Figure 4.

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.