Heterodimerization of Group I Ribozymes Enabling Exon Recombination through Pairs of Cooperative trans‐Splicing Reactions

Abstract

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.

A Hexameric Ribozyme Nanostructure Formed by Double‐Decker Assembly of a Pair of Triangular Ribozyme Trimers

“…In addition to mutual assistance which ribozyme modules perform in assembly of the double‐decker hexamer, cooperative effects may be generated among ribozyme units to further improve their catalytic ability. Such effects have been implicated in the catalytic properties of dimeric forms of the Tetrahymena ribozymes although they exhibited positive and negative cooperativity depending on the structural design of dimeric ribozymes [31,32] . To elucidate the relationship among structure, assembly and catalytic reaction of ribozyme‐based nanostructure catalysts, further structural and biochemical analyses are needed.…”

“…Such effects have been implicated in the catalytic properties of dimeric forms of the Tetrahymena ribozymes although they exhibited positive and negative cooperativity depending on the structural design of dimeric ribozymes. [31,32] To elucidate the relationship among structure, assembly and catalytic reaction of ribozyme-based nanostructure catalysts, further structural and biochemical analyses are needed.…”

An RNA Triangle with Six Ribozyme Units Can Promote a Trans-Splicing Reaction through Trimerization of Unit Ribozyme Dimers

“…Plasmids encoding the sequences of αand β-chains of kUrd RNAs were derived in two steps from plasmids encoding chimeric constructs of the wild-type Tetrahymena group I intron and its M1 variant-type intron [31]. PCR-based mutagenesis was used in each step of the stepwise plasmid construction.…”

“…In the intron form of the M1 type ∆P5 core ribozyme module on the opposite side of kUrd, 5′ and 3′ exons were provided by β-and α-chain RNAs, respectively (Figure S6B). The organization of α-and β-chain RNAs in the intron form of the ∆P5 core ribozyme modules would conduct trans-splicing reaction joining 5′ and 3′ exons, which were provided by two separate RNA chains, to produce a mature exon sequence (Figure S10A,B) [31,48]. Trans-splicing promoted by group I ribozyme has been recognized as a promising strategy to repair mRNAs of disease-related genes bearing undesirable mutations [48][49][50].…”

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

“…The cooperative splicing reactions of two group I intron ribozymes have also attracted attention in RNA-based synthetic biology because they may be applicable to the regulation of gene expression and gene therapy [ 27 , 28 , 29 ]. In these studies, structural and/or sequence engineering of the Tetrahymena intron ribozyme was performed to install cooperative functions between two monomeric introns.…”