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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 Ser-1423 Phosphorylation Is Required for BCLAF1 Recruitment and Target Gene Splicing following DNA Damage(A) BRCA1, pS1423-BRCA1, and BCLAF1 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP, BACH1, and EXO1 promoters irrespective of DNA damage and DNA damage-dependent enrichment of pS1423-BRCA1 and BCLAF1 on these promoters. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM.(B–D) Ratio of postspliced to prespliced ATRIP, BACH1, and EXO1 mRNA in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt-BRCA1), or S1423A Flag-BRCA1 (S1423A-BRCA1). Cells were mock treated or treated with etoposide. mRNA levels were assessed as described in Figure 4A. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM ATRIP, BACH1, and EXO1 splicing is upregulated in cells expressing wild-type BRCA1 but not S1423A-BRCA1, indicating that phosphorylation of BRCA1 S1423 is required for DNA damage-induced splicing.(E–G) FLAG and BCLAF1 ChIP-qRT-PCR analysis carried out in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt), or S1423A Flag-BRCA1 (S1423A). Cells were mock treated or treated with etoposide. Graphs represent the mean fold enrichment from three independent experiments ± SEM.(H) Representative western blots demonstrating wild-type and S1423A Flag-BRCA1 in HCC1937 cells used for splicing assays and ChIPs presented above. See also Figure S6.
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fig5: BRCA1 Ser-1423 Phosphorylation Is Required for BCLAF1 Recruitment and Target Gene Splicing following DNA Damage(A) BRCA1, pS1423-BRCA1, and BCLAF1 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP, BACH1, and EXO1 promoters irrespective of DNA damage and DNA damage-dependent enrichment of pS1423-BRCA1 and BCLAF1 on these promoters. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM.(B–D) Ratio of postspliced to prespliced ATRIP, BACH1, and EXO1 mRNA in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt-BRCA1), or S1423A Flag-BRCA1 (S1423A-BRCA1). Cells were mock treated or treated with etoposide. mRNA levels were assessed as described in Figure 4A. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM ATRIP, BACH1, and EXO1 splicing is upregulated in cells expressing wild-type BRCA1 but not S1423A-BRCA1, indicating that phosphorylation of BRCA1 S1423 is required for DNA damage-induced splicing.(E–G) FLAG and BCLAF1 ChIP-qRT-PCR analysis carried out in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt), or S1423A Flag-BRCA1 (S1423A). Cells were mock treated or treated with etoposide. Graphs represent the mean fold enrichment from three independent experiments ± SEM.(H) Representative western blots demonstrating wild-type and S1423A Flag-BRCA1 in HCC1937 cells used for splicing assays and ChIPs presented above. See also Figure S6.

Mentions: Taken together, our data suggest a model in which phosphorylated BRCA1, bound at a subset of gene promoters following DNA damage, recruits BCLAF1 and associated spliceosomal proteins, thereby facilitating DNA damage-induced mRNA splicing. To confirm that BRCA1, bound at the ATRIP, BACH1, and EXO1 promoters, is indeed phosphorylated at serine-1423 following DNA damage, we performed ChIP-qRT-PCR with BRCA1pSer-1423 antibodies. This revealed marked enrichment of BRCA1pSer-1423 at the ATRIP, BACH1, and EXO1 promoters only following DNA damage (Figure 5A). Also in support of this model, reconstitution of BRCA1 mutant cells (HCC1937) with wild-type BRCA1 restored their ability to upregulate ATRIP, BACH1, and EXO1 mRNA splicing following DNA damage, whereas reconstitution with BRCA1S1423A phosphomutant protein did not (Figures 5B–5D). Additionally, wild-type BRCA1 was able to recruit BCLAF1 to the ATRIP, BACH1, and EXO1 promoter regions following DNA damage, whereas the BRCA1S1423A phospho mutant was not (Figures 5E–5H). Consistent with this, inhibition of ATM and ATR (mediators of BRCA1S1423 phosphorylation) also abrogated DNA damage-induced ATRIP, BACH1, and EXO1 mRNA splicing and recruitment of BCLAF1 and U2AF65 to their promoters (Figures S6A–S6C).


