Study of the molecular mechanisms of alternative splicing. Identification of global changes in splicing during the epithelial-mesenchymal transition (EMT).
Alternative splicing, Post-transcriptional gene regulation, Exons, Introns, Epithelial Splicing Regulatory Proteins (ESRPs), Epithelial Mesenchymal Transition (EMT).
Description of Research
The focus of my laboratory is investigation of alternative splicing, whereby a single gene transcript can generate numerous spliced mRNAs, thereby greatly expanding ribonomic and proteomic diversity. We are particularly interested in the regulation of cell and tissue-specific splicing choices that are important during development and play roles in cancer progression. Our previous studies focused on alternative splicing of fibroblast growth factor receptor 2 (FGFR2) as a model system. Mutually exclusive splicing of two exons, IIIb and IIIc, gives rise to two functionally different receptors, FGFR2-IIIb and FGFR2-IIIc, in epithelial and mesenchymal cells, respectively. These exons encode the C-terminal half of an Ig-like domain in the receptor’s extracellular domain and the resulting receptor isoforms exhibit distinct binding preferences for the FGF family of ligands. The exquisite cell type-specific expression of these epithelial or mesenchymal specific splice variants is essential during vertebrate development. Furthermore, a switch in FGFR2 splicing occurs during the epithelial to mesenchymal transition (EMT), implying that this change in splicing is functionally involved in the EMT during development as well as in pathophysiologic conditions such as cancer metastasis and tissue fibrosis.
In collaboration with John Hogenesch in Pharmacology, we recently used luciferase-based splicing reporter assays to carry out a genome-wide, high throughput cDNA screen for factors that regulate FGFR2 splicing. These studies identified a number of novel mammalian splicing regulators, including two epithelial-specific factors that we named Epithelial Splicing Proteins 1 and 2 (ESRP1 and ESRP2). Expression of these splicing regulators is required for expression of the epithelial FGFR2 splice variant and ongoing work has shown that they regulate the splicing of an extensive epithelial-specific splicing program. These regulated targets include functionally relevant splicing switches that are implicated in the EMT and both ESRPs are transcriptionally inactivated during the EMT. We are currently carrying out massively parallel high throughput sequencing (RNA-seq) to identify an even more comprehensive epithelial splicing regulatory network (SRN). We predict that the proteins encoded by transcripts comprising this epithelial splicing signature will also define a protein interaction network that underlies important epithelial cell properties. Furthermore, we also have strong reasons to believe that, like FGFR2, many of these proteins will exhibit previously unrecognized isoform-dependent differences in function between epithelial and mesenchymal cells. These proteins and networks are likely to have biologically coherent functions that are relevant for the EMT in development and cancer metastasis as well as epithelial cell differentiation in diverse tissues and organs. In our ongoing studies we will further investigate the molecular mechanisms by which the ESRPs and several additional novel splicing regulators cause switches in splicing. We are also generating mice carrying conditional knockout alleles for Esrp1 and Esrp2 (and both) that will be used to create tissue-specific Esrp-knockout mice. These tools will allow us to clarify the roles of these splicing regulators during development and also allow us to profile ESRP-regulated splicing targets in vivo.
A number of potential projects to investigate the targets and functions of the ESRPs can be discussed. Such projects include biochemical characterization of the binding sites and mechanisms of function of these factors as well as work with the mouse knockout studies. We are also interested in performing similar studies with two other novel splicing regulators that we also recently identified. Projects to identify small molecules and compounds that regulate splicing using high throughput approaches are also available.
Tammy Yang-Graduate student
Kimberly Dittmar-Post-doctoral fellow
Laurie Heinicke-Post-doctoral fellow
Thomas Bebee-Post-doctoral Fellow
Katherine Sheridan-Research Specialist
Nick Rosculet-Undergraduate student
Dittmar, K. A.,Jiang, P.,Park, J. W., Amirikian, K., Wan, J., Shen, S.,Xing, Y., Carstens, R. P.: Genome-wide determination of a broad ESRP-regulated posttranscriptional network by high-throughput sequencing. Mol Cell Biol 32(8): 1468-1482, April 2012.
Warzecha, C.C.,Jiang, P.,Karine Amirikian,K., Dittmar,K.A.,Lu, H., Shen,S., Guo, W.,Xing,Y., and Carstens, R.P..: An ESRP-regulated splicing programme is abrogated during the Epithelial Mesenchymal Transition. EMBO Journal 29(19): 3286-3300, October 2010 Notes: Accompanied by a commentary in the same issue "A splicing mastermind for EMT" Tavanez, J.P. and Valcarcel, J. EMBO J. 2010 29(19):3286-3300.
Warzecha, C.C., Sato, T.K., Nabet, B., Hogenesch, J.B., and Carstens, R.P.: ESRP1 and ESRP2 are epithelial cell type-specific regulators of FGFR2 splicing. Molecular Cell 33(5): 591-601, March 2009 Notes: Accompanied by a preview in the subsequent issue "Getting under the skin of alternative splicing: identification of epithelial-specific splicing factors" Heyd, F and K.L. Lynch. Molecular Cell (2009) 33(6):674-676.
Warzecha, C.C., Shen, S., Xing, Y, and Carstens, R.P.: The epithelial splicing factors ESRP1 and ESRP2 positively and negatively regulate diverse types of alternative splicing events. RNA Biology 6(5): 546-62, November 2009.
Hovhannisyan, R.H. and Carstens, R.P.: Heterogeneous ribonucleoprotein M (Hnrnp M) is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons Journal of Biological Chemistry 282(50): 36265-36274, December 2007.
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Last updated: 02/11/2014
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