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Molecular and Cellular Biology, June 2008, p. 3850-3860, Vol. 28, No. 11
0270-7306/08/$08.00+0     doi:10.1128/MCB.02253-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Binding of DAZAP1 and hnRNPA1/A2 to an Exonic Splicing Silencer in a Natural BRCA1 Exon 18 Mutant{triangledown}

Elisa Goina, Natasa Skoko, and Franco Pagani*

International Center for Genetic Engineering and Biotechnology, Padriciano 99, Trieste 34012, Italy

Received 20 December 2007/ Returned for modification 30 January 2008/ Accepted 26 March 2008

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.

* Corresponding author. Mailing address: Human Molecular Genetics, International Center for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy. Phone: 0039-040-375-7342. Fax: 0039-040-226-555. E-mail: pagani{at}icgeb.org

{triangledown} Published ahead of print on 7 April 2008.

Molecular and Cellular Biology, June 2008, p. 3850-3860, Vol. 28, No. 11
0270-7306/08/$08.00+0     doi:10.1128/MCB.02253-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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