Analysis of group I intron splicing in the presence of naturally occurring methylxanthines

Recent advances in the understanding of RNA structure–function, intricate folding and its affinity to bind small molecules have led to the proposal that RNA can be a fastidious target for drug design. The revelation that RNA can act as enzymes as in group I intron and that has been recognized by small molecule ligands targeting the catalytic activity has necessitated our focus on group I intron as target for RNA binders. We studied the group I intron splicing of Tetrahymena in the presence of naturally occurring methylxanthines (theophylline, theobromine and caffeine) at 5–200 μmol/l concentration, and analyzed the spliced out products. For the first time the interference of splicing was ascertained on the basis of pre-rRNA accumulation. The gel mobility shift showed the binding of methylxanthines with group I intron RNA in a dose dependent manner. The densitometric analysis of pre-rRNA accumulation showed 50% of splicing interference at 200 μmol/l of theophylline and theobromine, whereas the structurally similar molecule caffeine does not alter splicing. The splicing interference measured from the accumulation of pre-rRNA in group I intron splicing is considered to be an uncomplicated or simple denominator for calculating the splicing interference or relative splicing activity in the presence of above RNA binders or splicing modulators.