RNA partitioning into stress granules is independent of G3BP. RNA partitioning into stress granules is independent of G3BP

Stress granules (SGs) are stress induced RNA-protein assemblies formed from untranslating mRNPs. Mammalian SGs are non-uniform and contain “cores”, which are stable in lysates and heterogenous in protein composition. The interactions defining RNA recruitment into SGs, and whether the RNA composition varies between different cores remains unclear. Herein, we determine the SG transcriptome through purification of both PABPC1 and G3BP SG cores during both arsenite and sorbitol stress. We observe that similar RNAs are recruited to SGs independent of the protein used for core purification, suggesting individual RNA-binding proteins (RBPs) may play a limited role in defining the SG transcriptome. Analysis of the sorbitol-induced SG transcriptome reveals G3BP1/2 has little effect on RNAs recruited to SGs suggesting RNAs localize to SGs independent of individual RBPs. Taken together, we suggest that partitioning of RNAs to SGs is independent of at least some proteins, consistent with SGs forming through intermolecular RNA-RNA interactions. Overall design: Stress granule core RNA was purified from U-2 OS cells via differential centrifugation and immunoprecipitation using a PABP antibody or GFP antibody (in the case of GFP-G3BP U-2OS cells). In addition, we collected total cytoplasmic RNA (nuclei were spun out) from stressed and unstressed cells. Stressed cells were either stressed with .5mM arsenite or .5M sorbitol.

应激颗粒（Stress granules, SGs）是由未翻译的mRNA-蛋白复合物（messenger ribonucleoprotein particles, mRNPs）组装形成的应激诱导型RNA蛋白组装体。哺乳动物细胞中的应激颗粒并非均质性结构，其包含“核心区域”，该区域在裂解液中保持稳定，且蛋白组成具有异质性。目前尚未明确介导RNA招募至应激颗粒的相互作用机制，以及不同核心区域间的RNA组成是否存在差异。本研究通过在亚砷酸盐（arsenite）与山梨醇（sorbitol）应激处理条件下，纯化结合多聚腺苷酸结合蛋白C1（poly(A)-binding protein C1, PABPC1）与G3BP蛋白（G3BP）的应激颗粒核心区域，从而解析应激颗粒的转录组。我们观察到，无论用于核心区域纯化的蛋白类型如何，相似的RNA均会被招募至应激颗粒，这提示单个RNA结合蛋白（RNA-binding proteins, RBPs）在界定应激颗粒转录组的过程中仅发挥有限作用。对山梨醇诱导的应激颗粒转录组的分析显示，G3BP1/2对招募至应激颗粒的RNA几乎无影响，这表明RNA定位于应激颗粒的过程不依赖于单个RNA结合蛋白。综上，我们认为RNA向应激颗粒的分选定位至少不依赖于部分蛋白，这与应激颗粒通过分子间RNA-RNA相互作用形成的假说相符。
整体实验设计：从U-2 OS细胞中纯化应激颗粒核心RNA，具体方法为差速离心结合免疫沉淀：使用多聚腺苷酸结合蛋白（poly(A)-binding protein, PABP）抗体；对于表达GFP-G3BP融合蛋白的U-2OS细胞，则使用绿色荧光蛋白（green fluorescent protein, GFP）抗体进行免疫沉淀。此外，我们从应激处理与未处理的细胞中收集了总细胞质RNA（细胞核已通过离心去除）。应激处理的细胞分别采用0.5mM亚砷酸盐或0.5M山梨醇进行应激诱导。