Fig. 6

Splicing targeting drugs affects 3’-end processing genes through intron retention and alternative last exon selection. A Venn diagram showing the overlap of regulated alternative last exon (ALE) and intron-retention (IR) events regulated by Indisulam (Ind), Pladienolide B (PladB) and THZ531 in 22Rv1 cells, according to our RNA-seq data. B, C Bar graph illustrating for genes regulated by IR and ALE by at least two drugs (see panel A) annotation of significantly enriched pathways in the Reactome database (B) and putatively regulatory transcription factors according to query of indicated ChiP-seq databases (C) (analysis performed with Enrichr tool, p-value < 0.05). D Heatmap showing the gene-expression fold-change for indicated genes of the cleavage and polyadenylation (CPA) machinery of 3’-end mRNA: CPA specificity factor (CPSF), cleavage factor I (CFI) and II (CFII), cleavage stimulation factor (CSTF) and other genes according to RNA-seq data of 22Rv1 treated with indicated drugs with respect to DMSO treated cells. Circles inscribed in a square indicate regulation by an ALE or IR event. E Schematic visualization in the EASANA database of the RNA-seq reads profile for CSTF3 (upper panel) and PCF11 (lower panel) genes in 22Rv1 cells treated with either Ind, Plad B, THZ531 with respect to the DMSO. P-value and log2 of the gene expression fold-change with respect to DMSO are indicated. F) qPCR analysis of CSTF3 and PCF11 full length transcripts in 22Rv1 cells treated (for 16 h) with indicated drugs compared to control. RPL34 was evaluated as loading control (mean ± SD, n = 3 one-way Anova). G, H Representative Western Blot analysis for CSTF3 (G) and PCF11 (H) in 22Rv1 cells treated for 24 h (G) or 16 h (H) with indicated drugs. ACTIN was evaluated as loading control. I) Bar graph showing the results of the densitometric analysis of the ratio of expression of indicated proteins with respect to ACTIN (mean ± SD, n = 3, one-way Anova)