Comparison of deadenylation rates and mRNA half-lives in HeLa cells. (A) Flag-β-globin and 5xFlag-EGFP mRNAs were analyzed by Northern blotting. HeLa cells were cotransfected with pFlag-CMV5/TO-Gl, pCMV-5xFlag-EGFP, pcDNA6-T7-TR, and either pCMV-5xMyc (lanes 1–4), pCMV-5xMyc-eRF3b (F66A) (lanes 5–8), pCMV-5xMyc-eRF3b (F75A) (lanes 9–12), pCMV-5xMyc-eRF3b (F66A/F75A) (lanes 13–16), or pCMV-5xMyc-eRF3b (lanes 17–20). One day later, Flag-β-globin mRNA was induced by tetracycline for 2.5 h, and cells were harvested at the specified times transcription was stopped. To analyze steady-state mRNAs, Flag-β-globin transcription was induced by treatment with tetracycline for 12 h (lanes 21,22). To mark deadenylated (A0) RNA, the steady-state mRNAs were digested with RNase H in the presence of oligo(dT) (lane 22). 5xFlag-EGFP mRNA served as a loading control. (B) Deadenylation rates and mRNA half-lives were calculated. (*) P < 0.05 compared with cells transfected with pCMV-5xMyc. The results in panel B are expressed as means ± SD of data from three independent experiments. (C) HeLa cells were transfected with pCMV-5xMyc (lanes 1,6,7), pCMV-5xMyc-eRF3b (lanes 2,8), pCMV-5xMyc-eRF3b (F66A) (lanes 3,9), pCMV-5xMyc-eRF3b (F75A) (lanes 4,10), or pCMV-5xMyc-eRF3b (F66A/F75A) (lanes 5,11). Cytoplasmic extracts were immunoprecipitated (IP) using anti-PABPC1 (lanes 7–11) or, as a control, preimmune serum (lane 6). The immunoprecipitates (lanes 6–11) and inputs (lanes 1–5) were analyzed by Western blotting using anti-PABPC1, anti-Myc, or anti-eRF3. Numbers immediately below the panel represent IP efficiencies of cellular eRF3 normalized to cellular PABPC1, where the normalized efficiency of cellular eRF3 calculated from lane 7 was defined as 100%. (N.D.) Indicates that cellular eRF3 was not detected. Results are representative of two independent experiments.
