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PMID: 12612063 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1.

Molecular and cellular biology ·Vol. 23 ·No. 6 ·2003-03-00 ·Pages 1874-84

Rooke N, Markovtsov V, Cagavi E, Black DL

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.

MeSH Terms
Animals Exons/genetics Eye Neoplasms/pathology Genes, src HeLa Cells/metabolism Heterogeneous Nuclear Ribonucleoprotein A1 Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology Humans Mice Models, Genetic Neoplasm Proteins/physiology Nuclear Proteins/physiology Polypyrimidine Tract-Binding Protein/physiology Protein Binding Protein Interaction Mapping RNA Precursors/metabolism RNA Splicing/physiology RNA-Binding Proteins Regulatory Sequences, Nucleic Acid Retinoblastoma/pathology Ribonucleoproteins Serine-Arginine Splicing Factors Tumor Cells, Cultured/metabolism
Chemicals
Heterogeneous Nuclear Ribonucleoprotein A1 Heterogeneous-Nuclear Ribonucleoprotein Group A-B Neoplasm Proteins Nuclear Proteins RNA Precursors RNA-Binding Proteins Ribonucleoproteins SRSF2 protein, mouse Polypyrimidine Tract-Binding Protein SRSF2 protein, human Serine-Arginine Splicing Factors
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Rooke Nanette
Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, 1602 Molecular Sciences Building, 405 Hilgard Avenue, Los Angeles, CA 90095, USA.
Markovtsov Vadim
Cagavi Esra
Black Douglas L
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.23.6.1874-1884.2003
Published
2003-03-00
Pages
1874-84
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC149473
Subset
IM
Grants
NIGMS NIH HHS · R01 GM 49662 · United States
External Links
PubMed source DOI: 10.1128/MCB.23.6.1874-1884.2003 PMC Full Text View journal details
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