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Updated: Feb 9, 2026
Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
Published on: August 9, 2019
Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin
G J Mulligan1, W Guo, S Wormsley
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724.
Abstract:
Previous studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7 (Guo, W., Mulligan, G. J., Wormsley, S., and Helfman, D. M. (1991) Genes & Dev. 5, 2095-2106). Using an RNA mobility-shift assay we have identified factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage. Here we present the purification to apparent homogeneity of a protein that exhibits these sequence specific RNA binding properties. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: 1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and 2) the intron regulatory element that is involved in the repression of exon 7. Our results demonstrate that the sequence requirements for PTB binding are different than previously reported and shows that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites.
Insights
Polypyrimidine tract binding protein (PTB) binds to regulatory elements in beta-tropomyosin pre-mRNA, influencing alternative splicing. This study reveals novel binding specificities and suggests PTB
Area of Science:
- Molecular Biology
- RNA Splicing
- Gene Regulation
Background:
- Alternative splicing of the rat beta-tropomyosin gene is regulated by factors in nonmuscle cells that block skeletal muscle exon 7 usage.
- Previous studies indicated the involvement of specific factors in this exon skipping mechanism.
Purpose of the Study:
- To identify and purify proteins from HeLa cell nuclear extracts that specifically bind to RNA sequences involved in beta-tropomyosin exon 7 repression.
- To characterize the binding properties and functional role of the purified protein, identified as polypyrimidine tract binding protein (PTB).
Main Methods:
- RNA mobility-shift assay to detect specific RNA-protein interactions.
- Protein purification to apparent homogeneity.
- Characterization of PTB binding to regulatory elements within beta-tropomyosin pre-mRNA introns.
Main Results:
- A protein identical to polypyrimidine tract binding protein (PTB) was purified.
- PTB specifically binds to two functional elements in intron 6 of beta-tropomyosin pre-mRNA, involved in branch point usage and exon 7 repression.
- PTB binding specificity is more complex than predicted by pyrimidine content alone.
- PTB binds to intron 6 but not introns 5 or 7 of beta-tropomyosin pre-mRNA.
Conclusions:
- PTB plays a role in the regulation of alternative splicing of beta-tropomyosin by binding to specific sequences in intron 6.
- PTB binding requirements are distinct from previous reports and cannot be solely based on pyrimidine richness.
- PTB's interaction with regulated introns provides new insights into its binding specificity and suggests a role beyond solely positive 3'-splice site recognition.
