G3BP1 promotes stress-induced RNA granule interactions to preserve polyadenylated mRNA

Abstract

G3BP1, a target of TDP-43, is required for normal stress granule (SG) assembly, but the functional consequences of failed SG assembly remain unknown. Here, using both transformed cell lines and primary neurons, we investigated the functional impact of this disruption in SG dynamics. While stress-induced translational repression and recruitment of key SG proteins was undisturbed, depletion of G3BP1 or its upstream regulator TDP-43 disturbed normal interactions between SGs and processing bodies (PBs). This was concomitant with decreased SG size, reduced SG-PB docking, and impaired preservation of polyadenylated mRNA. Reintroduction of G3BP1 alone was sufficient to rescue all of these phenotypes, indicating that G3BP1 is essential for normal SG-PB interactions and SG function.

Figure 1.

SG proteins and polyadenylated mRNA are well recruited to SGs despite impaired SG aggregation. (A–C) HeLa cells were transfected with the indicated siRNAs for 72 h. Coverslips were collected at 90 min after SA treatment (0.5 mM, 30 min). (A) Representative Western blot showing the efficiency of siRNA transfections. (B) Coverslips were labeled for eIF4G, eIF3A, HuR, Caprin1, or USP10 and the SG marker TIA-1. Bar, 25 µm. (C) Polyadenylated mRNA was visualized via fluorescence in situ hybridization with an oligo(dT) probe and HuR and TIA-1 labeling. Bar, 10 µm. Representative micrographs from ≥3 independent experiments are shown. Line scans (from the white lines) were used to assess colocalization (histograms).

Figure 2.

TDP-43 and G3BP1 depletion induce a defect in basal translation. (A–C) HeLa cells were transfected with the indicated siRNAs for 72 h. Cells were stressed with 0.5 mM SA for 30 min and collected at 90 (lanes 2, 5, 8, and 11) and 180 min (lanes 3, 6, 9, and 12) and before stress (lanes 1, 4, 7, and 10). All samples were incubated with 50 µM [³⁵S]methionine 30 min before cell lysis. Samples were prepared in Laemmli buffer and subjected to SDS-PAGE. (A) Experimental design. (B) Autoradiography of the cell lysates (left) and same gel stained with Coomassie blue to demonstrate equivalent protein loading (right). (C) The histogram shows densitometric quantification of the entire lane expressed relative to the Coomassie-stained gel. The means ± SEM (error bars) of ≥3 independent experiments are plotted. *, P < 0.05, compared with siControl; #, P < 0.05, compared with the unstressed sample.

Figure 3.

Increased mRNA correlates with alterations in PBs in basal conditions. (A–F) HeLa cells were transfected with the indicated siRNAs for 72 h. (A) Polyadenylated mRNA was visualized via FISH with an oligo(dT) probe. Total cellular fluorescence was quantified from at least 10 cells per experiment from ≥3 experiments (35 cells per sample in total). Bar, 10 µm. (B) Whole cell lysates were examined by immunoblotting for PB proteins Dcp1a and Xrn-1. Bands were quantified via densitometry and normalized to Actin. (C–F) Unstressed HeLa cells were labeled for Dcp1a (to visualize PBs) and TIA-1 (to visualize the nucleus). These data are extracted from the kinetics presented in Fig. S2 C. (C) Representative images of ≥3 experiments. Bar, 25 µm. (D) Percentage of cells with PBs in basal conditions. (E) Quantification of the number of individual PBs per cell. (F) Quantification of individual PB size. In all cases, the means ± SEM (error bars) of three independent experiments are plotted. *, P < 0.05.

Figure 4.

SG–PB docking is dependent on SG size. (A–C and F–H) HeLa cells were transfected with the indicated siRNAs for 72 h. (D and E) Cortical neurons treated with TDP-43 siRNA. (A) Representative images of docking in siControl and siTDP-43 cells. Cells were labeled for TIA-1 and Dcp1a to follow SGs and PBs, respectively. The boxed regions are enlarged on the right. Bar, 10 µm. (B and C) Quantification of docking between SGs and PBs in HeLa using TIA-1 and Dcp1a. PBs are considered docked when SGs and PBs were in contact. n = total number of events analyzed. (D) Representative images of unstressed transfected cortical neurons. Arrows indicate TDP-43^Low cells. Bar, 10 µm. (E) Quantification of SG–PB docking in cortical neurons. TDP-43^Low, n = 45 neurons; TDP-43^Endogenous, n = 48 neurons. (F) Size of SGs when docked with PBs. (G) Distribution analysis of SG size. SG size was separated between small (0.75–2 µm²) and large (2–5 µm²) SGs, as shown in the pie charts. (H) The number of stable SG–PB docking events (large SGs only) during a 10-min window, as captured with live stage imaging. All data are representative of ≥3 independent experiments and plotted as the mean ± SEM (error bars). *, P < 0.05.

Figure 5.

Decreased mRNA preservation after stress exposure. (A–C) Cells were transfected with the indicated siRNAs for 72 h and then subjected to two consecutive rounds of stress response induced by SA (SG assembly and disassembly). Coverslips were collected before stress, and after the first and the second round of SG assembly/disassembly. Polyadenylated mRNA was visualized via FISH with an oligo(dT) probe. (A) Experimental design. (B) Representative images of HeLa cells labeled with an oligo(dT) probe and quantification of total pixel intensity. (C) Representative images and quantification of a similar experiment performed in SH-SY5Y cells and examined only after the second SA stress exposure. All data are representative of >3 independent experiments and plotted as the mean ± SEM (error bars). *, P < 0.05. Bars, 10 µm.

Figure 6.

G3BP1 rescues SG–PB interactions and maintains polyadenylated mRNA levels. (A–C) HeLa cells transfected with control (C) or TDP-43 (T) siRNA for 48 h and transfected with GFP or G3BP1-GFP for 24 h, then treated with SA. (A) Representative confocal micrograph demonstrating low-level transient expression of G3BP1. The arrow indicates a typical example of a low-expressing cell that was quantified. Asterisks indicate cells with high levels of G3BP1 expression that were excluded from the analysis. Bar, 10 µm. (B) Coverslips were collected before and at 90 min after stress and then labeled for Dcp1a and TIA-1 to follow PBs and SGs, respectively. Docking was quantified as before in low fluorescence GFP-expressing cells. (C) Cells were subjected to two consecutive rounds of stress response induced by SA (SG assembly and disassembly). Coverslips were collected before stress, and after the first and second rounds of stress. Polyadenylated mRNA was visualized via FISH with a oligo(dT) probe and quantified as before. All data are representative of ≥3 independent experiments and plotted as the mean ± SEM (error bars). *, P < 0.05 compared with unstressed siControl; #, P < 0.05 compared with unstressed of the same sample.