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）是一类由未翻译的mRNA-蛋白质复合物（mRNPs）组装形成的应激诱导型RNA-蛋白质聚集体。哺乳动物来源的SGs呈现非均质性，包含稳定存在于细胞裂解液中的“核心”结构，且不同核心的蛋白质组成存在异质性。目前学界尚未明确决定RNA招募至SGs的分子互作机制，以及不同核心结构间的RNA组成是否存在差异。本研究通过在0.5mM亚砷酸盐与0.5M山梨醇两种应激条件下，分别纯化PABPC1与G3BP标记的SG核心结构，解析了SG的转录组谱。我们观察到，无论采用何种蛋白用于核心纯化，相似的RNA均会被招募至SGs，这提示单个RNA结合蛋白（RNA-binding proteins，RBPs）在调控SG转录组组成的过程中仅发挥有限作用。针对山梨醇诱导的SG转录组的分析显示，G3BP1/2对招募至SGs的RNA几乎无调控作用，表明RNA定位至SGs并不依赖于单个RBPs。综上，我们认为RNA向SGs的分配至少不依赖于部分蛋白质，这与SGs通过分子间RNA-RNA相互作用形成的理论相符。整体实验设计：从U-2 OS细胞中纯化应激颗粒核心RNA，具体方法为通过差速离心与免疫沉淀实验，分别使用PABP抗体或GFP抗体（针对GFP-G3BP U-2OS细胞系）富集目标RNA。此外，我们分别从应激处理与未处理的细胞中收集总细胞质RNA（已通过离心去除细胞核）。应激处理的细胞分别采用0.5mM亚砷酸盐或0.5M山梨醇进行诱导。