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Molecular and Cellular Biology, April 1999, p. 2699-2711, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Polypyrimidine Tract Binding Protein Functions as a Repressor To Regulate Alternative Splicing of alpha -Actinin Mutally Exclusive Exons

Justine Southby, Clare Gooding, and Christopher W. J. Smith*

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom

Received 16 November 1998/Accepted 18 December 1998

The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha -actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha -actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha -tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated alpha -TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of alpha -actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, alpha -actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB.

* Corresponding author. Mailing address: Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Old Addenbrookes Site, Cambridge CB2 1GA, United Kingdom. Phone: 44-1223-333655. Fax: 44-1223-766002. E-mail: cwjs1{at}mole.bio.cam.ac.uk.

Molecular and Cellular Biology, April 1999, p. 2699-2711, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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