title: Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1.
abstract: The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, 1602 Molecular Sciences Building, 405 Hilgard Avenue, Los Angeles, CA 90095, USA.
authors.forename: Nanette
authors.initials: N
authors.lastname: Rooke
authors.forename: Vadim
authors.initials: V
authors.lastname: Markovtsov
authors.forename: Esra
authors.initials: E
authors.lastname: Cagavi
authors.forename: Douglas L
authors.initials: DL
authors.lastname: Black
mesh_terms: D000818:Animals; D005091:Exons / Q000235:genetics*; D005134:Eye Neoplasms / Q000473:pathology; D016391:Genes, src; D006367:HeLa Cells / Q000378:metabolism; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000502:physiology*; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D009363:Neoplasm Proteins / Q000502:physiology; D009687:Nuclear Proteins / Q000502:physiology*; D038941:Polypyrimidine Tract-Binding Protein / Q000502:physiology; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D016601:RNA-Binding Proteins; D012045:Regulatory Sequences, Nucleic Acid; D012175:Retinoblastoma / Q000473:pathology; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C499372:SRSF2 protein, mouse; D038941:Polypyrimidine Tract-Binding Protein; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors