stage*

Maxim P. NekrasovDagger , Maria P. IvshinaDagger , Konstantin G. ChernovDagger , Elizaveta A. KovriginaDagger , Valentina M. EvdokimovaDagger , Adri A. M. Thomas§, John W. B. Hershey, and Lev P. OvchinnikovDagger ||

From the Dagger  Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russian Federation, the § Department of Developmental Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands, and the  Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616

Received for publication, September 6, 2002, and in revised form, January 23, 2003

    ABSTRACT
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The cytoplasmic messenger ribonucleoprotein particles of mammalian somatic cells contain the protein YB-1, also called p50, as a major core component. YB-1 is multifunctional and involved in regulation of mRNA transcription and translation. Our previous studies demonstrated that YB-1 stimulates initiation of translation in vitro at a low YB-1/mRNA ratio, whereas an increase of YB-1 bound to mRNA resulted in inhibition of protein synthesis in vitro and in vivo. Here we show that YB-1-mediated translation inhibition in a rabbit reticulocyte cell-free system is followed by a decay of polysomes, which is not a result of mRNA degradation or its functional inactivation. The inhibition does not change the ribosome transit time, and therefore, it affects neither elongation nor termination of polypeptide chains and only occurs at the stage of initiation. YB-1 induces accumulation of mRNA in the form of free messenger ribonucleoprotein particles, i.e. it blocks mRNA association with the small ribosomal subunit. The accumulation is accompanied by eukaryotic initiation factor eIF4G dissociation from mRNA. The C-terminal domain of YB-1 is responsible for inhibition of translation as well as the disruption of mRNA interaction with eIF4G.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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All mRNAs in eukaryotic cells are associated with proteins and form messenger ribonucleoprotein particles (mRNPs)1 (1-6). Some mRNA-associated proteins exhibit specificity for certain mRNA(s); others are universal. To date, two universal major proteins of cytoplasmic mRNPs tightly bound to mRNA are well characterized. One of them is poly(A)-binding protein that stabilizes mRNA (7, 8) and promotes protein synthesis at the initiation stage (9, 10). It is suggested that the protein biosynthesis promotion occurs due to binding of multimerized poly(A)-binding protein associated with poly(A) to eIF4G and 1) mRNA cyclization and facilitation of ribosomal recycling (11, 12) and/or 2) a conformational change in eIF4F and an increase in affinity of eIF4E for the 5'-cap (13).

The other common mRNP component is YB-1, a 36-kDa protein with abnormal mobility in SDS gel electrophoresis that is typical for this 50-kDa protein (14-16). According to its primary structure, YB-1 from rabbit reticulocytes was identified as a member of the Y-box (YB) transcription factor family: it was virtually identical (~98% identity) to human YB-1. YB-1 has been shown to participate in different steps of mRNA biogenesis, including mRNA transcription, processing, and transport from the nucleus into the cytoplasm (5, 17, 18) where it can regulate mRNA localization, translation, and mRNA stability (15, 16, 19). YB-1 displays DNA and RNA melting, annealing, and strand exchange activities, which probably underlies many functions of the YB protein family (20, 21).

The characteristic feature of these proteins is a central, highly conserved, cold shock domain (CSD) that consists of 80 amino acid residues and exhibits 43% identity to the major Escherichia coli cold shock protein CspA (4, 5). CSD comprises a five-stranded beta -barrel with RNP 1 and RNP 2 consensus motifs and provides some sequence specificity in nucleic acid binding (22, 23). YB proteins also contain an N-terminal alanine/proline (AP)-rich domain and a large C-terminal segment with four alternating clusters of basic and acidic amino acid residues. These domains are supposedly involved in nucleic acid- and protein-protein interactions (15, 24).

