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Molecular and Cellular Biology, January 1999, p. 69-77, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells

Min-Yuan Chou,1 Nanette Rooke,2 Christoph W. Turck,3 and Douglas L. Black1,2,*

Howard Hughes Medical Institute1 and Department of Microbiology and Molecular Genetics,2 University of California, Los Angeles, Los Angeles, California 90095, and Howard Hughes Medical Institute, Department of Medicine and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 941433

Received 28 July 1998/Returned for modification 25 August 1998/Accepted 13 October 1998

The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5' splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.

* Corresponding author. Mailing address: 5-748 MRL, Box 951662, 675 Circle Dr. South, Los Angeles, CA 90095-1662. Phone: (310) 794-7644. Fax: (310) 206-8623. E-mail: DougB{at}microbio.lifesci.ucla.edu.

Molecular and Cellular Biology, January 1999, p. 69-77, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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