UC San Diego

Group I introns are catalytic RNAs (ribozymes) capable of catalyzing their self-excision from precursor RNAs through two consecutive transesterification reactions. Although the ribozyme has evolved to perform this cis-splicing reaction in nature, man-made modifications to the 5' end of the ribozyme have allowed it to catalyze a trans- splicing reaction, in which it is able to replace the 3' portion of a substrate RNA with it's own 3' tail. The trans-splicing group I introns used in this thesis were variants of the Tth.L 1925 IC1 group I intron ribozyme found in Tetrahymena thermophila. The work contained in this dissertation aims to both utilize trans-splicing group I introns to further understand principles of RNA evolution, as well as develop and optimize a new trans- splicing variant of the ribozyme for future use in therapy. In the first study of this dissertation, a trans- splicing group I intron was used as a model system to examine the effect of selection pressure and recombination on evolving populations of RNA in a cellular environment. Four parallel evolutions were completed, two employing a low selection pressure, and two employing a high selection pressure. Ribozyme populations with higher efficiency, measured by cellular growth conferred by the ribozyme, resulted from evolutions performed at a low selection pressure. It was found that this increase in fitness was the result of a set of four mutations acting cooperatively. Fitness profiles of evolutionary intermediates revealed that a low selection pressure can increase the accessibility of evolutionary paths leading to the evolution of cooperative mutations. This finding not only adds to the understanding of natural RNA evolution, but also aids in the design of more efficient evolutions of RNA species. In the second study of this dissertation, a new trans-splicing variant of the group I intron was developed, capable of catalyzing the removal of internal sequences from pre-mRNA and joining the two flanking sequences, thereby generating a functional RNA. This group I intron has been termed the ̀spliceozyme' because its action is analogous to that of the spliceosome. The action performed by the spliceozyme give this system the ability to repair certain types of diseases caused by mis-splicing and therefore, the potential to be used therapeutically. To increase the efficiency and therapeutic potential of this system, the spliceozyme was evolved in E. coli cells, challenging it to more efficiently catalyze the removal of internal sequences. The most efficient variant contained a set of mutations resulting in increased product formation and decreased side product formation. This observed effect was seen in vitro, suggesting that this effect may increase spliceozyme efficiency in a range cell types. Future work will move this system into mammalian cells and optimize the spliceozyme for use in a mammalian system, thus developing it as a therapeutic tool