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 micromol/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 micromol/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.