Increased efficiency of evolved group I intron spliceozymes by decreased side product formation

Abstract

The group I intron ribozyme from Tetrahymena was recently reengineered into a trans-splicing variant that is able to remove 100-nt introns from pre-mRNA, analogous to the spliceosome. These spliceozymes were improved in this study by 10 rounds of evolution in Escherichia coli cells. One clone with increased activity in E. coli cells was analyzed in detail. Three of its 10 necessary mutations extended the substrate binding duplexes, which led to increased product formation and reduced cleavage at the 5 ′ -splice site. One mutation in the conserved core of the spliceozyme led to a further reduction of cleavage at the 5 ′ -splice site but an increase in cleavage side products at the 3 ′ -splice site. The latter was partially reduced by six additional mutations. Together, the mutations increased product formation while reducing activity at the 5 ′ -splice site and increasing activity at the 3 ′ -splice site. These results show the adaptation of a ribozyme that evolved in nature for cis-splicing to transsplicing, and they highlight the interdependent function of nucleotides within group I intron ribozymes. Implications for the possible use of spliceozymes as tools in research and therapy, and as a model for the evolution of the spliceosome, are discussed.

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

“…Through the reaction, the 3'-exono r5 'exon sequence of the ribozymes can replacet he downstream or upstream sequence, respectively,o ft he target site in the given RNA. [16,17] These examples suggested that it might be possible to engineer self-splicinga nd related reactions of GI ribozymes to achieve complex editing of two or more RNA sequences in ac ooperative manner. [12][13][14] Ribozymes engineered for trans-excision splicing act like spliceosomes because they are designedt or ecognize and to splice given target RNA sequences as introns to be removed.…”

“…[8,15] Recently,M üller and co-workersd eveloped an improved system based on the Tetrahymena GI ribozyme,w hich achieved trans-excision splicing in E. coli cells. [16,17] These examples suggested that it might be possible to engineer self-splicinga nd related reactions of GI ribozymes to achieve complex editing of two or more RNA sequences in ac ooperative manner.…”

Engineering of hairpin ribozyme variants for RNA recombination and splicing

“…The spliceosome and self‐splicing group II intron ribozymes share a common ancestor, suggesting that a ribozyme similar to today's self‐splicing group II introns performed analogous functions to today's spliceosomes. There are some reports in the literature on the engineering of spliceozymes based on the even simpler structure of group I introns . However, earlier stages of the RNA world may have relied on even simpler RNA structures than those of group I introns to mediate RNA splicing.…”

“…There are some reports in the literature on the engineering of spliceozymes based on the even simpler structure of group I introns. [47][48][49][50] However, earlier stages of the RNA world may have relied on even simpler RNA structures than those of group I introns to mediate RNA splicing. As described above, the hairpin ribozyme can efficiently support both phosphodiester bond cleavage and ligation and therefore again is a perfect candidate for a simple ancient spliceozyme.…”

Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

“…A trans -splicing group I intron ribozyme acting on two splice sites, a spliceozyme [ 8 ], was evolved in E. coli cells recently [ 68 ]. Following the lessons learned from previous studies, the most efficient splice site 258 was chosen as target site on CAT mRNA [ 8 , 51 ].…”