Aulas Anaïs, Fay Marta M, Szaflarski Witold, Kedersha Nancy, Anderson Paul, Ivanov Pavel
Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital; Department of Medicine, Harvard Medical School.
Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital; Department of Medicine, Harvard Medical School; Department of Histology and Embryology, Poznan University of Medical Sciences.
J Vis Exp. 2017 May 12(123):55656. doi: 10.3791/55656.
Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.
细胞常常面临突然的环境变化。应激颗粒（SGs）是在暴露于应激条件的细胞中形成的细胞质核糖核蛋白复合物，与细胞代谢和存活的各个方面都有关联。应激颗粒调节细胞信号通路、转录后基因表达和应激反应程序。这些含mRNA颗粒的形成与细胞翻译直接相关。应激颗粒的组装由翻译起始抑制触发，而应激颗粒的解体则由翻译激活或翻译延伸抑制促进。应激颗粒的组成进一步突出了这种关系。应激颗粒的核心成分是停滞的翻译起始前复合物、mRNA和选定的RNA结合蛋白（RBPs）。应激颗粒组装的目的是通过隔离翻译停滞的管家mRNA来保存细胞能量，从而增强应激反应蛋白的翻译。除了核心成分，如停滞的翻译起始前复合物外，应激颗粒还包含大量其他蛋白质和信号分子。应激颗粒形成的缺陷会损害细胞对应激的适应能力，从而促进细胞死亡。应激颗粒和类似的含RNA颗粒与许多人类疾病有关，包括神经退行性疾病和癌症，这引发了近期对RNA颗粒亚型进行分类和定义的兴趣。本方案描述了表征和定量哺乳动物应激颗粒的测定方法。
