Protein-protein interactions with G3BPs drive stress granule condensation and gene expression changes under cellular stress

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs condense into SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and stress-induced gene expression changes is incompletely understood. Here, we identified key residues for G3BP condensation such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that disruption of G3BP condensation corresponds to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.


Figure S1 :
Figure S1: Image analysis pipeline for segmentation of SGs in single cells.A. Image of U-2OS wild type cells forming stress granules under 200 µM NaAs for 2 hours captured by IF.SGs were stained with PABP.B. Single cell segmentation.C. Segmentation of PABP foci.D. Filtered PABP foci based on eccentricity.E. Distribution of SG eccentricity under water treatment as a control.SGs above the red vertical line were considered as artifacts.F. Distribution of SG eccentricity under 200 µM NaAs for 2 hours.SGs above the red vertical line were considered as artifacts.

Figure S2 :
Figure S2: Inhibition of PABP condensation by G3BP1/2 KO during the ISR. A. SGs stained with PABP by IF.Images are showing U-2OS wild type cells and G3BP1/2 KO cells under water or 200 µM NaAs for 2 hours.B. Percentage of cells with PABP foci from data shown in panel A. C. SGs stained with PABP by IF.Images are showing U-2OS wild type cells and G3BP1/2 KO cells under DMSO or 1 µM Tg for 2 hours.D. Percentage of cells with PABP foci from data shown in panel C. Plots B & D are showing mean ± SEM across Nreplicates = 3. * p < 0.05.

Figure S3 :
Figure S3: IDRs are critical for G3BP1 condensation under NaAs. A. Schematic of G3BP1 domains showing location of IDRs and S149 residue.B. Images of cells expressing mEGFP-G3BP1 variants at t = 0 hr and t = 2 hrs post-treatment with 200 µM NaAs.C. Percentage of cells with G3BP1 foci.Vertical red dashed line shows when NaAs was added to cells.D. Total area of G3BP1 foci per cell at 2 hours under NaAs.E. G3BP1 foci count per cell at 2 hours under NaAs.Plots C-E are showing mean ± SEM across Nreplicates ≥ 3. P-values were calculated based on whole cell populations (ncells ≥ 100 per replicate) relative to G3BP1 WT .* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.F. Schematic of the G3BP1 NTF2L domain (light blue) interacting with a Caprin-1 motif (gold), PDB ID 6TA7.Location of mutated residues are highlighted (aquamarine).G. Cytoplasmic GFP intensities as a proxy for G3BP1 expression across single cells between G3BP1 variants pre-treated with NaAs.Horizontal dashed red lines represent ± 25% from G3BP1 WT median cytoplasmic GFP intensity.

Figure S6 :
Figure S6: ISR activation is not affected by G3BP1 condensation under NaAs. A. Western blot showing G3BP1 expression in U2OS wild type, G3BP1/2 KO, G3BP1 WT , and G3BP1 V11A cells.B. Quantification of G3BP1 levels across cell lines from data shown in panel A. mean ± SD across Nreplicates = 3. C. Western blot showing a time course of eIF2α phosphorylation for G3BP1/2 KO cells expressing G3BP1 WT under 200 µM NaAs.D. Quantification of eIF2α phosphorylation across time from data shown on panel C. mean ± SD across Nreplicates = 3. E. Differential expression plots for Ribo-seq and total RNA-seq.G3BP1/2 KO cells or cells expressing either transgenic G3BP1 WT or G3BP1 V11A were compared between NaAs and Control conditions to show induced expression of canonical ISR factors under NaAs.F. Averaged ΔTE of ISR factors across cell lines.Significance was calculated relative to G3BP1/2 KO data.G. Differential expression plot for Ribo-seq and total RNA-seq of G3BP1 V11A vs G3BP1 WT under NaAs.H. Ribo-seq (left) and RNA-seq (right) LFC from data shown on panel G. of G3BP1 WT sensitive genes identified on Fig. 3D.I. GSEA for SG-associated mRNAs overlapping with differentially translated gene sets from G3BP1 WT (left) and G3BP1 V11A Ribo-seq profiles.

Figure S7 :
Figure S7: Condensation of G3BP1/2 paralogs during the ISR. A. Images of cells expressing G3BP-mEGFP paralogs at t = 0 hr and t = 2 hrs post-treatment with 200 µM NaAs.B. Percentage of cells with G3BP foci.Vertical red dashed line shows when NaAs was added to cells.C. Total area of G3BP foci per cell at 2 hours under NaAs.D. G3BP foci count per cell at 2 hours under NaAs.E. Images of cells expressing G3BP-mEGFP paralogs at t = 0 hr and t = 2 hrs post-treatment with 1 µM Tg.F. Percentage of cells with G3BP foci.Vertical red dashed line shows when Tg was added to cells.G.Total area of G3BP foci per cell at 2 hours under Tg.H. G3BP foci count per cell at 2 hours under Tg.Plots B-D and F-H are showing mean ± SEM across Nreplicates ≥ 3. P-values were calculated based on whole cell populations (ncells ≥ 100 per replicate) relative to G3BP1.* p < 0.05.

