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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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The BRCA1/BCLAF mRNA Splicing Complex Promotes the Splicing and Stability of ATRIP, BACH1, and EXO1 Transcripts following DNA Damage(A) Ratio of postspliced to prespliced ATRIP, BACH1, and EXO1 mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells mock treated or treated with etoposide. mRNA levels were assessed by qRT-PCR using exon 9-exon 10 (post-spliced-ATRIP) and exon 9-intron 9 (pre-spliced-ATRIP), exon 15-exon 16 (post-spliced-BACH1) and exon 15-intron 15 (pre-spliced- BACH1), and exon 1-exon 2 (post-spliced-EXO1) and exon 1-intron 1 (pre-spliced- EXO1) primers and normalized to ACTB mRNA. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM. Significance of changes in splicing ratios was assessed using Student’s two-tailed t test with significant changes indicated by ∗∗p < 0.01.(B) Semiquantitative PCR analysis of a cDNA generated from DNase-treated RNA, collected from control (siCtrl) and BRCA1- or BCLAF1-depleted cells, mock treated or treated with Etoposide. Primers targeting two independent intronic regions within ATRIP, BACH1, and EXO1 and a single intronic region within ATM and CHEK2 (control genes) were used for semiquantitative PCR analysis. Exon-spanning primers targeted against ACTB were used as a loading control.(C) Normalized expression of prespliced and postspliced mRNAs evaluated in (A).(D) Expression levels of postspliced and prespliced ATRIP mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells, transfected with control siRNA (siCtrl) or depleted of SMG1 (siSMG1), a key regulator of the non-sense-mediated decay pathway. Normalized expression levels were quantified as in (A). Graphs represent the mean normalized expression from three independent experiments ± SEM. See also Figures S4 and S5.
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fig4: The BRCA1/BCLAF mRNA Splicing Complex Promotes the Splicing and Stability of ATRIP, BACH1, and EXO1 Transcripts following DNA Damage(A) Ratio of postspliced to prespliced ATRIP, BACH1, and EXO1 mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells mock treated or treated with etoposide. mRNA levels were assessed by qRT-PCR using exon 9-exon 10 (post-spliced-ATRIP) and exon 9-intron 9 (pre-spliced-ATRIP), exon 15-exon 16 (post-spliced-BACH1) and exon 15-intron 15 (pre-spliced- BACH1), and exon 1-exon 2 (post-spliced-EXO1) and exon 1-intron 1 (pre-spliced- EXO1) primers and normalized to ACTB mRNA. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM. Significance of changes in splicing ratios was assessed using Student’s two-tailed t test with significant changes indicated by ∗∗p < 0.01.(B) Semiquantitative PCR analysis of a cDNA generated from DNase-treated RNA, collected from control (siCtrl) and BRCA1- or BCLAF1-depleted cells, mock treated or treated with Etoposide. Primers targeting two independent intronic regions within ATRIP, BACH1, and EXO1 and a single intronic region within ATM and CHEK2 (control genes) were used for semiquantitative PCR analysis. Exon-spanning primers targeted against ACTB were used as a loading control.(C) Normalized expression of prespliced and postspliced mRNAs evaluated in (A).(D) Expression levels of postspliced and prespliced ATRIP mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells, transfected with control siRNA (siCtrl) or depleted of SMG1 (siSMG1), a key regulator of the non-sense-mediated decay pathway. Normalized expression levels were quantified as in (A). Graphs represent the mean normalized expression from three independent experiments ± SEM. See also Figures S4 and S5.

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).


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)

The BRCA1/BCLAF mRNA Splicing Complex Promotes the Splicing and Stability of ATRIP, BACH1, and EXO1 Transcripts following DNA Damage(A) Ratio of postspliced to prespliced ATRIP, BACH1, and EXO1 mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells mock treated or treated with etoposide. mRNA levels were assessed by qRT-PCR using exon 9-exon 10 (post-spliced-ATRIP) and exon 9-intron 9 (pre-spliced-ATRIP), exon 15-exon 16 (post-spliced-BACH1) and exon 15-intron 15 (pre-spliced- BACH1), and exon 1-exon 2 (post-spliced-EXO1) and exon 1-intron 1 (pre-spliced- EXO1) primers and normalized to ACTB mRNA. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM. Significance of changes in splicing ratios was assessed using Student’s two-tailed t test with significant changes indicated by ∗∗p < 0.01.(B) Semiquantitative PCR analysis of a cDNA generated from DNase-treated RNA, collected from control (siCtrl) and BRCA1- or BCLAF1-depleted cells, mock treated or treated with Etoposide. Primers targeting two independent intronic regions within ATRIP, BACH1, and EXO1 and a single intronic region within ATM and CHEK2 (control genes) were used for semiquantitative PCR analysis. Exon-spanning primers targeted against ACTB were used as a loading control.(C) Normalized expression of prespliced and postspliced mRNAs evaluated in (A).(D) Expression levels of postspliced and prespliced ATRIP mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells, transfected with control siRNA (siCtrl) or depleted of SMG1 (siSMG1), a key regulator of the non-sense-mediated decay pathway. Normalized expression levels were quantified as in (A). Graphs represent the mean normalized expression from three independent experiments ± SEM. See also Figures S4 and S5.
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fig4: The BRCA1/BCLAF mRNA Splicing Complex Promotes the Splicing and Stability of ATRIP, BACH1, and EXO1 Transcripts following DNA Damage(A) Ratio of postspliced to prespliced ATRIP, BACH1, and EXO1 mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells mock treated or treated with etoposide. mRNA levels were assessed by qRT-PCR using exon 9-exon 10 (post-spliced-ATRIP) and exon 9-intron 9 (pre-spliced-ATRIP), exon 15-exon 16 (post-spliced-BACH1) and exon 15-intron 15 (pre-spliced- BACH1), and exon 1-exon 2 (post-spliced-EXO1) and exon 1-intron 1 (pre-spliced- EXO1) primers and normalized to ACTB mRNA. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM. Significance of changes in splicing ratios was assessed using Student’s two-tailed t test with significant changes indicated by ∗∗p < 0.01.(B) Semiquantitative PCR analysis of a cDNA generated from DNase-treated RNA, collected from control (siCtrl) and BRCA1- or BCLAF1-depleted cells, mock treated or treated with Etoposide. Primers targeting two independent intronic regions within ATRIP, BACH1, and EXO1 and a single intronic region within ATM and CHEK2 (control genes) were used for semiquantitative PCR analysis. Exon-spanning primers targeted against ACTB were used as a loading control.(C) Normalized expression of prespliced and postspliced mRNAs evaluated in (A).(D) Expression levels of postspliced and prespliced ATRIP mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells, transfected with control siRNA (siCtrl) or depleted of SMG1 (siSMG1), a key regulator of the non-sense-mediated decay pathway. Normalized expression levels were quantified as in (A). Graphs represent the mean normalized expression from three independent experiments ± SEM. See also Figures S4 and S5.
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).

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