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Figure 1 YB-1 is a potent mRNA stabilizer in vitro. Capped CAT and CAT-3' TNF mRNAs (0.1  g 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 g). 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' TNF mRNAs from three independent experiments were quantified using PhosphorImaging software and normalized to 18S rRNA (right panels).
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 | 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 g) was incubated in a HeLa extract with buffer alone (-) or in the presence of 0.5 g 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 g) 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.
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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 g/plate) and with vector (pcDNA3-HA) alone or pcDNA3-HA-YB-1 (pT7-HA-YB-1; 4 or 8 g/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.
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 | 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 g) was incubated in a rabbit reticulocyte lysate with buffer (control) or in the presence of 0.5 g 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 g) 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.
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Figure 5 YB-1 protects mRNA against degradation in a cap-dependent manner. (A) Polyadenylated LUC mRNAs (0.05 g) with differently modified 5' ends were incubated in a HeLa extract in the absence or presence of YB-1 (0.2 g). 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 g) 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.
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 | 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 g) 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 g 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 g of the indicated proteins. RNA–protein complexes were cross-linked by UV irradiation and detected as in (A).
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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 g) was incubated in a rabbit reticulocyte lysate with buffer alone (-) or in the presence of YB-1 (1 g), GDP (0.5 and 1 mM), m7GDP (0.5 and 1 mM), 4E-BP1 (1 and 4 g), pactamycin (PM) (2 and 10 g/ml), cycloheximide (CH) (10 and 50 g/ml) or poly(A) (40 and 80 g/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 and polyadenylated LUC mRNA (0.1 g) was incubated in the control or YB-1-depleted rabbit reticulocyte lysate in the presence of GDP (0.5 mM) or m7GDP (0.5 mM), as indicated in the figure. Total RNA was isolated at the times indicated and LUC mRNA and 18S rRNA were detected by sequential hybridization with the corresponding probes. (D) Kinetic analysis of LUC mRNA decay. The amounts of LUC mRNA from three independent experiments identical to that shown in (C) were quantified by PhosphorImaging software and normalized to 18S rRNA. (E) A western blot showing the efficiency of YB-1 immunodepletion from the rabbit reticulocyte lysate preincubated with either pre-immune or anti-YB-1 antibodies coupled to protein A–Sepharose. Western blot analysis was performed with pre-immune or YB-1-depleted reticulocyte lysate using anti-PABP antibodies as a loading control (upper panel) or anti-YB-1 antibodies (lower panel).
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 | Figure 8 Model: YB-1 functions as a 'sensor' to monitor eIF4E and mediate mRNA stabilization. Under standard growth conditions the mRNA m7GpppN cap (where m is a methyl group, and N is any nucleotide) is accessible to the eIF4F cap binding complex as well as to degradative enzymes, such as Dcp1 and PARN. YB-1 is anchored to the mRNA body due to its general non-specific RNA binding activity, and its interaction with the cap-proximal region is prevented by eIF4F. Under certain conditions, such as during early embryogenesis or stress, an increase in YB-1 activity or sequestration of eIF4E from the cap stimulates YB-1 binding at or near the cap, leading to general mRNA stabilization.
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