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Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Savage KI, Gorski JJ, Barros EM, Irwin GW, Manti L, Powell AJ, Pellagatti A, Lukashchuk N, McCance DJ, McCluggage WG, Schettino G, Salto-Tellez M, Boultwood J, Richard DJ, McDade SS, Harkin DP - Mol. Cell (2014)

Bottom Line: Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers.Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery.Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: k.savage@qub.ac.uk.

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BRCA1/BCLAF1-Mediated mRNA Splicing of ATRIP, BACH1, and EXO1 Promotes Resistance to DNA Damage and Efficient DNA Repair(A) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in BRCA1- or BCLAF1-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM.(B) Clonogenic survival assays demonstrating that depletion of BCLAF1 or U2AF65 induces sensitivity to IR in 293T cells. Mean surviving fraction of three independent experiments is plotted ± SEM.(C) Representative immunofluorescent staining of γ-H2AX marked DNA damage in untreated 293T cells depleted of either BCLAF1 or U2AF65 and 1 and 24 hr following 2Gy IR.(D. Quantification of three independent experiments described above (≥200 cells were scored/experiment). The mean fraction of cells containing ≥5 γ-H2AX foci is plotted ± SEM. Significant differences in the fraction of cells containing ≥5 γ-H2AX foci were assessed using Student’s two-tailed t test and are indicated by ∗∗∗p < 0.001.(E) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in U2AF65-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM. See also Figure S7.
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fig7: BRCA1/BCLAF1-Mediated mRNA Splicing of ATRIP, BACH1, and EXO1 Promotes Resistance to DNA Damage and Efficient DNA Repair(A) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in BRCA1- or BCLAF1-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM.(B) Clonogenic survival assays demonstrating that depletion of BCLAF1 or U2AF65 induces sensitivity to IR in 293T cells. Mean surviving fraction of three independent experiments is plotted ± SEM.(C) Representative immunofluorescent staining of γ-H2AX marked DNA damage in untreated 293T cells depleted of either BCLAF1 or U2AF65 and 1 and 24 hr following 2Gy IR.(D. Quantification of three independent experiments described above (≥200 cells were scored/experiment). The mean fraction of cells containing ≥5 γ-H2AX foci is plotted ± SEM. Significant differences in the fraction of cells containing ≥5 γ-H2AX foci were assessed using Student’s two-tailed t test and are indicated by ∗∗∗p < 0.001.(E) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in U2AF65-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM. See also Figure S7.

Mentions: Given that loss of BRCA1/BCLAF1-mediated splicing of ATRIP, BACH1, and EXO1 results in reduced but not absent protein levels following DNA damage, we examined whether reduction of these proteins could contribute to the DNA damage sensitivity observed in BRCA1/BCLAF1-depleted cells. Indeed, reduction but not complete absence of any of these proteins, using titrated siRNA concentrations, resulted in sensitization to IR induced DNA damage (Figures S7A and S7B). In contrast, ectopic expression of ATRIP, BACH1, or EXO1 alone was unable to rescue the sensitivity of BRCA1- or BCLAF1-depleted cells to IR (data not shown). However, the combined ectopic expression of ATRIP, BACH1, and EXO1 was able to partially rescue the IR sensitivity of both BRCA1- and BCLAF1-depleted cells, suggesting that BRCA1/BCLAF1-mediated splicing of these genes is required, at least in part, for BRCA1 and BCLAF1’s ability to mediate resistance to DNA damaging agents (Figures 7A and S7C).


Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Savage KI, Gorski JJ, Barros EM, Irwin GW, Manti L, Powell AJ, Pellagatti A, Lukashchuk N, McCance DJ, McCluggage WG, Schettino G, Salto-Tellez M, Boultwood J, Richard DJ, McDade SS, Harkin DP - Mol. Cell (2014)

BRCA1/BCLAF1-Mediated mRNA Splicing of ATRIP, BACH1, and EXO1 Promotes Resistance to DNA Damage and Efficient DNA Repair(A) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in BRCA1- or BCLAF1-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM.(B) Clonogenic survival assays demonstrating that depletion of BCLAF1 or U2AF65 induces sensitivity to IR in 293T cells. Mean surviving fraction of three independent experiments is plotted ± SEM.(C) Representative immunofluorescent staining of γ-H2AX marked DNA damage in untreated 293T cells depleted of either BCLAF1 or U2AF65 and 1 and 24 hr following 2Gy IR.(D. Quantification of three independent experiments described above (≥200 cells were scored/experiment). The mean fraction of cells containing ≥5 γ-H2AX foci is plotted ± SEM. Significant differences in the fraction of cells containing ≥5 γ-H2AX foci were assessed using Student’s two-tailed t test and are indicated by ∗∗∗p < 0.001.(E) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in U2AF65-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM. See also Figure S7.
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fig7: BRCA1/BCLAF1-Mediated mRNA Splicing of ATRIP, BACH1, and EXO1 Promotes Resistance to DNA Damage and Efficient DNA Repair(A) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in BRCA1- or BCLAF1-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM.(B) Clonogenic survival assays demonstrating that depletion of BCLAF1 or U2AF65 induces sensitivity to IR in 293T cells. Mean surviving fraction of three independent experiments is plotted ± SEM.(C) Representative immunofluorescent staining of γ-H2AX marked DNA damage in untreated 293T cells depleted of either BCLAF1 or U2AF65 and 1 and 24 hr following 2Gy IR.(D. Quantification of three independent experiments described above (≥200 cells were scored/experiment). The mean fraction of cells containing ≥5 γ-H2AX foci is plotted ± SEM. Significant differences in the fraction of cells containing ≥5 γ-H2AX foci were assessed using Student’s two-tailed t test and are indicated by ∗∗∗p < 0.001.(E) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in U2AF65-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM. See also Figure S7.
Mentions: Given that loss of BRCA1/BCLAF1-mediated splicing of ATRIP, BACH1, and EXO1 results in reduced but not absent protein levels following DNA damage, we examined whether reduction of these proteins could contribute to the DNA damage sensitivity observed in BRCA1/BCLAF1-depleted cells. Indeed, reduction but not complete absence of any of these proteins, using titrated siRNA concentrations, resulted in sensitization to IR induced DNA damage (Figures S7A and S7B). In contrast, ectopic expression of ATRIP, BACH1, or EXO1 alone was unable to rescue the sensitivity of BRCA1- or BCLAF1-depleted cells to IR (data not shown). However, the combined ectopic expression of ATRIP, BACH1, and EXO1 was able to partially rescue the IR sensitivity of both BRCA1- and BCLAF1-depleted cells, suggesting that BRCA1/BCLAF1-mediated splicing of these genes is required, at least in part, for BRCA1 and BCLAF1’s ability to mediate resistance to DNA damaging agents (Figures 7A and S7C).

Bottom Line: Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers.Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery.Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: k.savage@qub.ac.uk.

Show MeSH
Related in: MedlinePlus