The overall objective of the research proposed in this application is to elucidate molecular mechanisms used to regulate pre-mRNA splicing during development of a higher metazoa organism using a combination of molecular biology and biochemistry. The major portion of the applicant's efforts will be focused on understanding the biochemical mechanisms governing the tissue-specific pre-mRNA splicing of the P transposable element third intron (IVS3) in Drosophila melanogaster. It is anticipated that the results obtained from these studies will be useful in understanding mechanisms governing alternative pre-mRNA splicing events during human as well as Drosophila development. In order to accomplish the applicant's overall objectives, the applicant will: (A). Refine biochemical reconstitution assays to study the somatic repression of P element IVS3 pre-mRNA splicing in vitro; (B). Analyze RNA binding proteins that interact with P-element IVS3 and flanking exons; (C). Develop methods for the preparative purification of eukaryotic RNA binding proteins; (D). Characterize the 65kD Drosophila RNA binding protein, determine whether it interacts with 5' splice site sequences, and whether it functions as a splicing factor; (E). Purify and characterize the 97kK Drosophila RNA binding protein that specifically recognized the IVS3 5' exon and may be involved in inhibitory splicing of IVS3 pre-mRNA in somatic cells; (F). Analyze a variety of mutant IVS3 derivatives to test hypotheses regarding the mechanism of somatic IVS3 pre-mRNA splicing inhibition; (G). Attempt to identify other Drosophila genes whose pre-mRNA splicing patterns are regulated in a tissue-specific manner like P-element IVS3; (H). Attempt to develop Drosophila germ cell (pole cell) or ovarian cell lines to identify factors that may activate IVS3 pre-mRNA splicing in the germline.
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