Fig. 6: APA reporter assays indicate that CPA inhibition by JTE-607 correlates with PAS usage level. | Nature Communications

Fig. 6: APA reporter assays indicate that CPA inhibition by JTE-607 correlates with PAS usage level.

From: Elevated pre-mRNA 3′ end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation

Fig. 6: APA reporter assays indicate that CPA inhibition by JTE-607 correlates with PAS usage level.The alternative text for this image may have been generated using AI.

a The degree of 3′UTR lengthening elicited by JTE-607, as indicated by the relative expression difference of top two 3′UTR isoforms (Fig. 5a), is a function of aUTR size. All genes with detectable expression of 3′UTR isoforms (n = 2205) were divided into five equally sized bins based on their aUTR size. The aUTR size range for each bin is shown. Error bars are standard error of mean. b Scatter plot comparing 3′UTR isoform abundance difference between HepG2 and HeLa cells. Each dot represents a gene with two selected 3′UTR isoforms. Genes showing lengthened and shortened 3′UTRs in HepG2 compared to HeLa are in red and blue, respectively. c Schematic of the pTRE-RiG construct. TRE tetracycline response element, RFP red fluorescent protein, GFP green fluorescent protein, IRES internal ribosome entry site. As indicated, ratio of red to green fluorescent signals, or log2(R/G), reflects the relative usage of pPAS vs. dPAS. d Schematic showing two versions of pPAS, i.e., AD and AE, with different PAS motifs. e Percentage of long isoform expressed in HepG2 or HeLa cells after transfection of various constructs. f Reduction of log2(R/G) value in HepG2 and HeLa cells transiently transfected with pTRE-RiG-AD (weak pPAS, 1× GFP) after JTE-607 treatments. Linear regression is based on averaged values of two biological replicates. P value (t-test) comparing values for 10 μM JTE-607 data is indicated. g The degree of inhibition of pPAS usage by JTE-607, based on Δlog2(R/G) between 10 μM and 0.1 μM (y-axis), correlates with its usage level in the control condition (DMSO-treated), as indicated by the percent of short isoform expressed (x-axis). Polynomial regression is based on averaged values of two biological replicates. h Bar graph showing pPAS usage levels of pTRE-RiG-AD, indicated by log2(R/G), in HeLa and HepG2 cells. Data for episomal form (transient transfection) or genomic form (integrated via piggyBac transposase) of the reporter are shown. P values are based on t-test. i As in f, except that the construct was genome-integrated. j Schematic summarizing mode of action of JTE-607 (CPAi) in APA regulation. Source data are provided as a Source Data file.

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