Abstract 2466: A novel role for the transcription factor SNAIL in alternative splicing

The transcription factor SNAIL is a master regulator of the Epithelial to Mesenchymal Transition (EMT), a hallmark event during cancer metastasis. SNAIL repressed transcription of several epithelial cell-specific genes such as E-cadherin, leading to a mesenchymal phenotype that is less attached and more migratory. As a DNA-binding transcription factor, SNAIL binds consensus E-box motifs in gene promoters, and recruits epigenetic modifiers to repress or activate gene expression. Previous studies from our lab and others have indicated that SNAIL and its family member SLUG have several gene targets in common but also bind to unique gene targets. Whether SNAIL or SLUG share protein-interacting partners is unknown. To identify interactome partners of SNAIL and SLUG, we performed co-immunoprecipitation followed by iTRAQ mass spectrometry. The protein interactome of SNAIL, but not SLUG, was highly enriched for splicing proteins, of which the top hit was the Serine-Arginine Rich-Splicing Factor, SRSF1. SRSF1 participates in regulation of alternative splicing, a mechanism through which a cell can generate multiple proteins from the same gene. Alternative splicing events play a central role in EMT progression, and SRSF1 is overexpressed in many cancers. We explored the role of SNAIL and SRSF1 in driving alternative splicing events during EMT. First, we confirmed the association of SNAIL and SRSF1 by co-immunofluorescence, PLA assay and co-immunoprecipitation experiments during TGF-beta induction of EMT. RNA-IP (RIP) experiments demonstrated association of SNAIL with mesenchymal specific but not epithelial-specific splice isoforms, further suggesting an RNA binding role for SNAIL. Lastly, RNA-EMSA experiments suggest specificity for some but not all four Zinc fingers of SNAIL in binding to RNA. Based on these observations, we hypothesize that SNAIL regulates alternative splicing events during EMT at least in part through association with SRSF1 on RNA. As transcription and splicing are closely coupled, we predict that the regulation of splicing by transcription factors through RNA binding is universally utilized by other biological systems to alter phenotype in response to external stimuli. We are currently working on identifying the genome-wide changes in alternative splicing regulated by SNAIL, and the mechanism by which SNAIL interacts with the spliceosome to regulate alternative splicing. Citation Format: Atrayee Ray, Smruthi Rudraraju, Shawn Krueger, Janani kumar