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The major mRNA-associated protein YB-1 is a potent 5' cap-dependent mRNA stabilizer

2001; Springer Nature; Volume: 20; Issue: 19 Linguagem: Inglês

10.1093/emboj/20.19.5491

1460-2075

Valentina Evdokimova, Peter Ruzanov, Hiroaki Imataka, Brian Raught, Yuri V. Svitkin, Lev P. Ovchinnikov, Nahum Sonenberg,

RNA modifications and cancer

Article1 October 2001free access The major mRNA-associated protein YB-1 is a potent 5′ cap-dependent mRNA stabilizer Valentina Evdokimova Valentina Evdokimova Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Institute of Protein Research, Russian Academy of Sciences, Puschino, Moscow Region, Russian Federation, 142 292 Search for more papers by this author Peter Ruzanov Peter Ruzanov Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Institute of Protein Research, Russian Academy of Sciences, Puschino, Moscow Region, Russian Federation, 142 292 Search for more papers by this author Hiroaki Imataka Hiroaki Imataka Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Brian Raught Brian Raught Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Yuri Svitkin Yuri Svitkin Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Lev P. Ovchinnikov Lev P. Ovchinnikov Institute of Protein Research, Russian Academy of Sciences, Puschino, Moscow Region, Russian Federation, 142 292 Search for more papers by this author Nahum Sonenberg Corresponding Author Nahum Sonenberg Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Valentina Evdokimova Valentina Evdokimova Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Institute of Protein Research, Russian Academy of Sciences, Puschino, Moscow Region, Russian Federation, 142 292 Search for more papers by this author Peter Ruzanov Peter Ruzanov Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Institute of Protein Research, Russian Academy of Sciences, Puschino, Moscow Region, Russian Federation, 142 292 Search for more papers by this author Hiroaki Imataka Hiroaki Imataka Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Brian Raught Brian Raught Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Yuri Svitkin Yuri Svitkin Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Lev P. Ovchinnikov Lev P. Ovchinnikov Institute of Protein Research, Russian Academy of Sciences, Puschino, Moscow Region, Russian Federation, 142 292 Search for more papers by this author Nahum Sonenberg Corresponding Author Nahum Sonenberg Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Author Information Valentina Evdokimova1,2, Peter Ruzanov1,2, Hiroaki Imataka1, Brian Raught1, Yuri Svitkin1, Lev P. Ovchinnikov2 and Nahum Sonenberg 1 1Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 2Institute of Protein Research, Russian Academy of Sciences, Puschino, Moscow Region, Russian Federation, 142 292 *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5491-5502https://doi.org/10.1093/emboj/20.19.5491 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info mRNA silencing and storage play an important role in gene expression under diverse circumstances, such as throughout early metazoan development and in response to many types of environmental stress. Here we demonstrate that the major mRNA-associated protein YB-1, also termed p50, is a potent cap-dependent mRNA stabilizer. YB-1 addition or overexpression dramatically increases mRNA stability in vitro and in vivo, whereas YB-1 depletion results in accelerated mRNA decay. The cold shock domain of YB-1 is responsible for the mRNA stabilizing activity, and a blocked mRNA 5′ end is required for YB-1-mediated stabilization. Significantly, exogenously added YB-1 destabilizes the interaction of the cap binding protein, eIF4E, with the mRNA cap structure. Conversely, sequestration of eIF4E from the cap increases the association of endogenous YB-1 with mRNA at or near the cap, and significantly enhances mRNA stability. These data support a model whereby down-regulation of eIF4E activity or increasing the YB-1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization. Introduction