Active polysomes contain both major proteins, YB-1 and poly(A)-binding protein, whereas polysome-free inactive mRNPs contain predominantly YB-1 (25-29). It was shown that at a low YB-1/mRNA ratio YB-1 promoted translation at the stage of initiation (30), whereas an increase of this ratio strongly inhibited mRNA translation in vitro (16, 29, 31, 32) and in vivo (33). In initiation reactions reconstituted in vitro from purified translation components, YB-1 stimulated or inhibited formation of 48 S preinitiation complex depending on its amount bound to mRNA (34). It was also shown that inhibitory concentrations of YB-1 suppressed the interaction of eIF4E, eIF4A, and eIF4B with the mRNA cap structure (16). Experiments on the effect of separate YB-1 domains on mRNA translation and on interaction of the above initiation factors with mRNA cap structure gave a surprising result: AP-CSD as well as CSD alone displaced eIF4E, eIF4A, and eIF4B from the mRNA cap structure and increased the stability of mRNA but produced nearly no effect on its translational activity. In contrast, the C-terminal domain of YB-1 strongly inhibited protein synthesis without any effect on interactions of eIF4E, eIF4A, and eIF4B with the mRNA cap structure (16).

Although all these results suggest that YB-1 inhibits protein synthesis at the initiation stage, its effect on elongation and termination of protein synthesis cannot be ruled out. The exact initiation step inhibited by YB-1 cannot be specified.

In this study we demonstrate that YB-1 inhibits translation in a cell-free translation system only at the initiation stage, whereas elongation and termination of protein synthesis remain unaffected. YB-1 causes accumulation of intact, translationally competent mRNA in the form of free mRNPs. The full-length YB-1 as well as its inhibitory C-terminal domain displaces eIF4G from the mRNA, thereby causing an arrest of translation.

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Proteins and Antibodies-- Recombinant proteins were expressed in E. coli BL21(DE3). YB-1 and its truncated fragments were purified as described previously (16). All purified proteins were dialyzed against 150 mM KCl, 10 mM Hepes-KOH, pH 7.6, and stored at -70 °C in aliquots. Protein concentrations were determined by staining using a Micro BCA kit (Pierce).

Rabbit reticulocyte eIF4F was a gift from Dr. I. Shatsky (Moscow State University, Moscow, Russia). Rabbit anti-eIF4G antibodies were kindly provided by Dr. R. Rhoads (Louisiana State University, Department of Biochemistry and Molecular Biology).

RNA Preparations-- Globin mRNA was obtained from polysomes of rabbit reticulocytes as described previously (35). The ribosomal pellet was dissolved in 10 mM Tris-HCl, pH 9.0, 100 mM NaCl, 1% SDS with subsequent phenol/chloroform extraction followed by oligo(dT)-cellulose chromatography. 9 S globin mRNA was isolated by sucrose gradient centrifugation.

In Vitro Translation Assays-- The cell-free translation system derived from rabbit reticulocytes was described previously (30). Mixtures (15 µl) contained 7.5 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, a 25 µM concentration of each amino acid (except for the labeled ones), and 0.6 µCi of [14C]Leu (>300 mCi/mmol; Radioisotop, Obninsk, Russia) 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.

For ribosome transit time measurements, the volume of the translation mixtures was increased up to 150 µl, and 25-µl aliquots were taken at the indicated times, diluted with 125 µl of ice-cold stop buffer containing 10 mM Tris-HCl, pH 7.6, 100 mM KCl, 5 mM MgCl2, 0.1 mM cycloheximide (Sigma), and immediately placed on ice. The mixtures were layered onto a 30% glycerol cushion with 10 mM Tris-HCl, pH 7.6, 100 mM KCl, 5 mM MgCl2 and centrifuged at 100,000 rpm in a TLA-100.3 rotor (Beckman) for 40 min. The supernatant fraction and ribosomal pellet dissolved in 1% SDS were assayed for trichloroacetic acid-precipitable radioactivity.

mRNA-eIF4F Complex Assembly-- The globin mRNA-eIF4F complex was assembled as described previously (36) by incubating mRNA (23 pmol) with eIF4F (4 pmol) for 15 min at 30 °C in buffer containing 10 mM Hepes, pH 7.6, 2.5 mM Mg(OAc)2, 100 mM KOAc, 1 mM ATP, 2 mM dithiothreitol, 250 µM spermidine, and 25 units of RNasin (Promega). YB-1 was added to the incubation mixture as indicated in the figure legends. The samples were ice-cooled and analyzed by sedimentation in sucrose gradients.

