Key result
BRCA1 T6 mutation drives exon 18 exclusion via a splicing silencer binding hnRNPA1/A2 and DAZAP1.
Why the study?
A G-to-T transversion at position +6 of BRCA1 exon 18 induces exon exclusion by disrupting splicing regulation, but the precise mechanisms were unclear.
Answers are grounded in this paper and cite their sources.
The study demonstrates that a disease-causing BRCA1 mutation induces exon skipping by creating a silencer sequence that binds specific repressor proteins.
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.
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Goina et al. (2008) studied BRCA1 mutation. T6 BRCA1 exon 18 mutation vs. Wild-type BRCA1 was evaluated on Exon 18 inclusion/exclusion. The disease-causing T6 mutation in BRCA1 exon 18 creates a splicing silencer that binds hnRNPA1/A2 and DAZAP1, which is the primary determinant of exon 18 exclusion.
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