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 Ser-1423 Phosphorylation Is Required for BCLAF1 Recruitment and Target Gene Splicing following DNA Damage(A) BRCA1, pS1423-BRCA1, and BCLAF1 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP, BACH1, and EXO1 promoters irrespective of DNA damage and DNA damage-dependent enrichment of pS1423-BRCA1 and BCLAF1 on these promoters. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM.(B–D) Ratio of postspliced to prespliced ATRIP, BACH1, and EXO1 mRNA in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt-BRCA1), or S1423A Flag-BRCA1 (S1423A-BRCA1). Cells were mock treated or treated with etoposide. mRNA levels were assessed as described in Figure 4A. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM ATRIP, BACH1, and EXO1 splicing is upregulated in cells expressing wild-type BRCA1 but not S1423A-BRCA1, indicating that phosphorylation of BRCA1 S1423 is required for DNA damage-induced splicing.(E–G) FLAG and BCLAF1 ChIP-qRT-PCR analysis carried out in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt), or S1423A Flag-BRCA1 (S1423A). Cells were mock treated or treated with etoposide. Graphs represent the mean fold enrichment from three independent experiments ± SEM.(H) Representative western blots demonstrating wild-type and S1423A Flag-BRCA1 in HCC1937 cells used for splicing assays and ChIPs presented above. See also Figure S6.
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fig5: BRCA1 Ser-1423 Phosphorylation Is Required for BCLAF1 Recruitment and Target Gene Splicing following DNA Damage(A) BRCA1, pS1423-BRCA1, and BCLAF1 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP, BACH1, and EXO1 promoters irrespective of DNA damage and DNA damage-dependent enrichment of pS1423-BRCA1 and BCLAF1 on these promoters. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM.(B–D) Ratio of postspliced to prespliced ATRIP, BACH1, and EXO1 mRNA in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt-BRCA1), or S1423A Flag-BRCA1 (S1423A-BRCA1). Cells were mock treated or treated with etoposide. mRNA levels were assessed as described in Figure 4A. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM ATRIP, BACH1, and EXO1 splicing is upregulated in cells expressing wild-type BRCA1 but not S1423A-BRCA1, indicating that phosphorylation of BRCA1 S1423 is required for DNA damage-induced splicing.(E–G) FLAG and BCLAF1 ChIP-qRT-PCR analysis carried out in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt), or S1423A Flag-BRCA1 (S1423A). Cells were mock treated or treated with etoposide. Graphs represent the mean fold enrichment from three independent experiments ± SEM.(H) Representative western blots demonstrating wild-type and S1423A Flag-BRCA1 in HCC1937 cells used for splicing assays and ChIPs presented above. See also Figure S6.
Mentions: Taken together, our data suggest a model in which phosphorylated BRCA1, bound at a subset of gene promoters following DNA damage, recruits BCLAF1 and associated spliceosomal proteins, thereby facilitating DNA damage-induced mRNA splicing. To confirm that BRCA1, bound at the ATRIP, BACH1, and EXO1 promoters, is indeed phosphorylated at serine-1423 following DNA damage, we performed ChIP-qRT-PCR with BRCA1pSer-1423 antibodies. This revealed marked enrichment of BRCA1pSer-1423 at the ATRIP, BACH1, and EXO1 promoters only following DNA damage (Figure 5A). Also in support of this model, reconstitution of BRCA1 mutant cells (HCC1937) with wild-type BRCA1 restored their ability to upregulate ATRIP, BACH1, and EXO1 mRNA splicing following DNA damage, whereas reconstitution with BRCA1S1423A phosphomutant protein did not (Figures 5B–5D). Additionally, wild-type BRCA1 was able to recruit BCLAF1 to the ATRIP, BACH1, and EXO1 promoter regions following DNA damage, whereas the BRCA1S1423A phospho mutant was not (Figures 5E–5H). Consistent with this, inhibition of ATM and ATR (mediators of BRCA1S1423 phosphorylation) also abrogated DNA damage-induced ATRIP, BACH1, and EXO1 mRNA splicing and recruitment of BCLAF1 and U2AF65 to their promoters (Figures S6A–S6C).

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