FIGURE 3.

CPEB4 binds with CSAG2 mRNAs and induces its cytoplasmic polyadenylation and translation (A,B) HEK293T cells were transfected with 5 or 10 µg plasmids expressing vector or human CPEB4 as indicated, and the cell lysates were immunoprecipitated with IgG isotype control antibody or CPEB4 antibody. The protein level of CPEB4 in whole cell lysates and IP products was analyzed by Western blotting (A). Total RNAs bound with agarose beads were purified from immunoprecipitates and reversely transcribed to cDNA and analyzed by qRT-PCR to measure the level of CSAG2 transcript (B). The results in each sample represent the mean value of three replicates. The enrichment value relative to IgG group is shown. Data are mean ± SEM. **p < 0.01. (C–E) HEK293T cells were transfected with 5 or 10 µg plasmids expressing vector or human CPEB4 as indicated and cultured for 3 days. (C) Total RNAs were then extracted and the polyadenylation was measured by RNA ligation-coupled RT-PCR using (³²P) α-dATP and specific primers for CSAG2. Products were separated in a denaturing 8% polyacrylamide gel and visualized by autoradiography. *, non-adenylated RNAs; AA-, adenylated RNAs. (D) The whole cell lysates were analyzed by Western blotting for detecting the level of CSAG2. β-Actin was used as a loading control. Shown here are representative images. (E) The mRNA of CSAG2 was analyzed by qRT-PCR. β-Actin was used as a reference control. Data are mean ± SEM. n = 3. NS, not significant. (F) The cell lysates of SKOV3 (R) and CaOV3 (R) cells were immunoprecipitated with IgG isotype control antibody or CPEB4 antibody. The binding of 3′-UTR of CSAG2 mRNA with CPEB4 was analyzed as in (B). Data are mean ± SEM. **p < 0.01. (G–I) SKOV3 (R) and CaOV3 (R) cells stably expressing shRNA targeting control or human CPEB4 were routinely cultured for 3 days. The polyadenylation of CSAG2 mRNA (G), and the protein level (H) and mRNA level (I) of CSAG2 were analyzed as in (C–E). Data are mean ± SEM. n = 3. NS, not significant.