Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.
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.
Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: firstname.lastname@example.org.
- BRCA1 Protein/metabolism*
- DNA Repair*
- Genomic Instability*
- RNA, Messenger/metabolism*
- Adaptor Proteins, Signal Transducing/genetics/metabolism
- Basic-Leucine Zipper Transcription Factors/genetics/metabolism
- Cell Survival/radiation effects
- DNA Damage
- DNA Repair Enzymes/genetics/metabolism
- DNA-Binding Proteins/genetics/metabolism
- Fanconi Anemia Complementation Group Proteins/genetics/metabolism
- Genome, Human
- HEK293 Cells
- Protein Processing, Post-Translational
- RNA Splicing
- Radiation Tolerance
- Repressor Proteins/metabolism
- Tumor Suppressor Proteins/metabolism
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fig3: BRCA1/BCLAF1 Forms an mRNA Splicing Complex which Is Recruited to Target Gene Promoters and Transcripts following DNA Damage(A) Coimmunoprecipitation assays demonstrating that BCLAF1 interacts with the spliceosome proteins Prp8, U2AF65, U2AF35, and SF3B1 in both the presence and absence of DNA damage.(B) Coimmunoprecipitation assays demonstrating DNA damage-induced interaction between BRCA1 and the spliceosome proteins Prp8, U2AF65 U2AF35, and SF3B1 in response to DNA damage. Additionally, depletion of BCLAF1 results in abrogation of DNA damage-induced interaction between BRCA1 and these proteins.(C) BRCA1, BCLAF1, and U2AF65 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP, BACH1, and EXO1 promoters irrespective of DNA damage in control (siCtrl) cells. The ChIPs also demonstrate that BCLAF1 and U2AF65 are recruited to these promoters only in etoposide-treated cells and that depletion of BRCA1 or BCLAF1 results in loss of DNA damage-induced BCLAF1 and U2AF65 recruitment, respectively. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM.(D) BRCA1, BCLAF1, and U2AF65 RIP-qRT-PCRs demonstrating that BRCA1, BCLAF1, and U2AF65 only bind to ATRIP, BACH1, and EXO1 mRNAs in response to DNA damage. In addition, depletion of BCLAF1 results in loss of BRCA1 and U2AF65 mRNA binding to all three transcripts. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM. See also Figure S3.
Given this previously identified role for BCLAF1 in pre-mRNA splicing and interaction with a number of core splicing machinery proteins, we examined the ability of BCLAF1 and BRCA1 to interact with known components of the BCLAF1 interacting spliceosome (Merz et al., 2007). Coimmunoprecipitation confirmed that BCLAF1 constitutively interacts with a number of these core mRNA splicing proteins such as Prp8, U2AF65, U2AF35, and SF3B1, independently of DNA damage (Figure 3A). In contrast, BRCA1 coprecipitated with Prp8, U2AF65, U2AF35, and SF3B1 only in response to DNA damage (Figure 3B). Furthermore, depletion of BCLAF1 resulted in abrogation of the damage-induced interaction between BRCA1 and these proteins, suggesting that BCLAF1 mediates the interaction between phosphorylated BRCA1 and core components of the spliceosome in response to DNA damage (Figure 3B).