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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD028063-04
Application #
3329685
Study Section
Molecular Biology Study Section (MBY)
Project Start
1991-07-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Earth Sciences/Natur
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Labourier, E; Rio, D C (2001) Purification of Drosophila snRNPs and characterization of two populations of functional U1 particles. RNA 7:457-70
Crowder, S M; Kanaar, R; Rio, D C et al. (1999) Absence of interdomain contacts in the crystal structure of the RNA recognition motifs of Sex-lethal. Proc Natl Acad Sci U S A 96:4892-7
Rudner, D Z; Kanaar, R; Breger, K S et al. (1998) Interaction between subunits of heterodimeric splicing factor U2AF is essential in vivo. Mol Cell Biol 18:1765-73
Rudner, D Z; Breger, K S; Rio, D C (1998) Molecular genetic analysis of the heterodimeric splicing factor U2AF: the RS domain on either the large or small Drosophila subunit is dispensable in vivo. Genes Dev 12:1010-21
Lee, A L; Volkman, B F; Robertson, S A et al. (1997) Chemical shift mapping of the RNA-binding interface of the multiple-RBD protein sex-lethal. Biochemistry 36:14306-17
Adams, M D; Tarng, R S; Rio, D C (1997) The alternative splicing factor PSI regulates P-element third intron splicing in vivo. Genes Dev 11:129-38
Hammond, L E; Rudner, D Z; Kanaar, R et al. (1997) Mutations in the hrp48 gene, which encodes a Drosophila heterogeneous nuclear ribonucleoprotein particle protein, cause lethality and developmental defects and affect P-element third-intron splicing in vivo. Mol Cell Biol 17:7260-7
Kanaar, R; Lee, A L; Rudner, D Z et al. (1995) Interaction of the sex-lethal RNA binding domains with RNA. EMBO J 14:4530-9
Siebel, C W; Admon, A; Rio, D C (1995) Soma-specific expression and cloning of PSI, a negative regulator of P element pre-mRNA splicing. Genes Dev 9:269-83
Siebel, C W; Kanaar, R; Rio, D C (1994) Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI. Genes Dev 8:1713-25

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