Stress-induced APA can quantitatively modulate the expression of distinct mRNA isoforms. (A) IGV browser data displaying the 3′T-fill, mRNA, and pre-mRNA-seq data for the genes RORA (upper panel), SETD4 (middle panel), and SNX5 (lower panel) in stress (red) and control conditions (black) as examples for group I APA targets. The 3′T-fill lanes show poly(A) sites used under stressed (anisomycin) and unstressed (DMSO) conditions. Detected poly(A) sites are numbered and poly(A) sites that are significantly regulated relative to other poly(A) sites within the same gene are highlighted with green boxes. The mRNA-seq lanes display the expression of poly(A)+ mRNA and the pre-mRNA-seq lanes the expression of intronic regions. The bottom lanes schematically represent the mRNA transcripts of the respective genes as provided by Ensembl 73 with coding exons as filled boxes, 3′UTRs as open boxes, and introns as lines. The bar diagrams on the right display the mean 3′T-fill expression data for individual poly(A) sites (upper panel) and the quantification of total mRNA (middle panel) or pre-mRNA levels (lower panel) as obtained in mRNA- and pre-mRNA-seq sequencing analyses of three independent biological replicates. The gray and blue arrows indicate the primers used in the qRT-PCR displayed in panel C to specifically amplify the “short,” the “long,” or both (“short + long”) mRNA isoforms of the respective genes. (B) qRT-PCRs on cells treated with anisomycin, arsenite, or DTT using primers specific for the “short,” the “long,” or both (“short + long”) isoforms of the genes RORA, SETD4, SNX5, TRMT44, and TROAP. Pre-mRNA levels were assessed to confirm that changes in gene expression happened post-transcriptionally. Data were normalized to unstressed cells and GAPDH or preGAPDH in DTT- and anisomycin-treated cells. Because arsenite treatment influenced GAPDH pre-mRNA levels, pre-mRNA data were normalized to preCBFB in arsenite-treated cells. Bars represent mean + SEM, n ≥ 3. Two-sided Student's t-test was applied to calculate P-values.
