An ESRP-regulated splicing program is abrogated during the Epithelial Mesenchymal Transition

Alternative splicing achieves coordinated changes in post-transcriptional gene expression programs through the activities of diverse RNA binding proteins. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are cell type-specific regulators of transcripts that switch splicing during the Epithelial Mesenchymal Transition (EMT). To define a comprehensive program of alternative splicing that is regulated during the EMT, we identified an extensive ESRP-regulated splicing network of hundreds of alternative splicing events within numerous genes with roles in cell-cell adhesion, polarity, and migration. Loss of this global ESRP-regulated epithelial splicing program induces the phenotypic changes in cell morphology that are observed during the EMT. Components of this splicing signature provide novel molecular markers that can be used to characterize the EMT. Bioinformatics and experimental approaches revealed a high affinity ESRP binding motif and a predictive RNA map that governs their activity. This work establishes the ESRPs as coordinators of a complex alternative splicing network that adds an important post-transcriptional layer to the changes in gene expression that underlie epithelial-mesenchymal transitions during development and disease. Keywords: control / knockdown comparison and control / ectopic expression comparison siRNA knockdown of ESRP1 and ESRP2 in human prostate epithelial cells PNT2 and ectopic expression of mEsrp1 in human breast cancer cells MDA-MB-231 as described before (Warzecha et al., 2009, Molecular Cell 33:591 - 601). The efficiency of ESRP knockdown and ectopic expression was monitored by western blot using a monoclonal antibody which recognizes both proteins (Clone 23A7) prepared by Rockland Immunochemicals, Inc. (Gilbertsville, PA) using our GST-Esrp1 recombinant protein. We conducted HJAY exon and exon junction array profiling on RNAs from four siESRP1 + siESRP2 treated PNT2 samples vs. four siGFP treated PNT2 samples and four samples of MDA-MB-231 cells transduced with retrovirus expressing a cDNA for mEsrp1 vs. four samples of MDA-MB-231 cells transduced with retrovirus expressing a control empty vector.