Sucrose Gradient Analysis and Blot Assays-- Cell-free translation mixtures (60 µl) were layered onto 15-30% or 15-40% (w/v) linear sucrose gradients made in 10 mM Tris-HCl, pH 7.6, 100 mM KCl, 1 mM MgCl2. Centrifugation was carried out at 45,000 rpm in an SW-60 rotor (Beckman) for the times indicated. The mRNA-eIF4F complexes were resolved by centrifugation through 5-20% (w/v) linear sucrose gradients in 10 mM Hepes, pH 7.5, 1 mM Mg(OAc)2, 100 mM KOAc at 45,000 rpm for 5.5 h in a SW-60 rotor. All gradients were monitored for absorbance at 254 nm during their collection from the bottom. Fractions of 260 µl were diluted with 700 µl of ice-cold water and measured for [14C]Leu incorporation into protein.

For Northern hybridization, sucrose gradient fractions were supplemented with 1% SDS and deproteinized with an equal volume of phenol/chloroform. Total RNA was either filtered directly onto nitrocellulose membrane Hybond-N (Amersham Biosciences) or first separated by a 1.2% formaldehyde-agarose gel electrophoresis and then transferred onto the membrane. The membranes were hybridized with randomly primed 32P-labeled alpha -globin cDNA. For quantitation of hybridized RNA, each dot was excised, and radioactivity was measured by Cherenkov radiation in a Beckman LS-100C scintillation counter.

For Western blot analysis of the eIF4F and YB-1 distribution, proteins were precipitated from sucrose gradient fractions by trichloroacetic acid (5% final concentration), resolved by SDS-15% PAGE, and transferred onto a nitrocellulose membrane (Whatman). The membranes were blocked with 1% bovine serum albumin, 1% polyvinylpyrrolidone, 0.05% Tween 20 in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl and probed with anti-eIF4G (1:500) or anti-YB-1 (1:5000 dilution) polyclonal antibodies. Immunocomplexes were detected using alkaline phosphatase-coupled secondary antibodies (Promega) according to the manufacturer's recommendation.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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An Increase of the YB-1 Amount in Cell-free Translation Assays Causes Inhibition of Protein Synthesis-- The effect of the recombinant YB-1 on translation of endogenous and exogenous globin mRNA in the rabbit reticulocyte cell-free system was assayed. In both cases, increasing amounts of YB-1 caused the complete inhibition of protein synthesis (Fig. 1) that was observed after addition of 3 µg (80 pmol) of YB-1. In the cell-free system with 0.3 µg (1.4 pmol) of exogenous mRNA this provides a YB-1/mRNA mass ratio of about 10 and a molar ratio of about 60. This ratio far exceeds that in free mRNPs (mass ratio of about 3), and the amount of added exogenous YB-1 is ~6 times higher than the amount of endogenous YB-1 in reticulocyte lysates. To inhibit a system with more exogenous mRNA, a larger amount of YB-1 is required (29). A comparatively large amount of YB-1, required to perform the complete inhibition of translation, can be explained by titrating out part of YB-1 as a result of its nonspecific interaction with tRNA (32) and with ribosomal subunits (see Fig. 5). FRGY2, the Xenopus embryonic homolog of YB-1, was reported to be detected not only in mRNPs but also in 15 S complexes with nucleolin and with some other unidentified polypeptides (37). The amount of YB-1 that we used to inhibit translation in the cell-free system is likely to be present in the cell under some conditions. For example, the amount of Y-box proteins increases significantly in cancer cells (38, 39), and their mRNAs increase manifold during liver regeneration and cell growth stimulation with serum or interleukins (40, 41).


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Fig. 1.   Exogenous YB-1 inhibits mRNA translation in cell-free systems from rabbit reticulocytes. Rabbit reticulocyte lysates (7.5 µl/15 µl of translation mixture) with endogenous () and 1.4 pmol of exogenous (black-triangle) rabbit reticulocyte globin mRNA were translated with increasing amounts of YB-1. Translation reactions were carried out at 30 °C for 60 min and assayed for [14C]Leu incorporation as described under "Experimental Procedures."

