Binding of DAZAP1 and hnRNPA1/A2 to an exonic splicing silencer in a natural BRCA1 exon 18 mutant.
A disease-causing G-to-T transversion at position +6 of BRCA1 exon 18 induces exclusion of the exon from the mRNA and, as has been suggested by in silico analysis, disrupts an ASF/SF2-dependent splicing enhancer. We show here using a pulldown assay with an internal standard that wild-type (WT) and mutant T6 sequences displayed similar ASF/SF2 binding efficiencies, which were significantly lower than that of a typical exonic splicing enhancer derived from the extra domain A exon of fibronectin. Overexpression or small interfering RNA (siRNA)-mediated depletion of ASF/SF2 did not affect the splicing of a WT BRCA1 minigene but resulted in an increase and decrease of T6 exon 18 inclusion, respectively. Furthermore, extensive mutation analysis using hybrid minigenes indicated that the T6 mutant creates a sequence with a prevalently inhibitory function. Indeed, RNA-protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a splicing factor-dependent silencer. This sequence specifically binds to the known repressor protein hnRNPA1/A2 and to DAZAP1, the involvement of which in splicing inhibition we have demonstrated. Our results indicate that the binding of the splicing factors hnRNPA1/A2 and DAZAP1 is the primary determinant of T6 BRCA1 exon 18 exclusion.
Goina
Elisa
E
International Center for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.
Skoko
Natasa
N
Pagani
Franco
F
D017398:Alternative Splicing; D019313:BRCA1 Protein / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D020868:Gene Silencing; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors
D019313:BRCA1 Protein; C492913:BRCA1 protein, human; C412481:DAZAP1 protein, human; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C439768:hnRNP A2; C000619656:hnRNPA1 protein, human; D000068103:Serine-Arginine Splicing Factors