Maturation of Clk1 intron 3/4–retaining RNA induced by the inhibition of Clk1. (A) Clk1 inhibitor treatment. Positions of primer sets are illustrated above. TG003 was administered to NIH-3T3 cells at the concentrations of 0, 5, 10, and 20 µM for 30 min followed by RT-PCR analysis. In the drug-treated cells, the amount of Clk1 intron-retaining RNA was decreased (1,804 bp in a and 1,550 bp in b) as the mature mRNA was increased (546 bp in a). In addition, a maturation intermediate product, which retained only intron 4, was detected in the presence of TG003 (arrow in b). (bottom) Gapdh was used as an internal control. (B) Effect of a transcription inhibitor, α-amanitin, on the maturation of Clk1 mRNA. Maturation induced by TG003 (0, 10, and 50 µM for 30 min) was not influenced in the presence of a transcription inhibitor, α-amanitin (10 µg/ml at final concentration). The maturation intermediate product, which retained only intron 4, was also detected in the presence of TG003 (arrow in b). (C) Effect of the removal of Clk1 inhibitor. A scheme of the experimental time course and corresponding lane numbers are indicated above. NIH-3T3 cells (control; lane 1) were treated with 50 µM TG003 for 30 min (lane 2) and then washed with fresh medium twice. Compared with the culture without drug removal (lane 3), intron 3/4–retaining Clk1 RNA was restored to the control level within 60 min after the removal of TG003 (lane 4).