Inhibition of Protein Synthesis with Exogenous YB-1 Is Not a Result of mRNA Decay or Functional Inactivation-- To monitor the biological and physical state of mRNA in a cell-free translation system inhibited by YB-1, RNA was isolated from lysates incubated under cell-free translation conditions in the presence of YB-1. As shown in Fig. 2A, the amount and size of alpha -globin mRNA (determined by Northern hybridization with 32P-labeled alpha -globin cDNA) did not change as compared with control. RNA preserved its messenger activity in the cell-free translation system (Fig. 2B) and produced full-length globin chains (Fig. 2C). Thus, the YB-1-induced inhibition of protein synthesis is not caused by mRNA decay.


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Fig. 2.   YB-1 does not stimulate decay or functional inactivation of mRNA in the cell-free translation system. Translation reactions were carried out with endogenous mRNA in 30 µl of translation mixture and with an inhibiting amount (170 pmol) of YB-1 at 30 °C for 60 min. After incubation, total RNA was isolated by phenol/chloroform extraction and retranslated in fresh aliquots of nuclease-treated reticulocyte lysates. A, Northern blot hybridization with 32P-labeled alpha -globin cDNA of total RNA isolated from original lysate (lane 1) and lysate incubated under translational conditions without (lane 2) or with (lane 3) exogenous YB-1. B, [14C]Leu incorporation into trichloroacetic acid-precipitable material in a cell-free system. C, autoradiogram of [35S]Met-labeled translation products resolved by SDS-PAGE from a lysate with no RNA added (lane 1), lysates with endogenous RNA incubated in the absence (lane 2) and presence (lane 3) of exogenous YB-1, and nuclease-treated lysates with total RNA from untreated (lane 4) and YB-1-treated (lane 5) lysates.

Exogenous YB-1 Inhibits Translation at the Initiation Stage-- The phase of translation affected by an inhibitor can be determined by analyzing the polysome profile and measuring the elongation rate. The run-off of ribosomes from polysomes usually indicates inhibition of initiation, whereas maintenance of or an increase in polysome size suggests inhibition of elongation/termination. We have compared the polysome profiles of reticulocyte lysates incubated in the absence and presence of exogenous YB-1. Incubation of the cell-free system without YB-1 for 5 min did not produce any remarkable change in the polysome profiles (Fig. 3, compare A and B). However, incubation with YB-1 resulted in a complete polysomal decay (Fig. 3, compare A and B with C). The process was accompanied by dissociation of the radiolabeled polypeptide chain from the ribosomes, which once again indicates that decay of the major part of polysomes is not a result of mRNA fragmentation by ribonucleases since such cleavage of polysomes produces ribosomes associated with the growing polypeptide. Rather the run-off of ribosomes from polysomes implies that exogenous YB-1 mainly inhibited initiation.


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Fig. 3.   Exogenous YB-1 stimulates polysome decay in rabbit reticulocyte cell-free systems. A, the polysomal profile of the original lysate without any treatment or incubation (control). Cell-free systems with endogenous mRNA were incubated for 5 min at 30 °C without (B) and with 340 pmol of exogenous YB-1/60 µl of translation mixture (C). Lysates were subjected to centrifugation through 15-40% linear sucrose gradients for 55 min at 45,000 rpm in an SW-60 rotor. UV absorption profiles at 254 nm (---) and [14C]Leu incorporation into protein () are shown.

The average time of elongation + termination of polypeptide chains (transit time) can be quantitatively determined by measuring the kinetics of radioactive amino acid incorporation into total protein and into completed polypeptides released from the ribosome (42). We used this technique to determine the effect of YB-1-induced inhibition on the elongation + termination rate. The cell-free translation system was inhibited up to ~50% since a stronger inhibition would not produce reasonable (measurable) incorporation data. From the results shown in Fig. 4, the transit time for globin synthesis in the control (uninhibited lysate) is 1.3 min. In the experimental system with exogenous YB-1, the same transit time was obtained. Thus, YB-1 did not affect the polypeptide elongation or termination rate and only inhibited translation initiation.


