Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3'-terminal exon.
Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3'-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5' splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.
Lou
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Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA. hlou@bcm.tmc.edu
Helfman
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DM
Gagel
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RF
Berget
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SM
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D016384:Consensus Sequence; D006224:Cricetinae; D004742:Enhancer Elements, Genetic; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins; D038941:Polypyrimidine Tract-Binding Protein; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D000072668:Splicing Factor U2AF; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D038941:Polypyrimidine Tract-Binding Protein