Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA.
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.
Mulligan
G J
GJ
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724.
Guo
W
W
Wormsley
S
S
Helfman
D M
DM
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic; D014335:Tropomyosin / Q000235:genetics*
D012322:RNA Precursors; D016601:RNA-Binding Proteins; D014335:Tropomyosin; D038941:Polypyrimidine Tract-Binding Protein