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Fig. 4.   YB-1 does not affect the ribosome transit time. Reticulocyte lysates with endogenous mRNA were translated for the indicated times without (A) and with YB-1 (480 pmol/150 µl of translation mixture) (B). Transit times were obtained by multiplying by 2 the distance along the time axis between the "total" and "postribosomal" lines.

Translation Inhibition by YB-1 Causes Accumulation of mRNA in the Form of Free mRNPs-- To identify the YB-1-inhibited step in the initiation pathway, the cell-free translation systems with endogenous mRNA were incubated for 5 min without YB-1 or with the inhibiting amount of YB-1 (340 pmol of YB-1/60 µl of system), fractionated by centrifugation in sucrose gradients, and analyzed for alpha -globin mRNA distribution by Northern hybridization with 32P-labeled alpha -globin cDNA. Without YB-1, mRNA was found mainly in the region with a sedimentation coefficient exceeding 80 S, except for a small 20 S peak corresponding to free globin mRNPs (Fig. 5C). After incubation in the presence of YB-1, the major part of alpha -globin mRNA was found in the region of free globin mRNP sedimentation (~20 S) (Fig. 5D). This is similar to alpha -globin mRNA distribution after translation inhibition by the cap analogue m7GpppG (Fig. 5F), whereas after inhibition with edeine the major part of this mRNA was, as expected (43), a component of the 48 S preinitiation complexes (Fig. 5E).


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Fig. 5.   YB-1 and its C-terminal fragment displace eIF4G from mRNA in rabbit reticulocyte lysate. Reticulocyte lysates with endogenous mRNA were incubated under translational conditions for 5 min at 30 °C without any additions (A and C), with 80 pmol of YB-1 added (B and D), with edeine at a final concentration of 5 µM (E), with 2 mM m7GpppG (F), with 80 pmol of AP-CSD (G), or with 80 pmol of C-terminal (C-ter.) fragment (H). Samples were ice-cooled and subjected to 15-40% (A and B) or 15-30% (C-H) linear sucrose gradient centrifugation for 55 min (A and B) or 2 h and 45 min (C-H) at 45,000 rpm in an SW-60 rotor. UV absorbance profiles at 254 nm (---) and 32P radioactivity () are shown. The distribution of eIF4G and YB-1, as determined by Western blot, is shown in upper insets. For YB-1 bands in B and D, the reaction time was reduced to avoid overexposure in the presence of 6-fold exogenous YB-1.

Western blot analysis of YB-1 distribution showed that in the original lysate endogenous YB-1 was widely distributed both in the polysomal and postribosomal sedimentation regions (Fig. 5, A and C), which was consistent with our previous results (31). After incubation with edeine (Fig. 5E), cap analog (Fig. 5F), or exogenous YB-1 (Fig. 5, B and D) nearly all YB-1 sedimented in the postribosomal region with bimodal distribution: partly at 20 S (the region of free mRNPs with globin mRNA) and partly at 60-40 S. The absence of mRNA from the 60-40 S region when the translation is inhibited by the cap analog or YB-1 indicates that in this region YB-1 turns to be associated with ribosomal subunits. There was not a notable difference between YB-1 sedimentation distributions for the YB-1 and cap analog cases (Fig. 5, compare D and F). Interestingly in these two cases the bulk of ribosome dissociated into subunits, which in the edeine case was less pronounced.

The mRNA accumulation in the form of free mRNPs in the YB-1-inhibited cell-free system indicated that the protein synthesis inhibition occurred at the stage of mRNA association with the small ribosomal subunit. Since this association was preceded by the small subunit to Met-tRNAi binding, and this subunit, in its turn, interacted with YB-1, the latter interaction might suppress binding of Met-tRNAi to 40 S subunits and prevent formation of the 43 S preinitiation complex. However, a special experiment revealed that addition of YB-1 to the system did not affect the formation of the 43 S preinitiation complex with [35S]Met-tRNAi, and deacylation of this complex was not observed either (data not shown). This suggests that YB-1 inhibited translation either at the stage of mRNA association with 43 S complex or at a preceding stage of mRNA interaction with translation initiation factors.

