The broad long-term goal of the proposed work is to understand regulated alternative splicing in mammals. Many important questions remain to be answered in this area of RNA biology: What is the full encyclopedia of cis-acting elements and trans-acting factors that regulate splicing? How are alternative decisions coordinated to create networks of co-regulated transcripts? What are the molecular mechanisms by which exons are activated or silenced? How do mammals use these mechanisms to create tissue, organ and state-specific regulation of splicing? In this proposal we focus our study on the alternative splicing events that lead to the production of the IIIb and IIIc isoforms of the fibroblast growth factor receptor-2 (FGFR2). The choice between these isoforms is tightly regulated during development and is altered during prostate cancer progression. In order to understand this alternative splicing decision, and by analogy many other like-decisions, we propose the following three aims:
Specific aim no. 1: Characterization of the silencing of FGFR2 exon Illb. We have identified the cis-elements required for exon IIIb silencing and we have identified PTB as a trans-acting factor critical for this silencing. Here we propose to complete the characterization of the trans-acting factors that mediated this effect. We begin with a global analysis of PTB function to discover exons that may be silenced similarly to exon Illb. In the case of FGFR2, PTB does not act alone, however, and we intend to identify other trans-acting factors that collaborate with it to mediate silencing. Finally we propose to finish ongoing studies that examine exon IIIb silencing in tissues and organs.
Specific aim no. 2: Characterization of the mechanism for FGFR2 exon choice in epithelial cells. We propose to complete the characterization of trans-acting factors and to unravel the mechanism by which these activate exon Illb and repress exon IIIc in epithelial cells. We will focus on the role of homologues of the FOX-1 protein, which can activate exon Illb and repress exon IIIc.
Specific aim no. 3: Identification and characterization of cis-elements and trans-acting, factors that mediate activation of exon IIIc in mesenchymal cells. We will characterize the ISE required for exon IIIc inclusion. We will identify the trans-acting factors required for this inclusion using conventional biochemical methods and a new screen using siRNA libraries and fluorescence-based reporters of alternative splicing.
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