Control of mRNA stability plays an important role in the regulation of gene expression in eukaryotes (Ross, 1995; Caponigro and Parker, 1996; Schwartz and Parker, 2000). The 3′ poly(A) tail, in association with the poly(A) binding protein (PABP), is the best studied and characterized mRNA stability factor (Bernstein and Ross, 1989; Ross, 1995; Jacobson and Peltz, 1996). PABP was first identified as a protein tightly associated with mRNA in messenger ribonucleoprotein (mRNP) particles, together with a 50 kDa protein, first termed p50, and more recently, YB-1 (Blobel, 1972; Van Venrooij et al., 1977; for review see Evdokimova and Ovchinnikov, 1999). Active polysomal mRNPs possess both YB-1 and PABP, whereas YB-1 is the predominant protein component of translationally inactive mRNPs (Van Venrooij et al., 1977; Minich et al., 1989; Spirin, 1996). A large body of evidence indicates that YB-1 is a general translational repressor; an increase in YB-1 concentration results in an inhibition of translation initiation in vitro, and YB-1 overexpression in cells causes translational repression in vivo, via a mechanism that is poorly understood (Minich et al., 1993; Davydova et al., 1997). YB-1 belongs to the Y-box binding (YB) transcription factor protein family, so termed because of the ability of these proteins to bind to the Y-box promoter element (Wolffe et al., 1992). YB proteins are present in bacteria, plants and animals, and exhibit a wide variety of biological activities, ranging from transcriptional regulation of a large number of genes to ‘masking’ of mRNA in germinal cells. YB-1 is a nucleocytoplasmic shuttling protein (Matsumoto and Wolffe, 1998; Sommerville, 1999), and is highly conserved amongst vertebrates, including rabbit, rat, mouse, chicken, frog and human with ∼98% identity between the rabbit and human YB-1 proteins (Evdokimova et al., 1995). YB-1 consists of an alanine/proline (AP)-rich N-terminal domain followed by a cold-shock domain (CSD) and a C-terminal segment comprised of four alternating clusters of basic and acidic amino acids (Wolffe, 1994). The most conserved region in YB proteins is the 80 amino acid CSD; YB-1 exhibits >40% identity and >60% similarity to the major Escherichia coli cold-shock protein CspA (Matsumoto and Wolffe, 1998; Sommerville, 1999). The CSD is a five-stranded β-barrel containing RNP 1 and RNP 2-like consensus motifs, which binds to duplex and single stranded DNA and RNA (Wolffe et al., 1992; Schindelin et al., 1994; Wolffe, 1994; Bouvet et al., 1995). The N-terminal AP domain of YB-1 was reported to associate with actin microfilaments, presumably contributing to mRNA localization (Ruzanov et al., 1999). The C-terminal region of YB-1 and related proteins binds DNA and RNA in a sequence-independent manner and mediates protein–protein interactions (Wolffe, 1994; Sommerville and Ladomery, 1996). Although YB-1 has been implicated in specific stabilization of IL-2 mRNA following T-cell activation (Chen et al., 2000), its function in general mRNA stability has not been addressed. Several observations support a role for YB-1-related proteins in mRNA stabilization at early stages of metazoan development. For example, the Xenopus FRGY2 and the mouse MSY1 and MSY2 proteins are the major components of translationally inactive mRNPs, which are stored throughout gametogenesis (Richter and Smith, 1984; Murray et al., 1991; Gu et al., 1998). Consistent with a negative effect of YB proteins on protein synthesis, translational activation of stored mRNAs during embryogenesis coincides with massive degradation of ‘germinal’ YB proteins (Wolffe et al., 1992; Sommerville, 1999). In this study we demonstrate that, coincident with translation inhibition, YB-1 dramatically stabilizes mRNA. Strikingly, the CSD of YB-1 alone engenders mRNA stabilization, but not translational repression. On the contrary, the C-terminal domain strongly inhibits mRNA translation but does not affect mRNA stability, which indicates that these activities are separated, and