The cis-regulatory elements required for the intron retention of Clk1 RNA. (A) Scheme of β-globin minigene construction. Exons 1, 2, and 3 of β-globin are indicated by b1, b2, and b3, respectively. The highly conserved region of mouse Clk1 (indicated by a blue line in Fig. S3) was inserted into NotI–SpeI sites added in the second intron of the β-globin minigene expression vector. The sequence of insertion site in β-globin is indicated. The added sequence is indicated in the lowercase letters. (B) RT-PCR analysis of the reporter RNA splicing. Arrows indicate primer position. Retention of introns adjacent to the translocated exon 4 was observed (arrowheads). In the presence of TG003, a Clk1/4 inhibitor, the intron-retaining pre-mRNA (arrowheads) was decreased, and the splicing intermediate (arrows) and the exon 4–included spliced product were increased. Asterisks show the PCR products derived from the primary transcript and/or contaminated plasmid DNA. RT, reverse transcription; WT, wild type.