Figure 1

Insertion of BPS within theophylline-responsive riboswitch confers ligand-dependent control of splicing. (A) Schematic diagram of AdBPT pre-mRNAs. Underlined sequence and encircled adenosine residue represents the BPS and branch nucleotide, respectively. Open boxes represent exon sequences and horizontal lines between exons indicate introns. Arrow specifies 5' and 3' splice sites. The boxed residues within the secondary structure of theophylline binding aptamer are conserved for theophylline binding. The numbering system follows according to the reference [29]. (B) In vitro splicing of AdML 21AG and AdBPT pre-mRNAs. ³²P-labeled pre-mRNAs were incubated in HeLa nuclear extracts at 30°C for 2 h in the absence or presence of theophylline (for experimental details, see methods section). Total RNA isolated from each reaction was fractionated on a 13% polyacrylamide denaturing gel. The bands corresponding to pre-mRNA substrates, intermediates and spliced products are indicated on the right. M, Century™-plus RNA size marker (Ambion). Asterisk (*) indicates degraded lariat. (C) Histogram depicting the effect of theophylline on the splicing of AdML 21AG and AdBPT pre-mRNAs. Splicing was calculated as the ratio of spliced mRNA to the total and normalized to the control (no theophylline). Data represent mean ± standard error of the mean (SEM) of three independent experiments. Asterisk represents significant change as compared to the control (*, P < 0.0005). (D) Theophylline inhibits the splicing of AdBPT15AG pre-mRNA in a dose-dependent manner. ³²P-labeled AdBPT15AG pre-mRNA was incubated in HeLa nuclear extracts in the absence or with increasing concentration of theophylline for 2 h as described in (B). % Splicing calculated as described above and plotted against theophylline (mM). The values are expressed as mean ± SEM.