The broad long-term goal of the proposed work is to understand regulated alternative splicing in mammals. We focus our study on the alternative splicing of fibroblast growth factor receptor type 2 (FGF-R2) transcripts. This alternative splicing results in the production of two FGF-R2 isoforms, FGF-R2(IIIb) and FGF-R2(IIIc), which have very different ligand binding properties. The choice of FGF-R2(IIIb) vs. FGF-R2(IIIc) is highly regulated during development and is deregulated during prostate tumor progression. In order to further our knowledge of this regulated alternative splicing event we propose to accomplish the following four aims:
Specific aim no. 1: Characterization of cis-acting elements required for silencing of FGFR2 exon Illb. Through mutational analysis we have identified two intronic splicing silencer (ISS) elements upstream and downstream of exon IIIb, UISS and DISS, respectively. Here we propose to characterize UISS and DISS in detail. In addition, we will investigate the functional and physical interactions between these two splicing silencer elements.
Specific aim no. 2: Identification and characterization of trans-acting factors that mediate FGF-R2 exon IIIb silencing. We propose that the polypyrimidine tract binding protein (PTB) mediates IIIb silencing via the ISS elements. In this application we propose to test this and to investigate the mechanism by which PTB exerts its effect. A structure-function analysis of PTB is proposed. If our data indicate the existence of factors that cooperate with PTB to mediate silencing, we will search for and identify these.
Specific aim no. 3. Characterization of the cis-elements required for cell-type specific FGF-R2 exon choice. We have previously identified cis-elements required for cell-type specific regulation of FGF-R2 splicing. Here, we propose to dissect the intronic splicing activator and repressor (ISAR) found in intron 8, which lies between exons IIIb and IlIc. We also propose to characterize rIAS2, a second element within intron 8, and its functional and physical interactions with TSAR.
Specific aim no. 4: Identification of trans-acting factors that mediate cell-type specific FGF-R2 exon choice. Two alternative routes to identify cell-type specific trans-acting factors will be undertaken. First, we propose a search for ISAR binding factors. Second, we propose to identify cell-type specific PTB-interacting proteins, which we posit are good candidates for splicing regulators. These PTB-interacting proteins may be involved in the specific regulation of PTB action, contributing either to exon IIIb silencing or activation.
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