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.

Heterodimerization of Group†I Ribozymes Enabling Exon Recombination through Pairs of Cooperative trans-Splicing Reactions.

Group I (GI) self-splicing ribozymes are attractive tools for biotechnology and synthetic biology. Several trans-splicing and related reactions based on GI ribozymes have been developed for the purpose of recombining their target mRNA sequences. By combining trans-splicing systems with rational modular engineering of GI ribozymes it was possible to achieve more complex editing of target RNA sequences. In this study we have developed a cooperative trans-splicing system through rational modular engineering with use of dimeric GI ribozymes derived from the Tetrahymena group I intron ribozyme. The resulting pairs of ribozymes exhibited catalytic activity depending on their selective dimerization. Rational modular redesign as performed in this study would facilitate the development of sophisticated regulation of double or multiple trans-splicing reactions in a cooperative manner.