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. 2016 Sep;22(9):1441–1453. doi: 10.1261/rna.055657.115

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© 2016 Hollerer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

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FIGURE 3. — Transcriptional and post-transcriptional mechanisms target different groups of genes. (A) Genes were grouped according to the pattern of stress responses on the pre-mRNA and mRNA levels. Group I (blue) comprises post-transcriptionally regulated genes with regulated mRNA but unchanged or reversely regulated pre-mRNA levels. Group II (green) contains transcriptionally regulated genes with stress-induced mRNA and pre-mRNA levels. Group III (yellow) comprises genes with regulated pre-mRNA that are buffered at a post-transcriptional level and show unchanged or reversely regulated mRNA levels. (B) qRT-PCR validation of mRNA- and pre-mRNA-seq data of a subset of transcriptionally and post-transcriptionally up-regulated protein-coding genes. mRNA and pre-mRNA levels of genes randomly selected from group I, II, and III were compared to control genes (CBFB, HPRT). Data points represent mean + SEM of ≥ 3 independent qRT-PCR experiments normalized to GAPDH or preGAPDH, respectively. Two-sided Student's t-test was used to calculate P-values. (C,D) qRT-PCRs measuring mRNA and pre-mRNA levels of candidate genes (ZNF79, ZNF557, ZNF670, and RBM5) and not post-transcriptionally regulated control mRNAs (GADD45B, FOS, CBFB, and HPRT) after DTT (C) and arsenite treatment (D). Data were normalized to unstressed cells and GAPDH or preGAPDH in DTT-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. (E) Genes undergoing stress-induced APA were analyzed for total gene expression changes in stress and categorized into the groups I (blue), II (green), or III (yellow). The remaining genes either did not show a significant change in mRNA and pre-mRNA expression in stress (gray) or did not belong to any of the mentioned categories (black). These included genes in which stress treatment affected both mRNA and pre-mRNA expression but significantly regulated (P_adj < 0.01) only mRNA levels or vice versa.