G3BP2B stress granules regulate mRNA expression under ER stress [RNAseq_Exp2]. G3BP2B stress granules regulate mRNA expression under ER stress [RNAseq_Exp2]

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 G3BP paralogs, we performed Ribo-seq and total RNA-seq in U-2OS wild type cells, 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 G3BP1, G3BP2A, or G3BP2B 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.

应激颗粒（Stress granules, SGs）是一类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G3BPs等蛋白质的成核过程所驱动。G3BPs是一类RNA结合蛋白，在整合应激反应（integrated stress response, ISR）期间发生翻译停滞后，会通过相分离组装形成应激颗粒。目前已在脊椎动物中鉴定出3种G3BP旁系同源蛋白：G3BP1、G3BP2A与G3BP2B。本研究发现了一种可用于研究G3BP相分离形成应激颗粒的分子工具：通过定点突变G3BPs的V11残基。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。核糖体谱分析显示，G3BP相分离的破坏会影响整合应激反应期间的mRNA表达水平。此外，本研究发现G3BP2B可高效发生相分离，并在内质网（endoplasmic reticulum, ER）应激条件下显著改变mRNA表达谱。这表明G3BP旁系同源蛋白可差异性调控应激颗粒组装与基因表达程序。综上，本研究提示应激颗粒组装可通过改变基因表达程序，介导整合应激反应中的应激应答过程。

整体实验设计：为探究G3BP旁系同源蛋白对基因表达的调控差异，我们对三类细胞系开展了核糖体测序（Ribo-seq）与总RNA测序（total RNA-seq）：U-2OS野生型细胞、通过CRISPR-Cas9基因编辑构建的U-2OS G3BP1/2敲除细胞（该细胞系由Nancy Kedersha博士惠赠），以及分别稳定表达N端融合单体增强型绿色荧光蛋白（monomeric EGFP）的转基因G3BP1、G3BP2A或G3BP2B的G3BP1/2敲除细胞。所有细胞系均用二甲基亚砜（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，用于构建Ribo-seq与total RNA-seq文库。差异表达分析以G3BP1/2敲除细胞作为参照样本。此外，我们还通过比较毒胡萝卜素处理组与二甲基亚砜处理组的样本，验证了整合应激反应的激活。