Mapping the SF2/ASF binding sites in the bovine growth hormone exonic splicing enhancer.
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.
Dirksen
W P
WP
Department of Molecular Biology and Microbiology and the Department of Medicine, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4960, USA.
Li
X
X
Mayeda
A
A
Krainer
A R
AR
Rottman
F M
FM
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004742:Enhancer Elements, Genetic; D005091:Exons; D013006:Growth Hormone / Q000235:genetics*; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007438:Introns; D009687:Nuclear Proteins / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D013006:Growth Hormone