map to different regions of the protein. Although YB-1 stabilization of mRNA is sequence non-specific, it is strongly dependent on the presence of a blocked (m7GpppG or GpppG) mRNA 5′ end. Translation inhibitors that interfere with eIF4E cap binding function or disrupt the assembly of the eIF4F cap binding complex (such as cap analogs or the repressor protein 4E-BP1) promote an interaction between YB-1 and the mRNA 5′ cap structure, as determined by UV cross-linking experiments, and prevent mRNA degradation. Conversely, YB-1 depletion from cell extracts accelerates mRNA decay and abrogates mRNA stabilization by cap analogs. These data suggest that YB-1 is a potent, general cap-dependent mRNA stabilizer that protects mRNA against degradation when the association of eIF4E with the cap is impaired. Results YB-1 is a potent stabilizer of mRNAs in vitro and in vivo The effect of YB-1 on mRNA stability was examined in three different in vitro systems, a rabbit reticulocyte lysate, Krebs-2 ascites and HeLa cell extracts. Time-course experiments were performed to monitor the decay of a chloramphenicol acetyltransferase (CAT) mRNA, as well as the CAT mRNA fused to the 3′ UTR of the tumor necrosis factor α (TNFα) mRNA, which contains multiple destabilizing AU-rich elements (AREs). Equal amounts of total RNA were loaded in each lane, as determined by northern blot analysis of 18S rRNA (Figure 1). The half-life of CAT mRNA ranged from ∼55 min in the rabbit reticulocyte lysate (Figure 1A) to ∼35 min in the HeLa extract (Figure 1B). The half-life of CAT−3′ TNFα mRNA was somewhat shorter than that of the control CAT mRNA; ∼40 min in the reticulocyte lysate (Figure 1A) and ∼20 min in HeLa cell extract (Figure 1B). Since the difference in the half-lives of the CAT and CAT−3′ TNFα mRNAs is not large, the major decay pathway observed here is not likely to be ARE dependent. Strikingly, in the presence of YB-1 the decay rates of both the CAT and CAT−3′ TNFα mRNAs were dramatically decreased in all extracts tested, as the mRNAs remained stable even after a 2 h incubation (Figure 1). Similar results were obtained in an ascites Krebs-2 cell extract (data not shown). Taken together, these data demonstrate that YB-1 is a potent mRNA stabilizer in vitro, in several different cell extracts. Figure 1.YB-1 is a potent mRNA stabilizer in vitro. Capped CAT and CAT−3′ TNFα mRNAs (0.1 μg each) were incubated in a rabbit reticulocyte lysate (A) or HeLa extract (B) with buffer (control) or in the presence of YB-1 (0.5 μg). Total RNA was isolated at the times indicated, and CAT mRNA and 18S rRNA were detected by northern blot hybridization with the corresponding probes. CAT and CAT−3′ TNFα mRNAs from three independent experiments were quantified using PhosphorImaging software and normalized to 18S rRNA (right panels). Download figure Download PowerPoint To determine whether the effect on mRNA stability is peculiar to YB-1, or is a non-specific effect elicited by any RNA binding protein, several other RNA binding proteins [La autoantigen (Gottlieb and Steitz, 1989), PABP (Görlach et al., 1994) and nucleocapsid HIV-1 protein, NCp7 (Tanchou et al., 1995)] were tested in a HeLa extract. La autoantigen and NCp7 are promiscuous high-affinity RNA binding proteins (Kd < 10 nM) (Tanchou et al., 1995; Belsham and Sonenberg, 1996), whereas PABP exhibits sequence specificity toward poly(A) (Deo et al., 1999; Görlach et al., 1994). As expected (Bernstein et al., 1989), PABP increased the stability of capped and polyadenylated luciferase (LUC) mRNA (Figure 2A, lanes 6–10), albeit to a lesser degree than YB-1 (lanes 11–15). Neither La (lanes 16–20), nor NCp7 (lanes 21–25) significantly affected LUC mRNA stability. Thus, mRNA stabilization is not a general effect elicited by all RNA binding proteins. Figure 2.YB-1 uniquely mediates mRNA stabilization. (A) Effect of general mRNA binding proteins on mRNA stability. Capped and polyadenylated LUC mRNA (0.1 μg) was incubated in a HeLa extract with buffer alone (−) or in the presence of 0.5 μg of