Mapping the SF2/ASF binding sites in the bovine growth hormone exonic splicing enhancer

Dirksen, W. P., Li, X. L., Mayeda, A., Krainer, A. R., Rottman, F. M. (September 2000) Mapping the SF2/ASF binding sites in the bovine growth hormone exonic splicing enhancer. Journal of Biological Chemistry, 275 (37). pp. 29170-29177. ISSN 0021-9258

URL: http://www.ncbi.nlm.nih.gov/pubmed/10880506
DOI: 10.1074/jbc.M001126200

Abstract

Splicing of the last intron (intron D) of the bovine growth hormone pre-mRNA requires the presence of a downstream exonic splicing enhancer (ESE). This enhancer is contained within a 115-nucleotide FspI-PvuII (FP) fragment located in the middle of the last exon (exon 5). Previous work showed that the splicing factor SF2/ASF binds to this FP region and stimulates splicing of intron D in vitro. However, the precise sequences recognized by SF2/ASF within the FP region had not been determined. Here we used multiple strategies to map the SF2/ASF binding sites and determine their importance for ESE function. Taking advantage of the fact that SF2/ASF ultraviolet (UV) cross-links specifically to RNA containing the FP sequence, we first mapped a major SF2/ASF binding site by UV cross-linking and reverse transcription. This strategy identified a 29-nucleotide SF2/ASF binding region in the middle of the FP sequence containing the 7-nucleotide purine-rich motif described previously. Interestingly, this binding region is neither sufficient, nor absolutely required for SF2/ASF-mediated splicing, suggesting that additional SF2/ASF binding sites are present. The location of these additional sites was determined by electrophoretic mobility shift analysis of various subfragments of the FP sequence, Antisense 2'-O-methyl oligoribonucleotides complementary to selected SF2/ASF binding sites block bovine growth hormone intron D splicing. Thus, multiple SF2/ASF binding sites within the exonic splicing enhancer contribute to maximal enhancer activity.

Item Type: Paper
Uncontrolled Keywords: pre-messenger-rna small nuclear ribonucleoprotein protein-protein interactions t alternative exon sr-proteins premessenger rna recognition motifs 5'-splice-site recognition factors asf/sf2 factor sf2
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > exons > exon splicing
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > nuclear ribonucleoprotein
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > pre-mRNA
CSHL Authors:
Communities: CSHL labs > Krainer lab
Depositing User: Matt Covey
Date: September 2000
Date Deposited: 11 Dec 2013 16:34
Last Modified: 11 Dec 2013 16:34
Related URLs:
URI: http://repository.cshl.edu/id/eprint/28919

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving