Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 1 CstF1 interacts with both BARD1 and p53 to form a protein complex. (a) Diagram of BARD1, CstF1 and p53 derivatives. (b) Requirement of p53 C-terminal domain for both CstF1 and BARD1 interaction. Recombinant His-p53 or the indicated His-p53 derivatives were incubated with purified GST, GST-CstF1 or GST-BARD1. Protein samples were treated with RNase A. Bound proteins were eluted and analyzed by western blot with anti-p53 antibodies. Five percent of His-p53 or His-p53 derivatives used in the reaction are shown as input. (c) CstF1, BARD1 and p53 can coexist in complexes. Either GST-BARD1 or GST-CstF1 was immobilized in glutathione-Sepharose beads to test its ability to bind CstF1/BARD1 and p53 from NE of HeLa cells, while increasing concentrations of recombinant His-p53 (5, 10 and 20 ng) were added. Bound proteins were detected by immunoblotting with CstF1 and p53 antibodies. The pellets (PD) and the supernatant (SN) were analyzed. (d) p53, CstF and BARD1 co-immunoprecipitate from NEs of MCF7 cells treated with UV irradiation. CstF and p53 co-immunoprecipitation is irrespective of UV irradiation. Co-immunoprecipitation assays were performed using NEs of cells exposed or not to UV irradiation and allowed to recover for 2 h as described before. NEs were immunoprecipitated with either anti-CstF2, to ensure that any detected interactions were between p53 and intact CstF, or anti-p53 antibodies. Protein samples were treated with RNase A. Equivalent amounts of the pellets (IP) and the supernatants (SN) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and proteins were detected by immunoblotting with antibodies against BARD1, CstF2 and p53. Antibodies against Topo II were used as a control of specificity. Positions of Topo II, CstF2 and p53 are indicated. Twenty percent of the NE used in the immunoprecipitation reaction is shown as input.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Recombinant, Incubation, Western Blot, Irradiation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Control

Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 4 The S241F mutation in p53 disrupts the BARD1, CstF and p53 interaction and decreases the inhibitory effect of p53 on 30 cleavage, and all this is restored by WT-p53 expression. (a) CstF, p53 and BARD1 do not co-immunoprecipitate from NE of DLD-1 cells that express S241F mutant p53. NEs were immunoprecipitated with either anti-CstF2 or anti-p53 antibodies. Protein samples were treated with RNase A. Equivalent amounts of the pellets (IP) and the supernatants (SN) were resolved by sodium dodecyl sulfate– polyacrylamide gel electrophoresis and proteins were detected by immunoblotting with antibodies against BARD1, CstF2 and p53. Antibodies against Topo II were used as a control of specificity. Positions of Topo II, BARD1, CstF2 and p53 are indicated. Twenty percent of the NE used in the immunoprecipitation reaction is shown as input. (b) CstF, BARD1 and p53 co-immunoprecipitate from NEs of D-A2 cells following induction of WT-p53 expression. D-A2 cells were grown in the absence of Dox to induce the expression of WT-p53. Samples were analyzed as in (a). (c) The induced expression of WT-p53 in D-A2 cells inhibits pre-mRNA 30 cleavage. NEs from DLD-1 (left panel) and D-A2 cells (right panel) non-treated or treated with Dox and/or UV irradiation, and allowed to recover for the times indicated in the figure, were analyzed for L3 pre-mRNA 30 cleavage. Positions of pre-mRNA and the 50 cleavage product are indicated. (d) The S241F mutation in p53 abolishes the inhibition of 30 cleavage. NEs from HeLa cells were preincubated with no addition or increasing amounts of recombinant His-p53 or His-p53 (S241F) derivative (40, 80 and 120 ng). After 15 min, L3 pre-mRNA was added and incubation continued for 90 min. RNAs were purified and analyzed by denaturing PAGE. Positions of pre-mRNA and the 50 cleavage product are indicated.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Control, Irradiation, Inhibition, Recombinant, Incubation