the RNA binding protein indicated, and detected by northern blot hybridization. (B) Comparison of YB-1 and PABP mRNA stabilizing acitivities. LUC mRNA (0.1 μg) possessing or lacking poly(A) tail was incubated for 60 min in a HeLa extract with buffer (−) or in the presence of increasing amounts YB-1 or PABP, as indicated in the figure, and detected by northern blot hybridization. Lanes 1 and 14 (Input) show LUC mRNA isolated from the extract at 0 min of incubation. Download figure Download PowerPoint To examine the role of the poly(A) tail in YB-1-mediated mRNA stabilization, we studied the effects of YB-1 and PABP on the stability of LUC mRNA with or without a poly(A) tail. Both non-adenylated and polyadenylated LUC mRNAs incubated in a HeLa extract in the presence of increasing amounts of YB-1 were markedly stabilized during a 60 min incubation (Figure 2B, compare lanes 2 and 15 with lanes 3–7 and 16–20, respectively). It is noteworthy, however, that more YB-1 (0.6 μg) was required for full stabilization of non-adenylated LUC mRNA in comparison with the polyadenylated counterpart (0.1 μg YB-1). As reported previously (Bernstein et al., 1989), PABP protected exclusively polyadenylated mRNA (Figure 2B, lanes 22–26), but failed to significantly stabilize non-adenylated mRNA (lanes 9–13). Therefore, in contrast to PABP, which functions primarily via the poly(A) tail, YB-1 stabilizes mRNA mainly via a non-poly(A)-dependent mechanism. However, as the polyadenylated mRNA was more effectively protected by YB-1, a role of the poly(A) tail in YB-1-mediated mRNA stabilization is evident. To determine whether YB-1 also stabilizes mRNA in vivo, it was overexpressed in HeLa cells using the vaccinia virus T7 RNA polymerase system (Fuerst et al., 1986) (Figure 3). The vaccinia system was used to ensure high expression of exogenous over endogenous YB-1, which is abundant in eukaryotic cells (∼0.1% of total protein, 2.6 × 106 molecules/cell; Davydova et al., 1997). Cells were infected with a recombinant vaccinia virus encoding T7 RNA polymerase, followed by transfection with an expression vector containing the CAT cDNA under the control of the human cytomegalovirus early promoter (pCMV-CAT) alone, or together with the hemagglutinin (HA)-tagged YB-1 cDNA under the control of the T7 promoter (pT7-HA-YB-1). Assuming ∼25% transfection efficiency, and considering that the signal from the exogenously expressed YB-1 in a typical transient transfection (using 8 μg of YB-1 plasmid) was about four times stronger than that from the endogenous YB-1 (data not shown), the amount of the exogenously expressed YB-1 was estimated to be ∼16-fold greater than that of the endogenous protein. To determine the effect of YB-1 overexpression on the half-life of CAT mRNA, actinomycin D (ActD) was added 20 h post-transfection to block transcription, and total RNA was isolated at various time intervals. In the absence of the overexpressed YB-1 the decay of CAT mRNA was rapid, with an apparent half-life of <2 h, as determined by northern blotting (Figure 3A, lanes 7–12). The amount of CAT protein did not change significantly during the incubation with ActD over 10 h (Figure 3C, lanes 7–12), in spite of the reduction in mRNA level, because CAT protein is very stable (half-life of ∼50 h; Thompson et al., 1991). Co-transfection of pCMV-CAT with pT7-HA-YB-1 resulted in a dramatic stabilization of CAT mRNA (Figure 3A, lanes 13–18 and 19–24). Remarkably, the level of CAT mRNA was increased up to 50-fold (at 10 h of incubation) when 8 μg of pT7-HA-YB-1 was co-transfected (Figure 3A and B). Consistent with earlier data (Davydova et al., 1997), YB-1 overproduction also resulted in translational repression of CAT mRNA; CAT protein level was decreased ∼2-fold (Figure 3C, compare lanes 7 and 13). Thus, the overproduction of YB-1 in vivo potently stabilizes CAT mRNA, while moderately inhibiting its translation. Figure 3.Overexpression of YB-1 in HeLa cells results in mRNA stabilization and translational repression. (A) Northern blot analysis of CAT mRNA