Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress. Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress

The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of Growth Arrest and DNA Damage inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We established that the 3' untranslated region of PPP1R15A mRNA contains an active AU-rich element recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a key step in establishing the ISR molecular memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions. Overall design: Two replicates of Huh7 control cell clone #1 as well as two replicates of Brf1/TTP DKO cell clone #18 have been treated with DMSO for 3h prior to a 10-min treatment with 5 µg/ml actinomycin D in DMSO. Cells were harvested, total RNA extracted and sequenced.

整合应激反应（integrated stress response, ISR）是一类由环境扰动激活的关键细胞信号通路，其通过抑制蛋白质合成以恢复细胞稳态。为避免持续细胞损伤，ISR可通过上调生长阻滞与DNA损伤诱导蛋白34（Growth Arrest and DNA Damage inducible 34, GADD34）进行拮抗；该蛋白是蛋白磷酸酶1的应激诱导型调节亚基，可介导翻译重启与应激恢复。本研究揭示了一种全新的ISR调控机制，该机制通过转录后水平调控编码GADD34的PPP1R15A mRNA的稳定性。本研究证实，PPP1R15A mRNA的3'非翻译区（3' untranslated region, 3'UTR）包含一段可被ZFP36家族蛋白识别的功能性AU富集元件（AU-rich element, ARE），该元件可在正常培养条件下促进mRNA快速降解，而在应激条件下则维持mRNA稳定，以实现GADD34的高效表达。本研究明确，对PPP1R15A mRNA代谢的严格时序调控是构建ISR分子记忆的关键步骤，该调控可设定细胞的应答阈值，并介导细胞对反复应激条件的适应。实验设计：设置两组生物学重复，分别为Huh7对照细胞克隆#1与Brf1/TTP双敲除细胞克隆#18。两组细胞均先用二甲基亚砜（dimethyl sulfoxide, DMSO）处理3小时，随后以5 μg/ml放线菌素D的DMSO溶液处理10分钟后收集细胞，提取总RNA并进行测序。