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. 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.