decay in the absence or presence of overexpressed YB-1. HeLa cells infected with the recombinant vaccinia virus vT7F7-3 were co-transfected with pCMV-CAT (4 μg/plate) and with vector (pcDNA3-HA) alone or pcDNA3-HA-YB-1 (pT7-HA-YB-1; 4 or 8 μg/plate), as described in Materials and methods. Following addition of ActD for the times indicated, total cellular RNA was harvested and CAT mRNA and 18S rRNA were detected by hybridization with the corresponding probes. (B) Kinetic analysis of CAT mRNA decay. CAT mRNA from three independent experiments similar to that shown in (A) was quantified by PhosphorImaging software and normalized to 18S rRNA. (C) Western blot analysis of HA-YB-1 and CAT proteins. Five percent of HeLa extracts obtained from each time point in (A) were resolved by SDS–12% PAGE and analyzed with anti-HA or anti-CAT antibodies. Download figure Download PowerPoint The CSD of YB-1 confers mRNA stability, but not translational repression To determine which domains of YB-1 are responsible for mRNA stabilization and translational repression, the effects of different YB-1 fragments on the half-life and translation efficiency of the LUC mRNA were examined in a rabbit reticulocyte lysate. Unexpectedly, the CSD alone conferred stability on LUC mRNA almost as efficiently as the full-length YB-1 protein (Figure 4A, compare lanes 16–20 and 6–10). After 90 min of incubation, ∼100 and 60%, respectively, of LUC mRNA remained intact in the presence of the full-length YB-1 and the CSD fragment (Figure 4B). Although the CSD and the C-terminal portion of YB-1 (and related YB proteins) possess similar affinities for RNA (data not shown, see also Bouvet et al., 1995; Matsumoto et al., 1996), the C-terminal fragment failed to stabilize LUC mRNA (Figure 4A, lanes 21–25). Similarly, the AP domain, which also contributes to mRNA binding (Bouvet et al., 1995), did not affect mRNA stability (lanes 11–15). Thus, the non-specific RNA binding of YB-1 fragments is not sufficient to protect mRNA, and only the CSD exhibits stabilizing activity. Figure 4.The cold-shock domain of YB-1 confers mRNA stability, but not translational repression. (A) Top: schematic representation of YB-1 functional domains. The N-terminal AP-rich domain, the central CSD containing RNP 1 consensus motif, and the C-terminal domain with alternating basic (+) and acidic (−) amino acid clusters are indicated. Amino acids are numbered according to Evdokimova et al. (1995). (A) Bottom: effect of YB-1 fragments on mRNA stability. Capped and polyadenylated LUC mRNA (0.1 μg) was incubated in a rabbit reticulocyte lysate with buffer (control) or in the presence of 0.5 μg of YB-1 or its fragments. Total RNA was isolated at the times indicated and LUC mRNA and 18S rRNA were detected by northern blot hybridization with the corresponding probes. (B) Kinetic analysis of LUC mRNA decay. The amounts of LUC mRNA from three independent experiments similar to that shown in (A) were quantified by PhosphorImaging software and normalized to 18S rRNA. (C) Effect of YB-1 fragments on mRNA translation. Capped and polyadenylated LUC mRNA (0.1 μg) was incubated in a rabbit reticulocyte lysate with buffer alone (−) or increasing amounts of the protein indicated. Translation reactions were incubated at 30°C for 60 min and luciferase activity was monitored by luminometer. The luciferase activity in the presence of buffer alone (control) was set as 100%. Error bars denote the standard error from three independent experiments. Download figure Download PowerPoint To establish whether mRNA stabilization correlates with translational repression, the effects of full-length YB-1 or individual YB-1 domains on luciferase synthesis were monitored. In agreement with previous data (Matsumoto et al., 1996; Izumi et al., 2001), the AP domain and the CSD affected translation only slightly (∼10 and 40%, respectively, at the highest concentrations used), while the C-terminal domain of YB-1 repressed translation to almost the same degree as the full-length YB-1 protein (Figure 4C). Thus, although