Centre de recherche en cancérologie. Centre de recherche du CHU de Québec. Département de biologie moléculaire, biochimie médicale et pathologie, Faculté de médecine, Université Laval, Québec, PQ, Canada.
Complexe de diagnostic et d'épidémiosurveillance vétérinaires du Québec (CDEVQ) Université de Montréal, Montréal, Canada.
Sci Rep. 2017 Oct 23;7(1):13832. doi: 10.1038/s41598-017-14262-7.
Accumulation of unfolded and potentially toxic proteins in the endoplasmic reticulum (ER) activates a cell stress adaptive response, which involves a reprogramming of general gene expression. ATF4 is a master stress-induced transcription factor that orchestrates gene expression in cells treated with various ER stress inducers including those used to treat cancers. ER stress-induced ATF4 expression occurs mainly at the translational level involving the activity of the phosphorylated (P) translation initiation factor (eIF) eIF2α. While it is well established that under ER stress PeIF2α drives ATF4 expression through a specialised mode of translation re-initiation, factors (e.g. RNA-binding proteins and specific eIFs) involved in PeIF2α-mediated ATF4 translation remain unknown. Here we identified the RNA-binding protein named DDX3 as a promotor of ATF4 expression in cancer cells treated with sorafenib, an ER stress inducer used as a chemotherapeutic. Depletion experiments showed that DDX3 is required for PeIF2α-mediated ATF4 expression. Luciferase and polyribosomes assays showed that DDX3 drives ER stress-induced ATF4 mRNA expression at the translational level. Protein-interaction assays showed that DDX3 binds the eIF4F complex, which we found to be required for ER stress-induced ATF4 expression. This study thus showed that PeIF2α-mediated ATF4 mRNA translation requires DDX3 as a part of the eIF4F complex.
内质网 (ER) 中未折叠和潜在毒性蛋白质的积累会激活细胞应激适应性反应，涉及到一般基因表达的重新编程。ATF4 是一种主要的应激诱导转录因子，可协调各种 ER 应激诱导剂处理的细胞中的基因表达，包括用于治疗癌症的诱导剂。ER 应激诱导的 ATF4 表达主要发生在翻译水平，涉及磷酸化（P）翻译起始因子 (eIF) eIF2α 的活性。虽然已经证实，在 ER 应激下，PeIF2α 通过一种特殊的翻译重新起始模式驱动 ATF4 表达，但 PeIF2α 介导的 ATF4 翻译所涉及的因素（例如 RNA 结合蛋白和特定的 eIF）仍然未知。在这里，我们鉴定了一种名为 DDX3 的 RNA 结合蛋白，它是在用 ER 应激诱导剂索拉非尼处理的癌细胞中 ATF4 表达的促进剂，索拉非尼是一种用于化疗的药物。耗尽实验表明，DDX3 是 PeIF2α 介导的 ATF4 表达所必需的。荧光素酶和多核糖体测定表明，DDX3 在翻译水平上驱动 ER 应激诱导的 ATF4 mRNA 表达。蛋白相互作用测定表明，DDX3 结合 eIF4F 复合物，我们发现该复合物是 ER 应激诱导的 ATF4 表达所必需的。因此，本研究表明，PeIF2α 介导的 ATF4 mRNA 翻译需要 DDX3 作为 eIF4F 复合物的一部分。
