G3BP2B stress granules regulate mRNA expression under ER stress [RNAseq_Exp3]. G3BP2B stress granules regulate mRNA expression under ER stress [RNAseq_Exp3]

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Their function is not completely understood. Formation of these organelles is driven by the nucleation of proteins such as G3BPs. G3BPs are RNA-binding proteins that condense into SGs following translation shutoff during the integrated stress response (ISR). Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. Here, we found a molecular tool to study G3BP condensation into SGs by mutating residue V11 of G3BPs. This conserved amino acid potentiates the G3BP-Caprin-1 complex, hence promoting SG assembly. Ribosome profiling revealed that disruption of G3BP condensation impacts mRNA expression during the ISR. Moreover, we found that G3BP2B robustly condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. This indicates that G3BP paralogs differentially regulate SG assembly and gene expression programs. Together, this work suggests that stress granule assembly promotes changes in gene expression to mediate stress-response during the ISR. Overall design: To investigate differences in regulation of gene expression by G3BP2B condensation, we performed Ribo-seq and total RNA-seq in U-2OS G3BP1/2 KO cells (Cell line shared by Dr. Nancy Kedersha, which was developed via CRISPR Cas9 deletions), and G3BP1/2 KO cells stably expressing either transgenic G3BP2B WT, or G3BP2B V11A N-terminally tagged to monomeric EGFP. All cell lines were treated with either DMSO or 1 µM thapsigargin (Tg) for 2 hours and harvested using two replicates per sample for Ribo-seq and total RNA-seq library preparation. For differential expression analysis, cell lines were analyzed relative to G3BP1/2 KO as the reference sample. We also performed differential expression analysis by comparing Tg treated samples against DMSO treated samples to validate the activation of the integrated stress response. Furthermore, G3BP2B V11A was analyzed relative to G3BP2B WT to determine gene expression changes sensitive to stress granule deficiency.

应激颗粒（Stress granules，SGs）是在细胞应激条件下形成的大分子组装体，其功能尚未完全阐明。这类细胞器的形成由G3BP等蛋白的成核过程所驱动。G3BP是一类RNA结合蛋白（RNA-binding proteins），在整合应激反应（integrated stress response，ISR）期间翻译关闭后，会凝聚形成应激颗粒。脊椎动物中已鉴定出三种G3BP旁系同源蛋白（G3BP1、G3BP2A和G3BP2B）。本研究中，我们通过对G3BP的V11位点进行突变，开发出了一种研究G3BP凝聚形成应激颗粒的分子工具。该保守氨基酸可增强G3BP-Caprin-1复合物的活性，进而促进应激颗粒的组装。核糖体谱分析（Ribosome profiling）结果显示，G3BP凝聚的破坏会影响整合应激反应过程中的mRNA表达水平。此外，我们发现G3BP2B可在内质网（endoplasmic reticulum，ER）应激条件下高效凝聚，并促进mRNA表达的改变。这表明G3BP旁系同源蛋白可差异性调控应激颗粒的组装与基因表达程序。综上，本研究表明，在整合应激反应过程中，应激颗粒的组装可通过促进基因表达改变来介导应激应答。实验设计概述：为探究G3BP2B凝聚对基因表达调控的差异，我们在U-2OS细胞G3BP1/2双敲除（KO）细胞（该细胞系由Nancy Kedersha博士共享，通过CRISPR-Cas9基因编辑技术构建）以及稳定表达转基因野生型G3BP2B或N端融合单体增强型绿色荧光蛋白（monomeric EGFP）的G3BP2B V11A突变体的G3BP1/2 KO细胞中开展了核糖体谱测序（Ribo-seq）和总RNA测序（total RNA-seq）。所有细胞系均分别用二甲基亚砜（DMSO）或1 μM毒胡萝卜素（thapsigargin，Tg）处理2小时，每个样本设置两个生物学重复，用于核糖体谱测序和总RNA测序文库的构建。在差异表达分析中，我们以G3BP1/2 KO细胞作为参照样本，对其余细胞系进行分析。此外，我们通过比较毒胡萝卜素处理组与二甲基亚砜处理组的样本，开展差异表达分析以验证整合应激反应的激活。进一步地，我们以野生型G3BP2B细胞系为参照，对G3BP2B V11A突变体细胞系进行分析，以确定对应激颗粒缺陷敏感的基因表达变化。