YB-1 Inhibits mRNA Interaction with Translation Initiation Factor eIF4G-- Prior to mRNA interaction with the 43 S preinitiation complex, mRNA binds to eIF4F, eIF4A, and eIF4B (44). The first phase of this multistep process is ATP-dependent mRNA binding to eIF4F comprising the eIF4E, eIF4G, and eIF4A subunits. The YB-1 effect on interaction of eIF4F with endogenous mRNA in rabbit reticulocyte lysate was assayed. The mRNA-eIF4F complex formation was followed by sedimentational distribution of eIF4G using Western blot with monospecific antibodies to eIF4G.

In the control without YB-1 addition (Fig. 5A), a considerable portion of eIF4G sedimented in the region of polysomes. In the presence of edeine, the bulk of eIF4G was observed in the region of sedimentation of the 48 S complex, i.e. the small subunit-associated mRNA retained eIF4G in the presence of edeine (Fig. 5E). Inhibitory amounts of YB-1 causing mRNA accumulation as free mRNPs displaced eIF4G from mRNA completely. As a result, sedimentation of eIF4G was slower than that of free globin-containing mRNPs, that is, slower than 20 S (Fig. 5D).

The ability of YB-1 to displace eIF4F from mRNA was confirmed by experiments with isolated components of the translation system, eIF4F, mRNA, and YB-1, that were incubated in the presence of ATP. The results are given in Fig. 6. In the absence of mRNA, eIF4F distributed in the upper zone of the sucrose gradient (see Western blots, Fig. 6B); after addition of mRNA in both cap+ or cap- variants, almost all eIF4G (presumably as a constituent of eIF4F) was bound tightly to mRNA and sedimented somewhat faster than free mRNA (Fig. 6, C and H). Addition of YB-1 to the cap+ mRNA-eIF4F complex with a YB-1/mRNA molar ratio ranging from 6 to 36 resulted in an increase of mRNA sedimentation coefficient from 9 S (without YB-1) to 21 S, respectively; then all YB-1 co-sedimented with mRNA. Addition of YB-1 resulted in a nearly complete (at 12:1) or complete (at 36:1) displacement of eIF4G from cap+ mRNA (Fig. 6, D-F). In the case of uncapped mRNA (Fig. 6, G-J), the effect of YB-1 on eIF4G displacement was stronger: we could observe an almost complete displacement of eIF4G at as low a YB-1/mRNA molar ratio as 12:1 (Fig. 6, compare E, F, and J). These results are in good agreement with the finding that translation of uncapped mRNA is more sensitive to YB-1 inhibition (30). Note that the decay of the eIF4G-mRNA complex results from interactions between YB-1 and mRNA but not between YB-1 and eIF4G because, with eIF4G completely displaced from mRNA, YB-1 co-sediments with mRNA but not with eIF4G (Fig. 6, F and J).


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Fig. 6.   YB-1 disrupts the interaction of globin mRNA with eIF4F. RNA-protein complexes were assembled as described under "Experimental Procedures" and centrifuged in 5-20% sucrose gradients. mRNA distribution was determined using UV absorption profiles at 254 nm, and eIF4F distribution was analyzed on Western blots using anti-eIF4G antibodies (upper insets). A and G, 23 pmol of free globin RNA; B, 4 pmol of free eIF4F; C and H, globin mRNA incubated with eIF4F. Globin mRNA was incubated with eIF4F, and YB-1 (135, 270, and 810 pmol) was subsequently added to attain the YB-1/mRNA molar ratio of 6 (D and I), 12 (E and J), and 36 (F).

