FIGURE 2.

Non-stop mRNA is unstable, and its decay requires translation in HeLa cells. A, steady-state levels of β-globin non-stop mRNAs. HeLa cells were transfected with p5×FLAG-EGFP and either pFBG control (lane 1) or pFBG non-stop (lane 2). Numbers immediately below each lane represent the level of FBG mRNA normalized to the level of 5×FLAG-EGFP mRNA. B, degradation of β-globin non-stop mRNAs. HeLa cells were cotransfected with p5×FLAG-EGFP, pT7-TR, and either pFBG control (upper panel, lanes 1–5) or pFBG non-stop (lanes 6–10). 1 day later, FBG mRNA expression was induced by tetracycline for 2.5 h, and the cells were harvested at the specified times after stopping FLAG-β-globin mRNA transcription. The levels of wild-type FBG mRNAs, which were normalized to the levels of 5×FLAG-EGFP mRNAs, were quantitated with the levels of the mRNA from the 0-h time point defined as 100% (lower panel). C, translation is required for the fast degradation of β-globin non-stop mRNA. HeLa cells were cotransfected with p5×FLAG-EGFP, pT7-TR, and either pFBG non-stop (upper panel, lanes 1–10) or pIRE-FBG non-stop (lanes 11–20). The transcriptional pulse-chase analysis was performed as described for B, except that the cells were treated with 100 μg/ml cycloheximide (CHX; upper panel, lanes 6–10) or 50 μm hemin (lanes 16–20). The levels of FBG mRNAs were quantitated as described for B (lower panel). Error bars represent S.D. for three independent experiments.