The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly
- Howard Hughes Medical Institute, University of California, San Francis, United States
- University of California, Berkeley, United States
Figures
Figure 1 with 4 supplements
Translational regulation upon ER stress in mammalian cells.
(A) Translational and mRNA changes in HEK293T cells upon ER stress. HEK293T cells were treated with or without 1 μg/ml of Tm for 1 hr. The y-axis represents fold changes in ribosome-protected …
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Figure 1—source data 1
Read counts for all conditions and each individual transcript.
- https://doi.org/10.7554/eLife.05033.003
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Figure 1—source data 2
Source data for Figure 1.
(A) Significant translational changes in Tm-treated cells. List of genes that change more than twofold in translational efficiency after 1 hr of Tm treatment. mRNA (Tm/UT) represents log2-fold changes in mRNA levels between Tm and untreated samples, and Ribo-Seq (Tm/UT) represents log2-fold changes in RPFs between Tm- and un-treated samples (UT). Only genes with a log2-fold change >|1| and a FDR-corrected p-value <0.1 were included in the table. Known ISR-translational targets are highlighted in pink and targets containing previously identified uORFs are highlighted in green. (B) ISR-translational targets containing uORFs. List of genes that change more than twofold in translational efficiency after 1 hr of Tm treatment and contain previously identified uORFs. Ribo-Seq (Tm/UT) represents log2-fold changes in RPFs between Tm- and un-treated samples (UT). (C) Significant translational changes in Tm and ISRIB-treated cells. List of genes that change more than twofold in translational efficiency after 1 hr of Tm and ISRIB treatment. mRNA [(Tm + ISRIB)/UT] represents log2-fold changes in mRNA levels between Tm and ISRIB-treated and untreated samples and Ribo-Seq [(Tm + ISRIB)/UT)] represents log2-fold changes in RPFs between Tm and ISRIB-treated and untreated samples. Only genes with a log2-fold change >|1| and a FDR-corrected p-value < 0.1 were included in the table. (D) Significant translational changes in ISRIB-treated cells. List of genes that change more than twofold in translational efficiency after 1 hr of ISRIB treatment. mRNA (ISRIB/UT) represents log2-fold changes in mRNA levels between ISRIB-treated and untreated samples and[SPACE]Ribo-Seq (ISRIB/UT) represents log2-fold changes in RPFs between ISRIB-treated and untreated samples. Only genes with a log2-fold change >|1| and a FDR-corrected p-value < 0.1 were included in the table. (E) Alignment of sequencing libraries. Total number of reads and the percentage of reads for both mRNA-seq and ribo-seq libraries that align to the genome and ORF for all conditions (UT, Tm, Tm + ISRIB, ISRIB) and for each replicate.
- https://doi.org/10.7554/eLife.05033.004
Figure 1—figure supplement 1
Ribosome and mRNA densities in the 5’UTR of ATF4 and SLC35A4.
mRNA reads (y-axis) are represented along the sequence of each gene (x-axis) in the upper panel. Ribosome footprint (ribo) reads (y-axis) are represented along the sequence of each gene (x-axis) in …
Figure 1—figure supplement 2
Translational regulation of mTOR targets upon ER-stress.
(A) Cells were treated with or without 1 μg/ml of Tm for 1 hr. The y-axis represents fold changes in ribosome-protected fragments (Ribo-Seq) between Tm-treated and control samples. The x-axis …
Figure 1—figure supplement 3
Correlation plots for duplicate ribosome profiling experiments.
The detected ribosome (ribo) or mRNA density is plotted for each gene in each experimental condition (untreated [UT], tunicamycin [Tm], tunicamycin + ISRIB [Tm + ISRIB] and ISRIB). Correlation …
Figure 1—figure supplement 4
Mean mRNA abundance of all genes mapped.
The x-axis represents log2 (mean normalized count) for each mRNA mapped and the y-axis represents log2 fold changes in mRNA abundance in the different experimental conditions: Tm (panel A), Tm + …
Figure 2
ISRIB blocks stress granule formation induced by eIF2α phosphorylation.
(A) Immunofluorescence analysis (eIF3a) of U2OS cells treated with 200 nM Tg for 1 hr, 250 μM Ars for 30 min, or 100 nM Pat A for 30 min in the presence or absence of 200 nM ISRIB or 1 μM GSK797800 …
Figure 3 with 2 supplements
ISRIB addition rapidly dissolves pre-formed stress granules in live cells restoring translation.
(A) Live cell imaging of stress granules in U2OS cells stably expressing G3BP-GFP (SG marker). Cells were treated with 200 nM Tg for 40 min, 250 μM Ars for 30 min, 100 nM Pat A for 30 min, or 300 nM …
Figure 3—figure supplement 1
ISRIB dose response and inactive analog in stress granule assay.
(A) Live cell imaging of SGs in U2OS cells stably expressing G3BP-GFP. Cells were treated with 200 nM Tg and different doses of ISRIB (as indicated) or 2 μM of an inactive analog of ISRIB (ISRIBinact…
Figure 3—figure supplement 2
Representative SDS-PAGE gel of [35S]-methionine pulse as described in Figure 3D.
Top panel is an autoradiogram and bottom panel is total protein of the same gel as shown by Coomassie staining.
Videos
Video 1
ISRIB triggers stress granule disassembly.
U2OS cells stably expressing G3BP-GFP (SG marker) and Dcp1-RFP (P-body marker) were treated with 200 nM Tg for 40 min and then 200 nM ISRIB was added at t = 0 min to the well and imaged using an …
Video 2
ISRIB does not trigger disassembly of P-bodies.
Images of Dcp1-RFP (P-bodies) corresponding to the same field of cells as in Video 1 were collected every 30 s.
