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

Wessel P. Dirksen, Xinlan Li, Akira Maeda, Adrian R. Krainer, Fritz M. Rottman

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)29170-29177
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number37
DOIs
Publication statusPublished - 15-09-2000

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Introns
Binding Sites
Exons
Nucleotides
Oligoribonucleotides
Purine Nucleotides
Electrophoretic mobility
RNA Precursors
Transcription
Reverse Transcription
bovine growth hormone
RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Dirksen, Wessel P. ; Li, Xinlan ; Maeda, Akira ; Krainer, Adrian R. ; Rottman, Fritz M. / Mapping the SF2/ASF binding sites in the bovine growth hormone exonic splicing enhancer. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 37. pp. 29170-29177.
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Mapping the SF2/ASF binding sites in the bovine growth hormone exonic splicing enhancer. / Dirksen, Wessel P.; Li, Xinlan; Maeda, Akira; Krainer, Adrian R.; Rottman, Fritz M.

In: Journal of Biological Chemistry, Vol. 275, No. 37, 15.09.2000, p. 29170-29177.

Research output: Contribution to journalArticle

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