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Molecular and Cellular Biology, October 2000, p. 7388-7400, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

An Intronic Splicing Silencer Causes Skipping of the IIIb Exon of Fibroblast Growth Factor Receptor 2 through Involvement of Polypyrimidine Tract Binding Protein

Russ P. Carstens,1,2,3,dagger Eric J. Wagner,4 and Mariano A. Garcia-Blanco1,3,5,*

Department of Genetics,1 Division of Nephrology,2 Department of Medicine,3 Department of Microbiology,5 and Program in Molecular Cancer Biology,4 Duke University Medical Center, Durham, North Carolina 27710

Received 14 March 2000/Returned for modification 17 April 2000/Accepted 29 June 2000

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) transcripts involves the mutually exclusive usage of exons IIIb and IIIc to produce two different receptor isoforms. Appropriate splicing of exon IIIb in rat prostate cancer DT3 cells requires a previously described cis element (ISAR, for "intronic splicing activator and repressor") which represses the splicing of exon IIIc and activates the splicing of exon IIIb. This element is nonfunctional in rat prostate AT3 cells, which repress exon IIIb inclusion and splice to exon IIIc. We have now identified an intronic element upstream of exon IIIb that causes repression of exon IIIb splicing. Deletion of this element abrogates the requirement for ISAR in order for exon IIIb to be spliced in DT3 cells and causes inappropriate inclusion of exon IIIb in AT3 cells. This element consists of two intronic splicing silencer (ISS) sequences, ISS1 and ISS2. The ISS1 sequence is pyrimidine rich, and in vitro cross-linking studies demonstrate binding of polypyrimidine tract binding protein (PTB) to this element. Competition studies demonstrate that mutations within ISS1 that abolish PTB binding in vitro alleviate splicing repression in vivo. Cotransfection of a PTB-1 expression vector with a minigene containing exon IIIb and the intronic splicing silencer element demonstrate PTB-mediated repression of exon IIIb splicing. Furthermore, all described PTB isoforms were equally capable of mediating this effect. Our results support a model of splicing regulation in which exon IIIc splicing does not represent a default splicing pathway but rather one in which active repression of exon IIIb splicing occurs in both cells and in which DT3 cells are able to overcome this repression in order to splice exon IIIb.

* Corresponding author. Mailing address: Department of Genetics, Box 3053, Duke University Medical Center, Durham, NC 27710. Phone: (919) 613-8632. Fax: (919) 613-8646. E-mail: garci001{at}mc.duke.edu.

dagger Present address: University of Pennsylvania, Philadelphia, PA 19104.

Molecular and Cellular Biology, October 2000, p. 7388-7400, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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