Specificity in endoplasmic reticulum-stress signaling in yeast entails a step-wise engagement of HAC1 mRNA to clusters of the stress sensor Ire1
- San Raffaele Scientific Institute, Italy
- Howard Hughes Medical Institute, University of California, San Francisco, United States
- University of California, San Francisco, United States
- Center for Applied Medical Research, Spain
Figures
Figure 1
Oligomerization of Ire1's cytosolic domain is required for UPR signaling but not for Ire1 cluster formation or HAC1 mRNA recruitment.
(A) Schematic of S. cerevisiae Ire1. The ER-lumenal portion of Ire1 is divided in an N-terminal domain (I, gray), a core lumenal—ER-stress-sensing—domain (cLD, light blue), and BiP binding domain …
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Figure 1—Source data 1
- https://doi.org/10.7554/eLife.05031.004
Figure 2
The kinase and RNase domains of Ire1 are dispensable for foci formation and mRNA recruitment.
(A) Splicing reporter assay before or after ER-stress induction with 2 mM DTT for 2 hr (top), Western blot of Ire1 (middle), and viability assay under ER-stress conditions (0.2 µg ml−1 tunicamycin; …
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Figure 2—Source data 1
- https://doi.org/10.7554/eLife.05031.006
Figure 3
The cytosolic linker of Ire1 harbors a positively charged motif that is key for mRNA recruitment and splicing.
(A) Conservation of Ire1. Top: mapped onto a schematic of Ire1 domains bordered by residues of which the number is denoted, bar diagrams display relative conservation of the Saccharomyces cerevisiae …
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Figure 3—Source data 1
- https://doi.org/10.7554/eLife.05031.008
Figure 4
Three arginines in Ire1's linker are essential for mRNA docking.
(A) Splicing reporter assay before or after ER-stress induction with 2 mM DTT for 2 hr (top) and viability assay under ER-stress conditions (0.2 µg ml−1 tunicamycin; bottom). Assays were performed …
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Figure 4—Source data 1
- https://doi.org/10.7554/eLife.05031.010
Figure 5
Step-wise targeting and docking of mRNA are pre-requisite for activating Ire1.
(A) Localization of mCherry-tagged Ire1 and SpR Δ3′ BEU1A mRNA decorated with U1A–GFP. ER-stress was induced with 10 mM DTT for 45 min; imaging was performed in ire1Δ cells, complemented with …
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Figure 5—Source data 1
- https://doi.org/10.7554/eLife.05031.012
Figure 6
A model of step-wise activation of the Ire1/HAC1 mRNA signaling relay.
The steps are described in the Discussion. Color-coding is as in Figure 1, except that unfolded proteins are depicted in light green and mRNA in red. Ire1 domain interfaces are indicated by wiggly …
Additional files
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Supplementary file 1
Overview of all constructs used in this study.
- https://doi.org/10.7554/eLife.05031.014
