Mobile genetic elements or transposons are found in the genomes of all organisms. These elements can move via DNA or RNA intermediates. About 50% of the human genome is made up of transposable elements with ~ 2.7% corresponding to DNA-based transposons. Many of these putative transposons or transposase-related genes are uncharacterized. Our previous studies have focused on the P element family of DNA transposons in Drosophila. P element transposase functions as a tetramer, using GTP as a cofactor for transposition. N-terminal domain of the transposase corresponds to a C2CH THAP DNA binding domain, which is a member of a prevalent family of DNA binding domains found exclusively in animal genomes. One THAP gene, called THAP9, is homologous to the Drosophila P element transposase and is present in primates, Xenopus, zebrafish and Ciona, but is absent from rodents. Recent work from our lab has shown that the human and zebrafish THAP9 genes can mobilize the Drosophila and zebrafish P element transposons in human and Drosophila cells. This proposal is focused on understanding what role the human THAP9 gene may play in human embryonic stem cells and how the Drosophila P element transposase protein recognizes and assembles with the transposon ends, donor DNA, target DNA and GTP/Mg2+ to form an active protein-DNA complex. These studies are aimed at gaining mechanistic insights. Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in metazoans and is a conduit through which genomic sequence is transferred to proteomic information. Most eukaryotic genes are split and have the potential for alternative splicing, dramatically increasing proteomic diversity. Many human and mouse disease gene mutations affect the splicing process. Splicing silencers are a major type of RNA control element generating tissue- or cell type-specific alternative splicing patterns. Our previous work has focused on characterization of the tissue-specific Drosophila P element pre-mRNA exonic splicing silencer element. Recent work from our group has focused on how the action of the RNA binding proteins, PSI and hrp48. Using this information, we want to identify new Drosophila cellular splicing silencer elements that are controlled by these two splicing factors. The PSI protein also interacts with U1 snRNP and PSI mutant Drosophila strains that abolish this interaction exhibit male courtship behavior defects and altered pre-mRNA splicing of the Drosophila male-specific fruitless pre-mRNA isoforms. We want to investigate how the PSI protein controls fruitless pre-mRNA splicing and how it controls binding of U1 snRNP on the Drosophila transcriptome. U1 snRNP has distinct roles in U1 snRNP binding sites in PCPA (premature cleavage and polyadenylation), splicing at intron 5' splice sites and at potential new splicing silencers.
Mobile genetic elements or transposons are found in the genomes of all organisms and 50% of the human genome is made up of transposable elements with ~ 2.7% corresponding to DNA-based transposons. Alternative pre-mRNA splicing is a common mechanism for regulating gene expression in metazoans. The protein factors involved in controlling DNA transposition and patterns of alternative splicing have homologs, so results obtained in this system will have direct relevance and application to human health.
