Figure 7. An apoptotic alternative splicing program linked to cell cycle control.

(A) ASF/SF2 downregulation triggers apoptosis, and sensitizes cells to apoptosis induction by AURKA inhibition. HeLa cells were transfected with indicated siRNAs, incubated to allow factor depletion, then treated with varying concentrations of VX-680. Apoptosis was measured via cleavage of a fluorescent caspase-3 substrate. Depletion of ASF/SF2, but not SRPK1, SNRP70, or Sam68, sensitized cells to VX-680-induced apoptosis. This selective sensitization by ASF/SF2 depletion was not observed for staurosporine-induced apoptosis (see Figure S6D). Data reflect means of 3 independent transfections +/− SDs.

(B) Analysis in (A) was repeated using Annexin-V staining as the apoptosis marker.

(C) CASP9 and CASP2 AS were analyzed in HeLa cells by qPCR following transfection with the indicated siRNAs. Relative levels of pro-apoptotic isoform (L) normalized to negative controls are shown. Values are means of 3 independent measurements +/− SD.

(D) Results of AS profiling by real-time qPCR are shown in a heat-map of z-scores averaged across four biological replicates, along with unsupervised hierarchical clustering (see Figure S7 for an expanded view and validation of this data).

(E) A model of cell cycle arrest coupled to apoptosis via AS is shown. We propose anticipatory, pro-apoptotic splicing decisions in interphase that later promote apoptosis in arrested cells. Lightning bolts represent siRNAs, drugs, or other stimuli that disrupt the cell cycle and promote pro-apoptotic splicing of Bcl-x, Mcl1, CASP9, and other targets.