Fig 2: Identification of exons regulated by ZRANB2. (A) Types of AS events regulated by ZRANB2 depletion for 48 h in MCF7-DoxoR cells (RNA-seq analysis). (B) Direction of regulation of ASE events by ZRANB2 depletion. (C) ZRANB2-regulated ASEs with a nearby ZRANB2-binding site found by CLIP-seq. In bold, events that were validated by RT-PCR for regulation by ZRANB2 (all the tested events were validated). (D) Enriched functions (Ingenuity Pathway Analysis) in genes with AS regulation by ZRANB2. (E) Comparison of ZRANB2-regulated AS events with AS events regulated in MCF7-DoxoR versus MCF-7 cells. (F) RT-PCR validations of AS events regulated by siRNAs targeting ZRANB2 (whose depletion was verified in Figure 2B) and in MCF7-DoxoR versus MCF-7 cells. The effects of ZRANB2 siRNAs on FGFR1OP were weak but reproducible.

Fig 3: ZRANB2 and SYF2 splicing programs converge on the ECT2-Ex5+ splice variant. (A) Overlap between AS events regulated by ZRANB2 and SYF2 in RNA-seq. Events that were regulated in the opposite direction in MCF7-DoxoR versus MCF-7 cells are underlined, and those that were validated by RT-PCR are in bold. (B) RT-PCR validation of AS regulation in the MAST2, MRPL55 and ECT2 genes and RT-qPCR analysis of ECT2 isoforms and total mRNA levels in MCF7-DoxoR versus MCF-7 cells, and in MCF7-DoxoR cells transfected with siRNAs targeting ZRANB2 and SYF2 (whose depletion was verified in Figure 2B and C). (C, D) RT-qPCR analysis of ECT2 isoforms and total mRNA levels (C), and WST1 analysis of Doxo survival (D, normalized to untreated cells as in Figure 2) in MCF7-DoxoR cells transfected with siRNA pools #1 targeting ZRANB2 (siZ) and SYF2 (siS) alone and in combination (siZ+S). ZRANB2 and SYF2 deletion are shown in Supplementary Figure S4F. (E) CLIP-qPCR analysis of ZRANB2 and SYF2 association with ECT2 pre-mRNA at the indicated locations. IgG, control immunoglobulin.

Fig 4: An RNAi screen on splicing factors identifies ZRANB2 and SYF2 as mediators of Doxo resistance. (A) RNAi screen in MCF7-DoxoR cells. Following transfection and two-day recovery, cells were grown for three days with or without Doxo, and cell survival was assessed using a WST1 assay. For each siRNA, WST1 signal in the presence of Doxo was divided by WST1 signal in the absence of Doxo, and was expressed as percent of the data obtained with an siRNA that targets no gene (Ctl). (B, C) Validation of the effects of siRNAs targeting ZRANB2 (B) and SYF2 (C) on their protein levels (Western blot, bottom panels) and Doxo survival (WST1 assay as above, top panels) in MCF7-DoxoR cells. (D, E) Analysis of ZRANB2 and SYF2 expression levels in MCF7-DoxoR and MCF-7 cells by RT-qPCR (D) and Western blot (E). RT-qPCR data were normalized to 18S RNA levels.