Polypyrimidine Track-binding Protein Binding Downstream of Caspase-2 Alternative Exon 9 Represses Its Inclusion

doi:10.1074/jbc.M008924200

Polypyrimidine Track-binding Protein Binding Downstream of Caspase-2 Alternative Exon 9 Represses Its Inclusion*

From the Department of Pediatrics and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Abstract

We have been using the caspase-2 pre-mRNA as a model system to study the importance of alternative splicing in the regulation of programmed cell death. Inclusion or skipping of a cassette-type exon in the 3′ portion of this pre-mRNA leads to the production of isoforms with antagonistic activity in apoptosis. We previously identified a negative regulatory element (In100) located in the intron downstream of alternative exon 9. The upstream portion of this element harbors a decoy 3′ acceptor site that engages in nonproductive commitment complex interactions with the 5′ splice site of exon 9. This in turn confers a competitive advantage to the exon-skipping splicing pattern. Further characterization of the In100 element reveals a second, functionally distinct, domain located downstream from the decoy 3′ acceptor site. This downstream domain harbors several polypyrimidine track-binding protein (PTB)-binding sites. We show that PTB binding to these sites correlates with the negative effect on exon 9 inclusion. Finally, we show that both domains of the In100 element can function independently to repress exon 9 inclusion, although PTB binding in the vicinity of the decoy 3′ splice site can modulate its activity. Our results thus reveal a complex composite element that regulates caspase-2 exon 9 alternative splicing through a novel mechanism.

Footnotes

↵* This work was supported in part by National Institutes of Health grants (to J. Y. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.
↵§ Present address: Lady Davis Inst. for Medical Research, Montreal, Quebec H3T 1E2, Canada.
↵¶ Supported by a scholarship from the Leukemia and Lymphoma Society. To whom correspondence should be addressed: Dept. of Pediatrics and Dept. of Molecular Biology and Pharmacology, Washington University School of Medicine, 4938 Parkview Pl., MPRB Rm. 3107, St. Louis, MO 63110. Tel.: 314-286-2798; Fax: 314-286-2892; E-mail: jwu@molecool.wustl.edu.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M008924200
↵2 Z. Jiang and J. Y. Wu, unpublished results.
Abbreviations:

hnRNP

heteronuclear ribonucleoprotein

PTB

polypyrimidine track-binding protein

snRNP

small nuclear ribonucleoprotein

oligo

oligonucleotide

RT

reverse transcription

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

nPTB

brain-enriched form of polypyrimidine track-binding protein
- Received September 29, 2000.
- Revision received December 14, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.