Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.
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.
Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: email@example.com.Show MeSH
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Mentions: Consistent with this, we found that the mRNA splicing of ATRIP, BACH1, and EXO1 transcripts was significantly upregulated in response to DNA damage in both a BRCA1- and a BCLAF1-dependent manner using two independent siRNAs (Figures 4A and S5A). Additionally, saturating RT-PCR analysis with intron-targeted primers revealed the presence of introns in ATRIP, BACH1, and EXO1 transcripts following DNA damage in BRCA1- and BCLAF1-depleted cells, but not control cells, confirming that splicing of these transcripts following DNA damage requires BRCA1 and BCLAF1 (Figure 4B). In response to DNA damage, transcription of ATRIP, BACH1, and EXO1 is upregulated. However, BRCA1 or BCLAF1 depletion does not affect the transcription of these genes as indicated by similar levels of pre-mRNA production, in the absence or presence of DNA damage (Figure 4C). Similarly, RNA Pol II loading and activity on these gene promoters is unaffected by BRCA1 or BCLAF1 depletion (Figure S5B). In contrast, we observed a marked reduction in the production of postspliced ATRIP, BACH1, and EXO1 transcripts following depletion of BRCA1 or BCLAF1 (Figure 4C). Additionally, the increased ratio of postspliced/prespliced mRNA observed in BRCA1/BCLAF1-depleted cells following DNA damage is not due to increased decay of prespliced transcripts in these cells (Figure S5C). Instead, mRNA decay experiments revealed reduced levels of postspliced ATRIP, BACH1, and EXO1 transcripts, in comparison to prespliced transcripts, in BRCA1/BCLAF1-depleted cells following inhibition of transcription, which is consistent with a role for BRCA1/BCLAF1 in the cotranscriptional splicing of these genes (Figure S5C). Importantly, we did not observe changes in ATRIP, BACH1, or EXO1 splice variant expression following DNA damage, suggesting that DNA damage induced BRCA1/BCLAF1-mediated splicing of these genes does not facilitate alternative splicing (data not shown).
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.