FIGURE 4.

Inhibition of pol II elongation does not affect the relative rates of intron removal. Hep3B cells were transfected with the reporter minigene pUHC-EDA and treated with the ethanol vehicle (lane 1) or 25 μM DRB for 8 h (lane 2) following induction by withdrawal of tetracycline. (Lanes 3,4) Cells were cotransfected in the presence of tetracycline with the reporter minigene pUHC-EDA and expression vectors for human α-amanitin resistant pol II large subunits (hRpb1), WT (lane 3) and slow mutant (C4, lane 4). Minigene transcription was induced 18 h post-transfection by washing out tet and adding α-amanitin (10 μg mL⁻¹) to inhibit endogenous pol II transcription. Cells were harvested 20 h post-induction and used for subsequent assays. (A) Results of radioactive RT-PCR analysis of E33 isoforms and quantification of E33+/E33− ratios (gray bars). (B) Real-time RT-PCR analysis for the quantification of splicing intermediates calculated as %α-intermediate (black bars). Calculations show mean ± SD.