The stability of the G3BP protein is compromised by RNA lariats. (A) PKR+/+ and PKR−/− BJ cells were infected with VSV at an MOI of 0.01, or HSV-1-GFP at an MOI of 0.5 for 24 h, stimulated with polyI:C (2 µg/ml) for 5 h or heat stressed at 43°C for 40 min; immunofluorescence analysis was then performed for G3BP1. Scale bars, 20 µm. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) WT and shDBR1-transduced cells were transfected with lariat RNA for 8 h; immunofluorescence analysis was then performed for G3BP1-SGs. Scale bars, 20 µm. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with unpaired t tests. *, P < 0.05. (C) Lariat RNA was used to transfect shDBR1 HEK293T cells, and immunofluorescence analysis was then performed for G3BP1. Scale bars, 20 µm. Data representative of three independent experiments. (D) Fluorescence analysis of MCP (red) in HEK293T cells transfected with ID1/DKK1-8xMS2 -lariats (left). The percentage of cells with MCP and ID1/DKK1-8xMS2 -lariats foci was quantified (right), and at least 100 cells from each group were analyzed (n = 3 for each group). Scale bars, 50 µm. Data representative of six independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with unpaired t tests. ***, P < 0.001. (E) WB with the indicated antibodies following immunoprecipitation with an anti-FLAG (MCP) antibody on lysates of HEK293T cells in an overexpression system. Data representative of three independent experiments. (F) HEK293T cells were transfected with G3BP1/2-mNeonGreen, PKR-BFP or MCP-mScarlet separately, followed by immunofluorescence imaging. Scale bars, 20 µm. Data representative of three at least six independent experiments. (G) HEK293T G3BP1-mNenonGreen cells were transfected with ID1/DKK1-lariats, circRNA (circFNDC3B and circCAMSAP1) or polyI:C stimulation, and the formation of G3BP1 puncta was detected by immunofluorescence. Scale bars, 20 µm. Data representative of three independent experiments. (H) WB with the indicated antibodies following IP with an anti-FLAG (MCP) antibody for lysates of HEK293T PKR+/+ and PKR−/− cells in a stable overexpression system. Data representative of three independent experiments. (I) HEK293T PKR−/− cells with ID1/DKK1-MS2-Lariats/MCP-mScarlet/G3BP- mNeonGreen/shDBR1 transduction were imaged for the indicated fluorescence markers. Scale bars, 20 µm. Data representative of at least six independent experiments. (J) SG component analysis (log fold change) of shDBR1 versus scrambled cells, following VSV infection G3BP1 IP and mass spectrometry. (K) HEK293T shDBR1/G3BP2-mNeonGreen cells were treated with the proteasome inhibitor MG132 and the autophagy inhibitor CQ for 18 h, and FACS was performed to assess G3BP2-mNeonGreen fluorescence recovery. Data shown are representative of three independent experiments. (L) FACS was performed to assess G3BP2-mNeonGreen fluorescence recovery before and after the treatment of HEK293T G3BP2-mNeonGreen, shDBR1/G3BP2-mNeonGreen cells with various concentrations of the proteasome inhibitor MG132. Data shown are representative of three independent experiments. (M and N) HeLa scrambled control or shDBR1 cells were treated with MG132 for 4 h, and then were infected with VSV at an MOI of 0.1 (M) or HSV-1 at an MOI of 0.5 (N) for 24 h, and viral replication was then detected by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (O) WB with the indicated antibodies following immunoprecipitation with an anti-FLAG-(Flag-UB) antibody for the lysates of HEK239T cells in an overexpression system. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData FS4.
Figure S4.

The stability of the G3BP protein is compromised by RNA lariats. (A) PKR+/+ and PKR−/− BJ cells were infected with VSV at an MOI of 0.01, or HSV-1-GFP at an MOI of 0.5 for 24 h, stimulated with polyI:C (2 µg/ml) for 5 h or heat stressed at 43°C for 40 min; immunofluorescence analysis was then performed for G3BP1. Scale bars, 20 µm. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) WT and shDBR1-transduced cells were transfected with lariat RNA for 8 h; immunofluorescence analysis was then performed for G3BP1-SGs. Scale bars, 20 µm. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with unpaired t tests. *, P < 0.05. (C) Lariat RNA was used to transfect shDBR1 HEK293T cells, and immunofluorescence analysis was then performed for G3BP1. Scale bars, 20 µm. Data representative of three independent experiments. (D) Fluorescence analysis of MCP (red) in HEK293T cells transfected with ID1/DKK1-8xMS2 -lariats (left). The percentage of cells with MCP and ID1/DKK1-8xMS2 -lariats foci was quantified (right), and at least 100 cells from each group were analyzed (n = 3 for each group). Scale bars, 50 µm. Data representative of six independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with unpaired t tests. ***, P < 0.001. (E) WB with the indicated antibodies following immunoprecipitation with an anti-FLAG (MCP) antibody on lysates of HEK293T cells in an overexpression system. Data representative of three independent experiments. (F) HEK293T cells were transfected with G3BP1/2-mNeonGreen, PKR-BFP or MCP-mScarlet separately, followed by immunofluorescence imaging. Scale bars, 20 µm. Data representative of three at least six independent experiments. (G) HEK293T G3BP1-mNenonGreen cells were transfected with ID1/DKK1-lariats, circRNA (circFNDC3B and circCAMSAP1) or polyI:C stimulation, and the formation of G3BP1 puncta was detected by immunofluorescence. Scale bars, 20 µm. Data representative of three independent experiments. (H) WB with the indicated antibodies following IP with an anti-FLAG (MCP) antibody for lysates of HEK293T PKR+/+ and PKR−/− cells in a stable overexpression system. Data representative of three independent experiments. (I) HEK293T PKR−/− cells with ID1/DKK1-MS2-Lariats/MCP-mScarlet/G3BP- mNeonGreen/shDBR1 transduction were imaged for the indicated fluorescence markers. Scale bars, 20 µm. Data representative of at least six independent experiments. (J) SG component analysis (log fold change) of shDBR1 versus scrambled cells, following VSV infection G3BP1 IP and mass spectrometry. (K) HEK293T shDBR1/G3BP2-mNeonGreen cells were treated with the proteasome inhibitor MG132 and the autophagy inhibitor CQ for 18 h, and FACS was performed to assess G3BP2-mNeonGreen fluorescence recovery. Data shown are representative of three independent experiments. (L) FACS was performed to assess G3BP2-mNeonGreen fluorescence recovery before and after the treatment of HEK293T G3BP2-mNeonGreen, shDBR1/G3BP2-mNeonGreen cells with various concentrations of the proteasome inhibitor MG132. Data shown are representative of three independent experiments. (M and N) HeLa scrambled control or shDBR1 cells were treated with MG132 for 4 h, and then were infected with VSV at an MOI of 0.1 (M) or HSV-1 at an MOI of 0.5 (N) for 24 h, and viral replication was then detected by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (O) WB with the indicated antibodies following immunoprecipitation with an anti-FLAG-(Flag-UB) antibody for the lysates of HEK239T cells in an overexpression system. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData FS4.

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