The C-terminal Domain of YB-1 Suppresses the eIF4G Interaction with mRNA and Inhibits mRNA Translation-- YB-1 contains three domains: AP, CSD, and the C-terminal domain with basic and acidic residues. The C-terminal domain of YB-1 behaved in a manner similar to full-length YB-1: after incubation of cell-free translation system with the C-terminal fragment of YB-1, the major part of alpha -globin mRNA sedimented as free globin mRNPs, just as after addition of full-length YB-1. eIF4G was absent from this part of the gradient, the majority sedimenting as free eIF4F (Fig. 5H). Addition of the AP-CSD part of YB-1 to the system caused neither mRNA transition into the free mRNP region nor eIF4G displacement into the free factor sedimentation region (Fig. 5, compare G and C).

Fig. 7 illustrates the effect of the used YB-1 fragments on protein synthesis in the cell-free system. AP-CSD, which does not displace eIF4G from mRNA, affected translation only slightly even with large amounts of the protein used, whereas the C-terminal fragment dramatically suppressed translation and even exhibited a higher inhibitory activity than the entire YB-1. This is in agreement with previous results reported for YB-1 fragments (16) and for fragments of the homologous protein FRGY2 from Xenopus oocytes (45).


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Fig. 7.   The C-terminal domain of YB-1 is responsible for the inhibitory activity of YB-1. Rabbit reticulocyte lysate with endogenous mRNA was incubated with buffer alone (control) or with increasing amounts of YB-1, AP-CSD fragment, or the C-terminal (C-term) fragment as indicated and assayed for [14C]Leu incorporation as described under "Experimental Procedures."


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As reported previously and confirmed by the current study, YB-1 inhibits mRNA translation at a high YB-1/mRNA ratio. Here we show that the inhibition is not caused by mRNA degradation or its functional inactivation: there was no difference in size between mRNAs from lysates incubated with or without YB-1, and RNA preparations from these lysates displayed identical translational activities. Translation inhibition by YB-1 was accompanied by a complete decay of polysomes, and hence it occurred mostly or exclusively at the stage of initiation. Since the measured transit time of ribosomes was the same in the experiment with or without YB-1, YB-1 did not affect elongation or termination and inhibited protein synthesis at the stage of initiation only. Elongation was unaffected by YB-1 presumably because the ribosome-covered protein-coding mRNA region is inaccessible to YB-1. Another, still more probable, explanation is that the energy-supplied translating ribosomes can easily displace YB-1 from mRNA.

After polysome decay under the action of YB-1, mRNA sedimented as free mRNPs, i.e. YB-1 interfered with the interaction between mRNA and the 43 S preinitiation complex or with the preceding mRNA interaction with translation initiation factors eIF4F, eIF4A, and eIF4B. With model experiments, assembling a 48 S preinitiation complex from purified components, it was shown that inhibition by YB-1 could be overcome by increasing eIF4F concentration (34). This means that YB-1 blocks the first step in mRNA recruitment into initiation of translation, that is, YB-1 shuts off the interaction of mRNA with eIF4F. In vitro data showed that YB-1 inhibited binding of two eIF4F subunits, eIF4E, and eIF4A as well as the factor eIF4B with the mRNA cap structure (16). Surprisingly CSD and AP-CSD displaced these factors too (16), although protein synthesis was only inhibited slightly (Ref. 16 and Fig. 7 of the current paper) in spite of displacement of eIF4E. This can be explained by the fact that in rabbit reticulocyte lysate translation is relatively independent of the presence of this factor (32). In other words, binding of the above three factors (eIF4E, eIF4A, and eIF4B) to the mRNA cap structure is not so crucial for initiation of translation in this system.

The C-terminal domain of YB-1 is a strong inhibitor of protein synthesis (Ref. 16 and Fig. 7 of the current paper), although the interaction of eIF4A, eIF4B, or eIF4E with the cap structure was unaffected by this YB-1 domain (16). This suggested that YB-1 may interfere with eIF4G binding. The current study was focused on verification of this suggestion. We showed that YB-1, as well as its C-terminal domain, displaced eIF4G from mRNA in lysate. In a system with purified components (mRNA, eIF4F, and ATP), the result was the same, pointing to direct competition between eIF4G and YB-1. Thus, YB-1 displaces all subunits of eIF4F from the mRNA.

