Alternative splicing of FGFR2 exons IIIb and IIIc results in the cell type-specific expression of the FGFR2-IIIb isoform in epithelial cells and FGFR2-IIIc in mesenchymal cells and this splicing choice is essential during development. We previously hypothesized an epithelial cell-type specific splicing program in which FGFR2 and other transcripts is coordinated by epithelial-specific splicing regulators. In the first successful use of a genome-wide, high throughput cDNA expression screen for splicing factors, we discovered essential epithelial-specific FGFR2 splicing regulators and named them Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2). Ectopic expression of either protein in mesenchymal cells switches FGFR2 splicing to the epithelial pattern, whereas depletion of both factors in epithelial cells via RNA interference has the opposite effect. Further identification of CD44, ENAH, and p120-ctn as splicing targets of ESRP1 and ESRP2 implicates these proteins as the regulators of a broader epithelial post-transcriptional gene expression program. We will further characterize the mechanisms by which the ESRPs regulate splicing of FGFR2 and identify additional co-regulated alternatively spliced transcripts that constitute an epithelial-specific splicing signature. Expression of both ESRP1 and ESRP2 is turned off during the Epithelial Mesenchymal Transition (EMT) and therefore we will also investigate the role of ESRP downregulation and the corresponding changes in splicing of co-regulated transcripts that contribute to the EMT.
Specific Aim Number 1. Complete the identification of FGFR2 splicing regulators using established high throughput, cell-based splicing assays. We will complete genome-wide, high-throughput screens to establish a comprehensive set of splicing regulators that cooperate with the ESRPs in the general epithelial splicing program.
Specific Aim Number 2. Investigate the mechanisms by which ESRP1 and ESRP2 regulate epithelial cell type-specific splicing. The molecular mechanisms by which the ESRPs regulate splicing will be investigated through the identification of ESRP binding sites, essential protein domains, and functionally relevant protein-protein interactions.
Specific Aim Number 3. Identify an epithelial cell type-specific splicing signature of co- regulated alternative splicing events that are regulated by the ESRPs. Using splicing sensitive microarrays we will define a comprehensive set of biologically coherent transcripts that are co-regulated by the ESRPs in addition to FGFR2, CD44, ENAH, and p120-Ctn.
Specific Aim Number 4. Investigate the role of the ESRPs in the Epithelial Mesenchymal Transition (EMT). We will determine whether the loss of ESRPs expression is required for the EMT to occur and whether ESRP expression sufficient to induce a Mesenchymal to Epithelial Transition (MET).

Public Health Relevance

Alternative splicing is a fundamentally important process in which the same gene can generate different proteins that have different functions, and alterations in this process are implicated in numerous diseases, including cancer. We have identified an essential alternative splicing regulatory protein that is expressed in a specific cell type, the epithelial cell, and which is required for development of many organs. The loss of expression of this gene may be one process that contributes to the invasion and metastasis of many cancers.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cancer Molecular Pathobiology Study Section (CAMP)
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Haynes, Susan R
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University of Pennsylvania
Internal Medicine/Medicine
Schools of Medicine
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Cieply, Benjamin; Carstens, Russ P (2015) Functional roles of alternative splicing factors in human disease. Wiley Interdiscip Rev RNA 6:311-26
Bebee, Thomas W; Park, Juw Won; Sheridan, Katherine I et al. (2015) The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development. Elife 4:
Bhate, Amruta; Parker, Darren J; Bebee, Thomas W et al. (2015) ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation. Nat Commun 6:8768
Bebee, Thomas W; Cieply, Benjamin W; Carstens, Russ P (2014) Genome-wide activities of RNA binding proteins that regulate cellular changes in the epithelial to mesenchymal transition (EMT). Adv Exp Med Biol 825:267-302
Carstens, Russ P (2014) Networking in an alternative splicing world. Mol Cell 54:903-4
Heinicke, Laurie A; Nabet, Behnam; Shen, Shihao et al. (2013) The RNA binding protein RBM38 (RNPC1) regulates splicing during late erythroid differentiation. PLoS One 8:e78031
Lu, Hezhe; Liu, Jianglan; Liu, Shujing et al. (2013) Exo70 isoform switching upon epithelial-mesenchymal transition mediates cancer cell invasion. Dev Cell 27:560-73
Dittmar, Kimberly A; Jiang, Peng; Park, Juw Won et al. (2012) Genome-wide determination of a broad ESRP-regulated posttranscriptional network by high-throughput sequencing. Mol Cell Biol 32:1468-82
Shen, Shihao; Park, Juw Won; Huang, Jian et al. (2012) MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-Seq data. Nucleic Acids Res 40:e61
Warzecha, Claude C; Carstens, Russ P (2012) Complex changes in alternative pre-mRNA splicing play a central role in the epithelial-to-mesenchymal transition (EMT). Semin Cancer Biol 22:417-27

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