the precise mechanism by which YB-1 represses translation remains to be determined (see also Discussion), these data indicate that the two activities of YB-1, mRNA stabilization and translational inhibition, are distinct and map to separate domains of the protein. YB-1 stabilizes mRNA in a cap-dependent manner The 5′ cap structure and 3′ poly(A) tail are the primary targets for mRNA degradation pathways, and play critical roles in mRNA stability (Ross, 1995; Jacobson and Peltz, 1996; Schwartz and Parker, 2000). Because the poly(A) tail was not absolutely required for YB-1-mediated mRNA stabilization (Figure 2B), we next analyzed whether mRNA stabilization by YB-1 is dependent upon the presence of the cap structure. To assess the role of the cap in YB-1-mediated mRNA stabilization, polyadenylated LUC mRNAs possessing either a methylated (m7GpppG) or unmethylated (GpppG) cap, or lacking the cap structure altogether (pppG), were examined. Decay rates of the differentially cap-modified LUC mRNAs in the presence or absence of YB-1 in a HeLa extract are presented in Figure 5A. In agreement with previous data (e.g. Furuichi et al., 1977), LUC mRNAs possessing a blocked 5′ end (m7GpppG or GpppG) were more stable than uncapped mRNA (Figure 5A, compare lanes 1–5, 6–10 and 11–15). While YB-1 did not affect the stability of uncapped LUC mRNA, it dramatically stabilized the mRNAs harboring m7GpppG or GpppG cap structures (compare lanes 16–20 and 21–30), increasing their half-life by ∼4-fold (Figure 5B). In the presence of YB-1 a significant portion of the capped and polyadenylated LUC mRNAs remained intact even after 90 min of incubation, as compared with <10% for the uncapped mRNA counterpart. Figure 5.YB-1 protects mRNA against degradation in a cap-dependent manner. (A) Polyadenylated LUC mRNAs (0.05 μg) with differently modified 5′ ends were incubated in a HeLa extract in the absence or presence of YB-1 (0.2 μg). Total RNA was isolated at the times indicated and LUC mRNA and 18S rRNA were detected by northern blot hybridization with the corresponding probes. (B) Kinetic analysis of LUC mRNA decay. LUC mRNA from three independent experiments similar to that shown in (A) was quantified by PhosphorImaging software and normalized to 18S rRNA. (C) Effect of YB-1 on mRNA decapping. 32P-cap-labeled and polyadenylated LUC mRNA (0.05 μg) was incubated in a HeLa extract for 60 min with buffer alone (−) or in the presence of increasing amounts of either YB-1, AP/CSD or AP/CSD mutant, as indicated, and analyzed by 4% acrylamide–7 M urea gel electrophoresis and autoradiography. Lane 1 (Input) shows LUC mRNA isolated from the extract at 0 min of incubation. Download figure Download PowerPoint Next, the effect of YB-1 on mRNA cap structure stability was assayed directly. LUC mRNA was 32P-labeled exclusively in the 5′ cap structure and incubated in a HeLa extract in the presence of increasing amounts of YB-1. The mRNA cap structure was almost completely removed after 60 min of incubation (Figure 5C, compare lanes 1 and 2, 8, 14), but YB-1 significantly inhibited mRNA decapping and degradation, even at the lowest amount added (0.1 μg; lane 3). Increasing the YB-1 concentration to 0.5 μg (lane 7) resulted in complete retention of the cap structure during the incubation time. To determine whether the CSD can confer cap protection as well, a YB-1 fragment (AP/CSD) and a form bearing two point mutations within the RNP 1 motif (GYGFI → GAGAI) were tested. The RNP 1 motif is a probable contact site between YB-1 and RNA; these mutations were based on previous reports showing an essential role in RNA binding for conserved aromatic amino acids in the RNP 1 motif (Bouvet et al., 1995; Schroder et al., 1995). We chose to use the two AP/CSD domains of YB-1 in this assay, instead of just the CSD, to minimize the effects of the point mutations on the general RNA binding capacity of the fragments. Strikingly, in a manner similar to the full-length protein, the AP/CSD fragment was sufficient to protect the cap-labeled mRNA, while