Figure 2

Theophylline-dependent inhibition of pre-mRNA splicing is specific. (A) The diagram of MAdBPT15AG pre-mRNA. The nucleotide residues shown in bold have been mutated to prevent the binding of theophylline. The RNA secondary structure is adapted from Zucker's M-FOLD program [62] (see Additional File 2). (B) Theophylline failed to inhibit the splicing of a substrate (MAdBPT15AG) that contains mutations within aptamer core. ³²P-labeled MAdBPT15AG pre-mRNA was subjected to in vitro splicing in the absence (lane 2) or presence of theophylline (lanes 3) for 2 h. The extracted RNA was fractionated on a 13% polyacrylamide denaturing gel. The bands corresponding to splicing substrates, intermediates and spliced products are indicated on right. The % splicing calculated as in Fig. 1 and values are expressed as mean ± SEM. (C) AdT+10, a pre-mRNA in which theophylline-binding aptamer was inserted upstream of BPS was constructed as described in methods. ³²P-labeled AdT+10 pre-mRNA was subjected to in vitro splicing in the absence (lane 1) or presence of theophylline (lane 2) for 2 h. The extracted RNA was fractionated on a 13% polyacrylamide denaturing gel (details as in Fig. 1B).