Exploring intron mobilization and detection of an intron gain event via intron transposition using a novel intron gain and loss reporter
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Eukaryotic nuclear genes are discontinuous with the presence of intervening sequences termed spliceosomal introns. Once the DNA coding sequences are transcribed into pre-mRNA, these spliceosomal introns must be removed within the ribonucleoprotein complex called the spliceosome. The processed mRNA is exported from nucleus to cytoplasm where it is translated into protein. Therefore the removal of spliceosomal introns, pre-mRNA splicing, is an essential process for appropriate gene expression in eukaryotes. Given the importance of pre-mRNA splicing and diversity of intron densities across eukaryotic genomes, numerous studies have been conducted to understand the origin and evolution of spliceosomal introns. Although several models based on the phylogenetic analyses exist which address the molecular mechanism of the intron gain and loss, validation of these models is restricted due to the lack of experimental evidence. In this dissertation, we report the use of a novel strategy which detects selected intron gain and loss events. Our reporter is designed to produce an intronic RNA containing a selectable marker that detects its incorporation into the yeast genome. We have experimentally verified the first demonstration of intron gain via intron transposition in any organism. The intron RNA derived from the reporter was perfectly transposed in the yeast gene RPL8B and remains stable and intact within the genome. This novel allele, RPL8Bint, is functional when overexpressed in a deletion strain of RPL8A, a paralog of RPL8B, demonstrating that the newly formed intron is successfully removed by the spliceosome. To address the mechanism of this intron transposition, we investigated the involvement of the known cellular genes in intron transposition using the intron gain and loss reporter. A number of deletion strains of the spliceosome-related genes and recombination-related genes were employed in addition to the conditional mutants of splicing helicases. The results from these mutational analyses provided evidence to further understand the mechanism of intron mobilization with highlighting the importance of RAD52 and Ty transposable elements. Altogether this dissertation describes the development and validation of a novel reporter detecting in vivo intron gain and loss and the utilization of the reporter in understanding the mechanism of intron mobilization in S. cerevisiae.
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