FIG 7.

The CUGBP1–ph-S51-eIF2α complex represses p53 translation in cultured cells and in livers of dnp300 mice. (A) Diagram showing GFP-CUGBP1 constructs used in these studies. (B) Interactions of transfected CUGBP1 mutants with total eIF2α and ph-S51-eIF2α. GFP-CUGBP1 constructs were transfected into Hep3B2 cells, and CUGBP1 was immunoprecipitated with Abs to GFP. The IPs were probed with Abs to total eIF2α and to ph-S51-eIF2α. Bar graphs show ratios of eIF2α levels to CUGBP1 levels in CUGBP1 IPs. B, beads. (C) The CUGBP1 S302A mutant represses translation of p53 mRNA through the CUGBP1-binding site located in the 5′ UTR of p53 mRNA. The top shows 5′ sequences of luciferase constructs containing WT and mutant CUGBP1-binding sites from the 5′ UTR of p53 mRNA. Bar graphs show results of cotransfections of WT and mutant CUGBP1 with WT and mutant p53-luciferase constructs. Data represent means ± SD (n = 5). *, P < 0.05. (D) CUGBP1 shows a stronger interaction with p53 mRNA in livers of dnp300 mice than in livers of WT mice. (Top) Levels of CUGBP1 and eIF2α in cytoplasmic extracts after CCl₄ treatments. (Middle) Results of UV cross-linking assays of immunoprecipitated CUGBP1 with an RNA oligomer corresponding to the WT p53 probe (Fig. 6E). Coomassie stain of the UV-cross-linked membrane is shown at the bottom. (Bottom) Image showing results from two-dimensional gel electrophoresis of CUGBP1 from livers of WT and dnp300 mice 48 h after CCl₄ treatments. Red arrows on the top show the positions of phosphorylated CUGBP1, which works as an activator of translation. Black arrows show the positions of unphosphorylated CUGBP1, which represses the translation of mRNAs. (E) Amounts and associations of CUGBP1–ph-S51-eIF2α complexes with p53 mRNA are increased in livers of dnp300 mice after CCl₄ treatments. Inactive ph-S51-eIF2α was immunoprecipitated from protein extracts at different time points after CCl₄ injection, and CUGBP1 protein and p53 mRNA levels in these IPs were determined by Western blotting. (Top) Western blotting with Abs to CUGBP1. The IgG signals are shown at the bottom. (Middle) Amounts of CUGBP1 shown as ratios to the amount of IgG. (Bottom) Bar graphs showing amounts of p53 mRNA in the same ph-S51-eIF2α IPs as a summary of data from three independent experiments.