Figure 7.

Ubiquitination of CstF50 is catalyzed by BRCA1. (A) BRCA1 knockdown impairs CstF50 ubiquitination. Whole cell extracts from HEK293T cells transfected with control or BRCA1 siRNAs were immunoprecipitated with a control or an anti-ubiquitin resin. Western blots were analysed with the indicated antibodies. β-catenin was used as control for BRCA1-independent ubiquitination and is highlighted by an arrow. The asterisk indicates non-specific bands. Upper panel: Quantification of CstF50 ubiquitination upon BRCA1 silencing. Quantification was performed on three independent immunoprecipitation experiments. Immunoprecipitated CstF50 protein was normalized on β-catenin. Error bars represent standard deviations. Lower panel: representative western blot. (B) Overexpression of a catalytically inactive mutant BRCA1 protein impairs CstF50 ubiquitination. HEK293T cells were cotransfected with plasmids expressing CstF50, BARD1 and either wild type BRCA1 or the BRCA1-ΔN mutant. Ubiquitinated proteins were immunoprecipitated with an anti-ubiquitin resin as described in ‘Materials and Methods’ and analysed by western blotting with the indicated antisera. Upper panel: Quantification was performed on three independent immunoprecipitation experiments. Immunoprecipitated CstF50 protein was normalized on IgG bands that are indicated in the Ponceau Staining. Error bars represent standard deviations. *P < 0.05. Lower panel: representative western blot. The arrow indicates the bands corresponding to the IgGs. (C) BRCA1 knockdown favors inclusion of the distal ALE. Quantification of the amount of proximal and distal ALE usage in the indicated cell lines. Data are representative of three independent experiments. Error bars represent standard deviation. ***P < 0.001.