Pairwise Engineering of Tandemly Aligned Self-Splicing Group I Introns for Analysis and Control of Their Alternative Splicing.

Alternative splicing is an important mechanism in the process of eukaryotic nuclear mRNA precursors producing multiple protein products from a single gene. Although group I self-splicing introns usually perform regular splicing, limited examples of alternative splicing have also been reported. The exon-skipping type of splicing has been observed in genes containing two group I introns. To characterize splicing patterns (exon-skipping/exon-inclusion) of tandemly aligned group I introns, we constructed a reporter gene containing two Tetrahymena introns flanking a short exon. To control splicing patterns, we engineered the two introns in a pairwise manner to design pairs of introns that selectively perform either exon-skipping or exon-inclusion splicing. Through pairwise engineering and biochemical characterization, the structural elements important for the induction of exon-skipping splicing were elucidated.