Figure S8 :
Figure S8: G3BP1 condenses differently across NaAs and Tg stress.A. Images of cells expressing mEGFP-G3BP1 WT at t = 0 hr and t = 2 hrs post-treatment with DMSO, 200 µM NaAs, and 1 µM Tg.B. Percentage of cells with G3BP1 foci.Vertical red dashed line shows when treatments were applied to cells.C. Total area of G3BP1 foci per cell at 2 hours under treatment.D. G3BP1 foci count per cell at 2 hours under treatment.Plots B-D are showing mean ± SEM across Nreplicates ≥ 3. P-values were calculated based on whole cell populations (ncells ≥ 100 per replicate) relative to DMSO.**** p < 0.0001.E. Cytoplasmic GFP intensities as a proxy for G3BP1/2 expression across single cells between N-terminal tagged paralogs pre-treated with NaAs and Tg.F. Cytoplasmic GFP intensities as a proxy for G3BP1/2 expression across single cells between C-terminal tagged paralogs pre-treated with NaAs and Tg.Horizontal dashed red lines represent ± 25% from G3BP1 median cytoplasmic GFP intensity.

Figure S9 :
Figure S9: Endogenous G3BPs condense similarly during the ISR. A. SGs stained against G3BP1 and G3BP2 by IF.Images are showing U-2OS wild type cells and G3BP1/2 KO cells under water or 200 µM NaAs for 2 hours.B. Percentage of cells with G3BP1/2 foci from data shown in panel A. C. SGs stained against G3BP1 and G3BP2 by IF.Images are showing U-2OS wild type cells and G3BP1/2 KO cells under DMSO or 1 µM Tg for 2 hours.D. Percentage of cells with G3BP1/2 foci from data shown in panel C. Plots B & D are showing mean ± SEM across Nreplicates = 3.

Figure S10 :
Figure S10: Comparing G3BP1/2 expression across transgenic cell lines.A. Western blot showing expression of G3BPs and GFP across cell lines.B. Quantification of GFP levels across transgenic cell lines from data shown on panel A. C. Quantification of G3BP1 levels from data shown on panel A. D. Quantification of G3BP2 levels from data shown on panel A. For plots B-D, mean ± SD.E. RNA-seq TPMs of G3BP1 gene across cell lines.F. RNA-seq TPMs of G3BP2 gene across cell lines.G. Ribo-seq TPMs of G3BP1 gene across cell lines.H. Ribo-seq TPMs of G3BP2 gene across cell lines.I. Ribo-seq TPMs of total G3BP expression across cell lines.

Figure S15 :
Figure S15: ISR activation is not affected by G3BP2B condensation under Tg. A. Images of cells expressing mEGFP-G3BP2B variants at t = 0 hr and t = 1 hrs post-treatment with 200 µM NaAs.B. Percentage of cells with G3BP2B foci.Vertical red dashed line shows when NaAs was added to cells.C. Total area of G3BP2B foci per cell at 1 hour under NaAs.D. G3BP2B foci count per cell at 1 hour under NaAs.Plots B-D are showing mean ± SEM across Nreplicates ≥ 3. P-values were calculated based on whole cell populations (ncells ≥ 100 per replicate).****p < 0.0001.E. Cytoplasmic GFP intensities as a proxy for G3BP2B variant expression across single cells pre-treated with NaAs (upper) and Tg (lower).Horizontal dashed red lines represent ± 25% from G3BP2B median cytoplasmic GFP intensity.F. Western blot showing G3BP2 expression and eIF2α phosphorylation in U2OS wild type, G3BP1/2 KO, G3BP2B WT , and G3BP2B V11A cells in both DMSO and Tg treated conditions.G. Quantification of G3BP2 levels across cell lines from data shown in panel F. mean ± SD across Nreplicates = 3. H. Quantification of eIF2α phosphorylation across cell lines from data shown in panel F. mean ± SD across Nreplicates = 3. Significance was calculated relative to wild type cells.I. Differential expression plots for Ribo-seq and total RNAseq.G3BP1/2 KO cells or cells expressing either transgenic G3BP2B WT or G3BP2B V11A were compared between Tg and control conditions to show induced expression of canonical ISR factors under Tg.J. Averaged ΔTE of ISR factors across cell lines.Significance was calculated relative to G3BP1/2 KO data.

Figure S16 :
Figure S16: G3BP2B impacts the expression of select mRNAs under Tg. A. ΔTE LFC correlations between G3BP2B WT and G3BP2B V11A under Tg.B. Differential expression plot for Ribo-seq and total RNA-seq of G3BP2B V11A vs G3BP2B WT under Tg.C. Ribo-seq (left) and RNA-seq (right) LFC from data shown on panel B. of G3BP2B WT sensitive genes identified on Fig. 6E.D. GO of RNA abundance down genes identified in data of panel B. E. RNA-seq coverage tracks of DDX3X of G3BP1/2 KO, G3BP2B WT , and G3BP2B V11A expressing cells under Tg.F. GO of RNA abundance up genes identified in data of panel B. G. GSEA for SG-associated mRNAs overlapping with differentially translated gene sets from G3BP2B WT (left) and G3BP2B V11A (right) Ribo-seq profiles.