Most eukaryotic pre-mRNAs contain introns that must be efficiently and accurately spliced out to allow production of functional proteins. The splicing of many pre-mRNAs is further complicated in that the cell must distinguish not only between intron and exon sequences, but also between alternatively spliced exons. This proposal seeks to further understand the mechanisms by which alternatively spliced introns are removed using the a- tropomyosin (a-TM) gene as a model system. In particular, the focus will be on the regulated splicing of a-TM exons 2 and 3 where exon 3 is used in all cells except smooth muscle cells which retain exon 2. A repeated RNA sequence element surrounding exon 3 has been mapped that is responsible for negatively regulating the splicing of this exon in smooth muscle cells. This proposal seeks to determine the identity of this protein and determine its role in regulating alternative splicing. While it is possible that the protein will be specific to smooth muscle, it is also possible that previously identified splicing factors might affect the splicing of a-TM exon 3. Thus, four mammalian constitutive splicing factors (PTB, PSF, hnRNP A1, and U2AF) will be tested for their ability to alter the splicing of a-TM in vivo and in vitro. Each of these proteins binds the polypyrimidine tract which has been shown to be the major determinant in the default selection of exon 3. The binding of each of these proteins to the polypyrimidine tract will be determined as well as a functional analysis of polypyrimidine strength. For PSF, further domain analysis will be carried out to map the functional regions of this newly identified essential pre-mRNA splicing factor.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050418-02
Application #
2188258
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1994-08-01
Project End
1998-07-31
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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Dye, B T; Buvoli, M; Mayer, S A et al. (1998) Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2. RNA 4:1523-36
Buvoli, M; Mayer, S A; Patton, J G (1997) Functional crosstalk between exon enhancers, polypyrimidine tracts and branchpoint sequences. EMBO J 16:7174-83
Perez, I; McAfee, J G; Patton, J G (1997) Multiple RRMs contribute to RNA binding specificity and affinity for polypyrimidine tract binding protein. Biochemistry 36:11881-90
Perez, I; Lin, C H; McAfee, J G et al. (1997) Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo. RNA 3:764-78
Coolidge, C J; Seely, R J; Patton, J G (1997) Functional analysis of the polypyrimidine tract in pre-mRNA splicing. Nucleic Acids Res 25:888-96
Hirano, K; Erdodi, F; Patton, J G et al. (1996) Interaction of protein phosphatase type 1 with a splicing factor. FEBS Lett 389:191-4