Determination of a comprehensive alternative splicing regulatory network and the combinatorial regulation by key factors during the epithelial to mesenchymal transition

The epithelial to mesenchymal transition (EMT) is a process by which epithelial cells transdifferentiate into mesenchymal cells. It is essential for embryonic development and implicated in cancer metastasis. While the transcriptional regulation of EMT has been well-studied, the role of post-transcriptional regulation, particularly alternative splicing (AS) regulation in EMT, remains relatively uncharacterized. We previously showed that the epithelial cell-type-specific proteins ESRP1 and ESRP2 are important for regulation of many AS events that altered during EMT. However, the contributions of the ESRPs and other splicing regulators to the splicing regulatory network in EMT require further investigation. In the first part of my thesis, we used a robust in vitro EMT model to comprehensively characterize splicing switches during EMT in a temporal manner. These investigations revealed that the ESRPs are responsible for a large number of AS events during EMT. We determined that RBM47 transcript is down-regulated during EMT and RBM47 depletion regulates many EMT-associated AS events. We also determined that Quaking (QKI) broadly promotes mesenchymal splicing patterns for numerous EMT-associated AS events. Our study highlights the broad role of post-transcriptional regulation and the important role of combinatorial regulation by different splicing factors to fine tune gene expression programs during the EMT. In the second part of my thesis, we determined that Esrp1 generates both nuclear and cytoplasmic isoforms, due to the use of two competing alternative 5’ splice sites in exon 12. We carried out a detailed characterization of the Esrp1 nuclear localization signal (NLS) that represents a novel class of NLS. Furthermore, we identified splice variants encoding nuclear and cytoplasmic isoforms of Fusilli, the Esrp1 orthologue in D. Melanogaster. Our observations demonstrate that the production of both nuclear and cytoplasmic Esrp1 isoforms through alternative splicing is highly conserved among species, strongly suggesting it’s biologically significant. Thus, while the first part of thesis has characterized extensive regulation by nuclear Esrp1 to promote epithelial splicing patterns, it will be of great interest to study the contribution of cytoplasmic Esrp1 in the maintenance of epithelial cell functions