Figure 5.
Trans-splicing of COT pre-mRNAs in vitro. Transcription plasmids (Bluescript SK+) with the corresponding PCR fragments were obtained as described in Materials and Methods. A Pre-mRNA was 220 nucleotides (nt) long: 55 nt from the plasmid polylinker region (PL) + exon 2 (136 nt) + 25 nt of the 5′ end of intron 2 + 4 nt PL; B Pre-mRNA was 278 long: 23 nt PL + 115 nt of the 3′ end of intron 1 + exon 2 (136 nt) + 4 nt PL. BP indicates branch-point. The 32P-labeled A pre-mRNA was mixed with increasing concentrations of unlabeled B pre-mRNA and incubated in trans-splicing conditions. Lane 2 shows incubation reaction of labeled A pre-mRNA with 50 ng of B pre-mRNA without ATP. Lane 3 shows the incubation reaction with A pre-mRNA but without construct B. A pre-mRNA was incubated with increasing amounts of B pre-mRNA (10 ng, 25 ng, 50 ng, 100 ng, and 200 ng). The trans-splicing product (AxB) (lanes 4–8) was confirmed by RT-PCR and sequencing. The exon 2 band might correspond to the intermediate exon 2 without the attached intron.