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

Group I introns are pre-mRNA introns that do not require the spliceosome for their removal. Instead, they fold into complex three-dimensional structures and catalyze two transesterification reactions, thereby excising themselves and joining the flanking exons. These catalytic RNAs (ribozymes) have been modified previously to work in trans, whereby the ribozymes can recognize a splice site on a substrate RNA and replace the 5- or 3-portion of the substrate. Here we describe a new variant of the group I intron ribozyme from Tetrahymena that recognizes two splice sites on a substrate RNA, removes the intron sequences between the splice sites, and joins the flanking exons, analogous to the action of the spliceosome. This group I spliceozyme functions in vitro and in vivo, and it is able to mediate a growth phenotype in E. coli cells. The intron sequences of the target pre-mRNAs are constrained near the splice sites but can carry a wide range of sequences in their interior. Because the splice site recognition sequences can be adjusted to different splice sites, the spliceozyme may have the potential for wide applications as tool in research and therapy.

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 trans-splicing, 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.

Understanding the evolution of functional RNA molecules is important for our molecular understanding of biology. Here we tested experimentally how two evolutionary parameters, selection pressure and recombination, influenced the evolution of an evolving RNA population. This was done using four parallel evolution experiments that employed low or gradually increasing selection pressure, and recombination events either at the end or dispersed throughout the evolution. As model system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 rounds of selection and amplification, including mutagenesis and recombination. The low selection pressure resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombination did not have a strong effect. Five mutations were responsible for the highest efficiency. The first mutation swept quickly through all four evolving populations, whereas the remaining four mutations accumulated later and more efficiently under low selection pressure. To determine why low selection pressure aided this evolution, all evolutionary intermediates between the wild type and the 5-mutation variant were constructed, and their activities at three different selection pressures were determined. The resulting fitness profiles showed a high cooperativity among the four late mutations, which can explain why high selection pressure led to inefficient evolution. These results show experimentally how low selection pressure can benefit the evolution of cooperative mutations in functional RNAs.

Group I introns are pre-mRNA introns that do not require the spliceosome for their removal. Instead, they fold into complex three-dimensional structures and catalyze two transesterification reactions, thereby excising themselves and joining the flanking exons. These catalytic RNAs (ribozymes) have been modified previously to work in trans, whereby the ribozymes can recognize a splice site on a substrate RNA and replace the 5'- or 3'-portion of the substrate. Here we describe a new variant of the group I intron ribozyme from Tetrahymena that recognizes two splice sites on a substrate RNA, removes the intron sequences between the splice sites, and joins the flanking exons, analogous to the action of the spliceosome. This 'group I spliceozyme' functions in vitro and in vivo, and it is able to mediate a growth phenotype in E. coli cells. The intron sequences of the target pre-mRNAs are constrained near the splice sites but can carry a wide range of sequences in their interior. Because the splice site recognition sequences can be adjusted to different splice sites, the spliceozyme may have the potential for wide applications as tool in research and therapy.