Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada.
Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.
J Biol Chem. 2022 Aug;298(8):102180. doi: 10.1016/j.jbc.2022.102180. Epub 2022 Jun 23.
The integrated stress response is a network of highly orchestrated pathways activated when cells are exposed to environmental stressors. While global repression of translation is a well-recognized hallmark of the integrated stress response, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD box 1 (DDX1) protein accumulates at DNA double-strand breaks during genotoxic stress and promotes DNA double-strand break repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors, including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing, we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs are increased during stress in a DDX1-dependent manner. Even though DDX1's RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.
整合应激反应是细胞暴露于环境应激源时激活的高度协调途径的网络。虽然翻译的全局抑制是整合应激反应的一个公认标志，但在应激过程中 mRNA 稳定性的调节知之甚少。DEAD 盒蛋白是一类参与 RNA 代谢各个方面的 RNA 解旋/重塑酶。我们之前表明，DEAD 盒蛋白 1（DDX1）蛋白在遗传毒性应激时在 DNA 双链断裂处积累，并通过同源重组促进 DNA 双链断裂修复。在这里，我们研究了 DDX1 在应对环境应激中的作用。我们表明，DDX1 被募集到细胞中暴露于各种环境应激源（包括亚砷酸盐、过氧化氢和 thapsigargin）时的应激颗粒（SG）中。我们还表明，DDX1 耗竭会延迟亚砷酸盐诱导的 SG 的解决。使用 RNA 免疫沉淀测序，我们鉴定了与内源性 DDX1 结合的 RNA 靶标，包括先前涉及应激反应的基因转录的 RNA。我们表明，当细胞暴露于应激时，与 DDX1 结合的靶 RNA 数量增加，并且这些 RNA 的总水平在 DDX1 依赖性方式下在应激期间增加。尽管 DDX1 的 RNA 结合特性对于维持其靶 mRNA 水平至关重要，但我们发现 RNA 结合对于 DDX1 到 SG 的定位不是必需的。此外，DDX1 敲低似乎不会影响 RNA 到 SG 的定位。总之，我们的结果揭示了 DDX1 在维持暴露于氧化应激的细胞中细胞质 mRNA 水平方面的新作用。
