The novel splicing factors ESRP1 and ESRP2 orchestrate an epithelial cell type-specific alternative splicing network

Growing evidence suggests post-transcriptional processing events have a significant role in the regulation of critical gene expression programs. Alternative splicing represents one mechanism of achieving coordinated changes in gene expression through RNA binding proteins that bind and regulate the splicing of a host of transcripts in different cell types or cellular milieus. Although alternative splicing mechanisms have been studied extensively, only recently have we begun to appreciate the full diversity and complexity of alternative splicing events and the protein isoforms generated during these events. Furthermore, satisfactory models to explain regulated alternative splicing requires the discovery of splicing regulators with more limited expression than most defined to date. This thesis describes the use of several novel genome-wide strategies to study the alternative splicing patterns present in epithelial and mesenchymal cell types. We developed a luciferase-based splicing reporter that was used as a readout in a high-throughput, cell-based cDNA expression screen to identify factors that promote splicing of the epithelial splice variant of the FGFR2 gene. Among the novel splicing regulators identified in this screen were epithelial cell type-specific regulators of FGFR2 alternative splicing, which we named Epithelial Splicing Regulatory Protein 1 and 2 (ESRP1 and ESRP2). We then used a combination of splicing sensitive exon microarrays and exon junction microarrays to determine if the ESRPs regulate a broader network of epithelial-specific alternative splicing events. These studies revealed that the ESRPs regulate the splicing of several hundred candidate transcripts, including all known types of alternative splicing events. Many of the regulated transcripts encode proteins involved in biologically coherent processes. Most importantly, we discovered that loss of ESRP expression during the epithelial-to-mesenchymal transition (EMT) is accompanied by a change in splicing of ESRP target genes. We propose that the ESRPs regulate the expression of functionally distinct epithelial versus mesenchymal splicing isoforms that are critical in defining cell morphology and behavior during the EMT as it pertains to both normal development and as a hallmark of cancer metastasis.