the AP/CSD mutant exhibited much less of a protective effect (Figure 5C, compare lanes 9–13 and 15–19). The AP domain alone and the C-terminal fragment of YB-1 failed to prevent mRNA decapping and degradation, although they were stable during the incubation time (data not shown, see also Figure 4A). Therefore, the CSD and, in particular, the RNP 1 motif, appear to play an important role in protection of the cap structure and mRNA against degradation. Taken together, these data suggest that YB-1 functions as a cap-protective protein that modulates the stability of capped mRNAs. YB-1 destabilizes the interaction of eIF4E with the cap structure To elucidate the mechanism by which YB-1 prevents the decapping and degradation of mRNA, the effect of YB-1 on the interaction of the cap binding protein eIF4E with the mRNA cap structure was studied. LUC mRNA possessing a 32P-labeled cap was incubated in a reticulocyte lysate to allow for the assembly of RNA–protein complexes, and then subjected to UV irradiation. The translation initiation factors eIF4B (80 kDa), eIF4A (46 kDa) and eIF4E (24 kDa) were cross-linked to the capped RNA (Figure 6A, lane 1). The assignment of the cross-linked proteins is based on numerous previous reports (e.g. Pelletier and Sonenberg, 1985). Cross-linking of eIF4E to the cap structure is dependent on direct binding of eIF4E to the cap, whereas eIF4A and eIF4B interaction with the cap is a secondary event (Gingras et al., 1999). The non-specific cross-linking of a 65 kDa protein has also been reported previously (Pelletier and Sonenberg, 1985). Addition of increasing amounts of YB-1 resulted in a gradual inhibition of eIF4E, eIF4A and eIF4B cross-linking to the cap-labeled mRNA. This was accompanied by the appearance of a major cross-linked band of ∼50 kDa (Figure 6A, lanes 2–6). The ∼50 kDa cross-linked polypeptide was identified as YB-1 by immunoprecipitation (data not shown; see also Figure 7B). It is also noteworthy that YB-1 failed to decrease the non-specific cross-linking of p65. Thus, YB-1-mediated stabilization of capped mRNAs is accompanied by a specific inhibition of eIF4E (and associated initiation factors) interaction with the cap. Figure 6.YB-1 inhibits the interaction of eIF4E with the cap. (A) Cross-linking of proteins to the mRNA cap structure. 32P-cap-labeled and polyadenylated LUC mRNA (0.1 μg) was incubated in a rabbit reticulocyte lysate for 15 min with buffer alone (−) or in the presence of increasing amounts of YB-1, as indicated. RNA–protein complexes were covalently cross-linked by UV irradiation, treated with RNase A and subjected to SDS–12% PAGE. (B) Coomassie Blue staining of YB-1 or its fragments (2 μg each). (C) Cross-linking of YB-1 fragments to the cap structure. 32P-cap-labeled and polyadenylated LUC mRNA was incubated in a rabbit reticulocyte lysate as in (A) with buffer alone (−) or in the presence of 1 μg of the indicated proteins. RNA–protein complexes were cross-linked by UV irradiation and detected as in (A). Download figure Download PowerPoint Figure 7.YB-1 cross-links to the cap and promotes mRNA stabilization when the eIF4E interaction is reduced. (A) Effect of the translation inhibitorson YB-1 cross-linking. 32P-cap-labeled and polyadenylated LUC mRNA (0.1 μg) was incubated in a rabbit reticulocyte lysate with buffer alone (−) or in the presence of YB-1 (1 μg), GDP (0.5 and 1 mM), m7GDP (0.5 and 1 mM), 4E-BP1 (1 and 4 μg), pactamycin (PM) (2 and 10 μg/ml), cycloheximide (CH) (10 and 50 μg/ml) or poly(A) (40 and 80 μg/ml). Reaction mixtures were incubated for 15 min at 30°C, UV irradiated, treated with RNase A and subjected to SDS–12% PAGE. (B) Cross-linked proteins obtained as in (A) in the presence of m7GDP (0.5 mM) were immunoprecipitated (IP) with either pre-immune or anti-YB-1 antibodies as described in Materials and methods and subjected to SDS–12% PAGE. (C) Northern blot analysis of LUC mRNA decay in a rabbit reticulocyte lysate pre-incubated with either pre-immune (control) or anti-YB-1 (YB-1-depleted) antibodies. Capped an