IN VIVO ANALYSIS OF METAZOAN TRNA INTRON SPLICING

Pre-tRNA processing is an essential step in generating a supply of functional mature tRNAs. In some instances, this processing event includes removal of an intron. Recent work from our lab has shown that these introns are cut out of pre-tRNAs and ligated into circular RNAs, called tricRNAs, in the fruit fly Drosophila melanogaster. To study the mechanism of tricRNA biogenesis in Drosophila, I generated a series of splicing reporters adapted for expression in human and fly cells. Using these reporters, I discovered specific cis-acting elements important for proper spicing, including a conserved base pair found in all metazoan pre-tRNAs to date. I also identified candidate tRNA processing factors in Drosophila via sequence homology to human factors, and verified these candidates using an in vivo cellular splicing assay. My results show that Drosophila use the “direct ligation”-type tRNA ligation pathway also found in archaea and human cells. I also examined Drosophila tRNA processing factors in animals. Using available stocks, I observed striking neurological phenotypes when mutating or depleting these enzymes. These data correlate with a known human disease called Pontocerebellar hypoplasia (PCH), caused by mutations in human tRNA processing factors. I further found a tissue-specific requirement for the CLP1 ortholog cbc in both viability and locomotion, consistent with previous data from a mouse model. In addition to my cellular and animal work, I also developed a method to express circular RNAs using tRNA splicing. This method utilizes standard molecular biology techniques and can generate circular RNAs for a variety of functions. There are many potential applications to this technology. My work is the first characterization of tricRNA splicing in a metazoan model organism. My analysis of cis-acting elements and trans-acting factors yielded findings that are consistent with previously published data. Furthermore, the method I have developed has a wide range of potential uses, and my analysis of processing factor mutants provides new context to understanding a human neurological disease. Overall, my investigation of tricRNA biogenesis adds a new dimension to an established field.Doctor of Philosoph