Genotoxic stress triggers the selective activation of IRE1α-dependent RNA decay to modulate the DNA damage response

The molecular connections between homeostatic systems that sustain genome integrity and proteostasis are poorly understood. Here we identified the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to engage repair programs and sustain cell survival. DNA damage engages IRE1 signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) and not its canonical output mediated by the transcription factor XBP1. IRE1α controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The protective role of IRE1α under genotoxic stress is conserved in evolution as demonstreated using fly and mouse models. Altogether, our results uncovered a novel intersection between the molecular pathways that ensure genome stability and proteostasis. Affymetrix gene expression data were pre-processed using ‘affyPLM’ packages of the Bioconductor Software. Genes with the strongest evidence of differential expression were obtained using a linear model fit. Data obtained from untreated wild type or IRE1αcKO after ploy I:C treatment liver tissues were used as reference for tunicamycin (Tm) and etoposide (Eto) treatment respectively. Custom chip definition file version 22 from Brainarray based on Entrez ID’s was used. A false positive rate of a=0.05 with FDR correction and a fold change greater 1.5 was taken as the level of significance. Liver tissue were obteined from a conditional knockout (cKO) mice controlled by the Mx-Cre system for the deletion of IRE1a gene. Poly[I:C] was injected to induce Cre expression, and three weeks later animals were treated with a single dose of either etoposide or tunicamycin, followed by the analysis of gene expression of liver tissues. WT IRE1 = Mice with the full expression of IRE1a (ERN1), KO IRE1= Mice wiht IRE1a (ERN1) deletion, WT IRE1 Eto 50ug 16h= Mice with the full expression of IRE1a (ERN1) treated with Etoposide 50ug/kg for 16 hours, WT IRE1 Tm 1ug 16h= Mice with the full expression of IRE1a (ERN1) treated with Tunicamicyn 1ug/kg for 16 hours, Fi/Fi Cre WT Eto50ug 6h=Mice with the full expression of IRE1a (ERN1) treated with Etoposide 50ug/kg for 6 hours,Fi/Fi Cre WT Tm1ug 6h=Mice with the full expression of IRE1a (ERN1) treated with Tunicamicyn 1ug/kg for 6 hours, KO IRE1 Eto 50ug 16h= Mice wiht IRE1a (ERN1) deletion,treated with Etoposide 50ug/kg for 16 hours, KO IRE1 Tm 1ug 16h= Mice wiht IRE1a (ERN1) deletion,treated with Tunicamicyn 1ug/kg for 16 hours, Fi/Fi Cre KO Eto 50ug 6h= Mice wiht IRE1a (ERN1) deletion,treated with Etoposide 50ug/kg for 6 hours,Fi/Fi Cre KO Tm 1ug 6h=Mice wiht IRE1a (ERN1) deletion, treated with Tunicamicyn 1ug/kg for 6 hours, WT IRE1 untreat = Mice with the full expression of IRE1a (ERN1) with DMSO, KO IRE1 untreat= Mice wiht IRE1a (ERN1) deletion with DMSO, Fi/Fi Cre WT untreat= Mice with the full expression of IRE1a (ERN1) with DMSO, Fi/Fi Cre KO untreat= Mice wiht IRE1a (ERN1) deletion with DMSO

维持基因组完整性的稳态系统与蛋白质稳态系统之间的分子关联机制目前仍未得到充分阐释。本研究发现，基因毒性应激下未折叠蛋白应答转导因子肌醇需求酶1α（IRE1α）会被选择性激活，从而启动修复程序并维持细胞存活。DNA损伤可在未检测到内质网（ER）应激特征的情况下激活IRE1α信号通路，仅触发调控性IRE1α依赖降解（RIDD）的活化，而非由转录因子X盒结合蛋白1（XBP1）介导的经典下游效应。IRE1α可调控参与DNA损伤应答的mRNA稳定性，进而影响DNA修复、细胞周期阻滞与细胞凋亡过程。基因毒性应激下IRE1α的保护作用在进化过程中具有保守性，该结论通过果蝇与小鼠模型得到验证。综上，本研究揭示了保障基因组稳定性与蛋白质稳态的分子通路之间存在全新的交叉互作节点。

Affymetrix基因表达芯片数据使用生物导体（Bioconductor）软件的'affyPLM'包进行预处理。通过线性模型拟合筛选得到差异表达最显著的基因。分别以未经处理的野生型小鼠肝脏组织，或经聚肌胞苷酸（poly I:C）处理后的IRE1α条件性敲除（IRE1αcKO）小鼠肝脏组织作为衣霉素（Tm）与依托泊苷（Eto）处理组的参照样本。本研究使用基于Entrez基因ID（Entrez ID）的Brainarray定制芯片注释文件版本22。本研究以经过错误发现率（FDR）校正后的假阳性率α=0.05，且折叠变化大于1.5作为显著性阈值。

本研究的肝脏组织取自通过Mx-Cre系统介导IRE1α基因敲除的条件性敲除（cKO）小鼠。通过注射聚肌胞苷酸（poly[I:C]）诱导Cre重组酶表达，三周后分别给予小鼠单次剂量的依托泊苷或衣霉素，随后对肝脏组织的基因表达进行分析。
WT IRE1：完整表达IRE1α（ERN1）的野生型小鼠；
KO IRE1：IRE1α（ERN1）基因敲除小鼠；
WT IRE1 Eto 50ug 16h：以50μg/kg剂量的依托泊苷处理16小时的完整表达IRE1α（ERN1）的野生型小鼠；
WT IRE1 Tm 1ug 16h：以1μg/kg剂量的衣霉素处理16小时的完整表达IRE1α（ERN1）的野生型小鼠；
Fi/Fi Cre WT Eto50ug 6h：以50μg/kg剂量的依托泊苷处理6小时的完整表达IRE1α（ERN1）的野生型小鼠；
Fi/Fi Cre WT Tm1ug 6h：以1μg/kg剂量的衣霉素处理6小时的完整表达IRE1α（ERN1）的野生型小鼠；
KO IRE1 Eto 50ug 16h：以50μg/kg剂量的依托泊苷处理16小时的IRE1α（ERN1）基因敲除小鼠；
KO IRE1 Tm 1ug 16h：以1μg/kg剂量的衣霉素处理16小时的IRE1α（ERN1）基因敲除小鼠；
Fi/Fi Cre KO Eto 50ug 6h：以50μg/kg剂量的依托泊苷处理6小时的IRE1α（ERN1）基因敲除小鼠；
Fi/Fi Cre KO Tm 1ug 6h：以1μg/kg剂量的衣霉素处理6小时的IRE1α（ERN1）基因敲除小鼠；
WT IRE1 untreat：经二甲基亚砜（DMSO）处理的完整表达IRE1α（ERN1）的野生型小鼠；
KO IRE1 untreat：经二甲基亚砜（DMSO）处理的IRE1α（ERN1）基因敲除小鼠；
Fi/Fi Cre WT untreat：经二甲基亚砜（DMSO）处理的完整表达IRE1α（ERN1）的野生型小鼠；
Fi/Fi Cre KO untreat：经二甲基亚砜（DMSO）处理的IRE1α（ERN1）基因敲除小鼠。