SRP409216 PRJNA904279 GSE218545 Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation [ChrRNA-seq] Cleavage and polyadenylation (CPA) defines the 3' end of almost all eukaryotic mRNAs. CPA inhibition, or CPAi, leads to transcriptional readthrough. Here, we show that the CPSF-73 inhibitor JTE-607 globally perturbs gene expression, especially for those with a high GC content and located in high gene density regions. Based on regulated alternative polyadenylation (APA) events, we found that more frequently used CPA sites are inhibited by JTE-607 to a greater extent. Consistently, cells with elevated CPA activities, as indicated by preferential usage of proximal APA sites, display greater transcriptional readthrough and gene expression disturbance upon JTE-607 treatment. Remarkably, overexpression of the core CPA factor FIP1 enhances global CPA activity in the cell and leads to greater JTE-607 sensitivity. Taken together, our data indicate that CPAi selectively impacts genes based on their genomic features and the CPA activity of a cell is a key determinant of sensitivity to CPAi. Overall design: For chromatin RNA sequencing, HeLa, HepG2, U937 and U937MP cells were treated with DMSO or 1 µM JTE-607 for 2 hrs before cells were collected for chromatin RNA extraction. GSE218545 pubmed 37528120 parent_bioproject PRJNA904308