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

Abstract

The smooth muscle (SM) and nonmuscle (NM) isoforms of α-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat α-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 α-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated α-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 α-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, α-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.

ACKNOWLEDGMENTS

We thank Paul Kemp for the genomic library, Jim Patton for the recombinant His-tagged PTB clone, and Sarah Hunt and Richard Jackson for the PTB antibodies, preimmune serum, and recombinant unr. We also thank Gavin Roberts for critically reading the manuscript.

This work was supported by a Wellcome Travelling Research Fellowship to J.S. and grants from the Wellcome Trust (052968) and Medical Research Council (G9417692) to C.W.J.S.