The displacement of eIF4G occurred at a YB-1/mRNA molar ratio of 12-36 (mass ratio 2-6) (Fig. 6), which is close to that in free mRNPs. The higher mass ratio (about 10) required for complete suppression of protein synthesis in lysates (Fig. 1) can possibly be explained by the presence of various YB-1-binding competitors; the major competitors among these are ribosomal subunits. The functional meaning of the interaction between YB-1 and the ribosomal subunits remains to be studied.

Taken together, the current and previous results can be presented as an overall diagram of the effect of YB-1 on translation initiation (Fig. 8). At a relatively low YB-1/mRNA ratio (Fig. 8A), YB-1 stimulates initiation of translation by associating with mRNA over its entire length, presumably displacing RNA-binding factors from nonspecific binding sites. This may lead to concentration of these factors around the cap structure that is their specific binding site (29, 34). It is also possible that YB-1 additionally facilitates movement of the small ribosomal subunit in complex with initiator Met-tRNA up to the initiation codon by melting the secondary structure of mRNA 5'-untranslated region. This is supported by data on RNA melting activity reported in Refs. 14 and 20.


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Fig. 8.   A possible mechanism of the effect of YB-1 and its two domains on interactions between translation initiation factors and mRNA. A and C, translatable mRNA; B and D, untranslatable mRNA. 4A, eIF4A; 4E, eIF4E; 4G, eIF4G; C-ter., C-terminal.

At a higher YB-1/mRNA ratio (Fig. 8B), YB-1 inhibits initiation of translation and stabilizes mRNA as a result of eIF4F displacement from mRNA. The AP-CSD domains displace eIF4E and eIF4A from the cap structure (Fig. 8C), thereby stabilizing mRNA (16) without significantly affecting its translational activity. The C-terminal domain displaces eIF4G from the mRNA (Fig. 8D), thus suppressing initiation of translation. So two different functions of YB-1 are distinctly attributed to its different domains.

Translation of uncapped mRNA was inhibited by lower concentrations of YB-1 as compared with translation of capped mRNA (32). Our results provide a simple explanation for this observation: the YB-1 C-terminal domain easily annihilates binding of eIF4G that is not additionally stabilized by the eIF4G-eIF4E-cap bridge.

In cytoplasm in vivo, concentration of YB-1, and hence the YB-1/mRNA ratio, may vary due to both a changing total amount of YB-1 in the cell (38, 46) and YB-1 redistribution between the nucleus and cytoplasm in response to environmental stimuli (47-50). This should affect the rate of translation initiation. On the other hand, eIF4F competes with YB-1 for binding to mRNA, and an increase of its concentration in the cell may cause displacement of YB-1 from the mRNA 5'-untranslated region and activate initiation of translation. Thus, the translational status and stability of mRNA is determined not only by the YB-1/mRNA ratio but also by the YB-1/eIF4F ratio.

    ACKNOWLEDGEMENTS

We are grateful to Prof. I. Shatsky for kindly providing rabbit eIF4F, Prof. R. Rhoads for anti-eIF4G antibodies, and Dr. P. Ruzanov for plasmids with recombinant YB-1 fragments.

    FOOTNOTES

* This work was supported in part by the Civilian Research and Development Foundation (Grant RB1-282), the International Association for the Promotion of Cooperation with Scientists from the New Independent States of the Former Soviet Union (Grant 97-open-501), the Russian Foundation for Basic Research (Grants 98-04-48015, 00-15-97903, and 01-04-49038), and a grant from the Russian Academy of Sciences (Project on "Protein Biosynthesis and its Regulation").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: 7-095-924-04-93; E-mail: ovchinn@vega.protres.ru.

Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M209145200

    ABBREVIATIONS

The abbreviations used are: mRNP, messenger ribonucleoprotein particle; eIF, eukaryotic initiation factor; YB, Y-box; CSD, cold shock domain; AP, alanine/proline; tRNAi, initiator tRNA.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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