| NSF Org: |
MCB Division of Molecular and Cellular Biosciences |
| Recipient: |
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| Initial Amendment Date: | July 2, 1999 |
| Latest Amendment Date: | April 3, 2001 |
| Award Number: | 9904565 |
| Award Instrument: | Continuing Grant |
| Program Manager: |
Susan Porter Ridley
MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences |
| Start Date: | September 1, 1999 |
| End Date: | August 31, 2002 (Estimated) |
| Total Intended Award Amount: | $405,000.00 |
| Total Awarded Amount to Date: | $410,000.00 |
| Funds Obligated to Date: |
FY 2000 = $140,000.00 FY 2001 = $140,000.00 |
| History of Investigator: |
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| Recipient Sponsored Research Office: |
10900 EUCLID AVE CLEVELAND OH US 44106-4901 (216)368-4510 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
10900 EUCLID AVE CLEVELAND OH US 44106-4901 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | EUKARYOTIC GENETICS |
| Primary Program Source: |
01000102DB NSF RESEARCH & RELATED ACTIVIT app-0199 |
| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.074 |
ABSTRACT
The long term goal of this work is to understand the molecular mechanisms that regulate alternative RNA splicing in a multicellular organism. The question of how splicing is regulated is particularly important because alternative splicing has proven to be a common means of regulating gene expression. Here the sex-specific splicing regulation of the Drosophila sex determination gene Sex-lethal (Sxl) is used as a model system. In this award, the focus is on the role of the sans-fille (snf) gene in promoting female-specific splicing of the Sxl pre-mRNA. Genetic studies have established that the non-sex specific snf gene product functions with the female-specific SXL RNA binding protein to direct splicing of the Sxl pre-mRNA. Remarkably, cloning of the snf gene identified it as the homolog of two well studied human spliceosomal proteins, UlA and U2B". Like UlA, SNF is a Ul snRNP protein. And, like U2B", SNF is a U2 snRNP protein.s. This provides the first example of an snRNP protein that plays a role in regulating RNA splicing. The specific aims are: 1. Determine if SNF's presence in the U2 snRNP is both necessary & sufficient for Sxl splicing regulation. We have established that the snf5MER allele, encodes a mutant protein that preferentially incorporates in the Ul snRNP and is not detected in the U2 snRNP in vivo. Our characterization of this mutant phenotype is consistent with the hypothesis that SNF's role Sxl splicing regulation requires that it be a component of the U2 snRNP. Here we propose to: a) Continue our biochemical characterization of the snf5MER allele to establish which properties of the protein are necessary for its function in Sxl splicing regulation.b) Test the hypothesis that SNF's presence in the U2 snRNP is sufficient for Sxl splicing regulation by making directed snf mutations that are predicted to preferentially incorporates into the U2 snRNP's and be left out of U1 snRNPs. If U2 snRNP incorporation is sufficient then we expect that these new mutant snf alleles will not be defective in Sxl splicing regulation. 2. Identify the protein domains of SNF necessary for Sxl splicing regulation. We have established that the C-terminal RRM, while not essential for snf+ function can compensate for mutations in the N-termin`us. Preliminary studies, however, suggest that the N-terminal RRM alone is not sufficient for snf+ function. Here we propose to: Identify critical functional domains of the protein by making transgenic animals that express the minimum N-terminal sequences sufficient for both snf's vital function and its sex determination function. As part of this characterization, we will need to generate antibodies that recognize the N-terminal end of the protein and use these antibodies to determine whether the new SNF mutant proteins are incorporated into Ul and/or U2 snRNPs. 3. Identify and analyze other gene products that function in Sxl splicing autoregulation. To continue to elucidate the mechanism by which Sxl splicing is regulated, we propose to identify other critical gene products that function with SNF in regulating Sxl splicing. To accomplish this: a) we have used a dosage-sensitive modifier assay to survey known splicing factors and we have identified U2AF35 and hel (the UAP56 homolog) as likely players in Sxl splicing regulation. In mammalian cells, both of these gene products are required for the recruitment of the U2 snRNP to the pre-mRNA, suggesting that the genetic interaction is due to a direct effect on Sxl splicing. Here we propose to test this hypothesis by determining if either or both these proteins interact with SNF, either directly or indirectly, as a member of the SXL containing complex. b) we are using the yeast two-hybrid system to identify proteins that interact with SNF. We will confirm the in vivo relevance of these interactions by both biochemical assays and genetic interaction tests.
The studies described in this award exploit the power of Drosophila genetics to gained from the experiments in this award will provide critical information on two understand the function of this conserved snRNP protein in vivo. The information fronts. First, we will add to our understanding of how a general splicing factor (SNF) and a tissue-specific splicing factor (SXL) function together to control RNA splicing. Second, we will contribute to our overall understanding of how the different protein components of the spliceosome contribute to the accuracy of splicing. Given the evolutionary conservation of the components of the RNA splicing machinery, the information gained from our studies will be directy applicable to mammalian systems and will be a significant advance in our understanding of regulated pre-mRNA splicing.
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