A functional unfolded protein response is required for normal vegetative development in Arabidopsis
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ABSTRACT: The Unfolded Protein Response (UPR) is activated in plants in response to ER stress and plays an important role in mitigating stress damage. We have analyzed an ire1a ire1b bzip17 triple mutant in Arabidopsis with defects in stress signaling and found that the mutant is also impaired in vegetative plant growth under conditions without externally applied stress. This raises the question as to whether the UPR functions in plant development in same manner as it does in responding to stress. bZIP17 is an ER-associated membrane transcription factor that is mobilized to the nucleus in response to stress. Through the analysis of a mobilization defective bZIP17 mutant, we found that bZIP17 must be capable of mobilization to support normal plant development. Likewise, through the analysis of ire1 mutants, we found that protein kinase and ribonuclease activities of IRE1 must be functional to support normal development. These findings demonstrate that the UPR is active, albeit at low levels, during unstressed conditions, and that these activities are required for normal development.
Project description:Autophagy is a conserved process in eukaryotes that contributes to cell survival in response to stress. Previously, we found that ER stress induces autophagy in a manner dependent upon IRE1b, an ER membrane-associated factor involved in the splicing of bZIP60 mRNA. IRE1 is a dual protein kinase and ribonuclease, and here we studied the involvement of the protein kinase catalytic domain, nucleotide binding and RNase domains of IRE1b in activating autophagy. Autophagy was assessed by quantifying the numbers of autophagosomes in transgenic Arabidopsis seedlings bearing mutations in the various IRE1b domains. The results showed that nucleotide binding and RNase activity of IRE1b are required for ER stress-mediated autophagy. The RNase activity is involved in IRE1b’s mRNA splicing function, but its principal splicing target, bZIP60, is not involved in IRE1b’s activation of autophagy. We therefore considered other roles for IRE1b in the activation of autophagy. Clustering of ER localized IRE1b-YFP was observed when seedlings were subjected to ER stress, and so we investigated whether IRE1b clustering induced autophagy. However, the RNase knockout mutation in IRE1b still undergoes clustering, suggesting that IRE1b clustering does not induce autophagy. In response to ER stress, the RNase of IRE1 has been found to engage in another activity called Regulated Ire1-Dependent Decay of Messenger RNA (RIDD), which is the promiscuous degradation of other mRNA in response to ER stress. By analyzing the RNA-seq data, 12 RIDD target genes were picked up for testing their role in inhibiting autophagy, and glucosidase 21 and peroxidase 14 are proved to be degraded to support the induction of autophagy by ER stress.
Project description:The NGS-associated mRNA-seq analysis was conducted to survey transcriptome changes responding to three UPR inducers (tunicamycin, DTT, & Azetidine-2-cytosine) by four double mutant of three UPR-associated transcription factors (bZIP17, bZIP28, & bZIP60) and two activators (S1P & S2P).
Project description:The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of the mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic ?-cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded however, the ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in the cell population. We used microarray gene expression arrays to analyze the IRE1-dependent activation of genes in response to misfolded proinsulin expression in an inducible mutant proinsulin (C96Y) insulinoma cell line by inhibiting the IRE1 endoribonucleas activity with a specific inhibitor, 4u8c. Insulinoma cells with doxycycline inducible C96Y-proinsulin expression were either untreated, treated with doxycycline alone or treated with dox and 4u8c. This was done with two biological replicates.
Project description:The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of the mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic -cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded however, the ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in the cell population. We used microarray gene expression arrays to analyze the IRE1-dependent activation of genes in response to misfolded proinsulin expression in an inducible mutant proinsulin (C96Y) insulinoma cell line by inhibiting the IRE1 endoribonucleas activity with a specific inhibitor, 4u8c.
Project description:The unfolded protein response (UPR) allows the endoplasmic reticulum (ER) to recover from the accumulation of misfolded proteins, in part by increasing its folding capacity. IRE1 promotes this remodeling by detecting misfolded ER proteins and activating a transcription factor, XBP-1, through endonucleolytic cleavage of its mRNA. We found that IRE1 independently mediated the rapid degradation of a specific subset of mRNAs. The arrays deposited here show the effects of depletion of IRE1 and XBP-1 on UPR induction in S2 cells. We have characterized the IRE1-dependent repressive branch of this response. Keywords: stress response, RNAi, DTT
Project description:Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation (ChIP) analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention.
Project description:One major component of cellular proteome resides in the endoplasmic reticulum (ER), the key organelle for protein biogenesis in the secretory pathway. Disruption of ER proteostasis leads to ER stress, which subsequently activates multiple adaptive stress response pathways, collectedly termed as the unfolded protein response (UPR). One UPR branch is mediated by IRE1(inositol-requiring enzyme 1). Activation of the IRE1 UPR branch generates a potent transcriptional factor: spliced X-box–binding protein-1 (XBP1s). Since activation of this UPR branch has been shown to be neuroprotective in brain ischemia/stroke, it is critical to identify the XBP1s-regulated genes in neurons in vivo and thus help determine the molecular mechanisms underlying the beneficial effects exerted by this UPR branch. In this study, we performed RNA-Seq analysis on the hippocampus from XBP1s conditional transgenic mice.
Project description:Eukaryotic IRE1 mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, mammalian RIDD seemingly targets only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human breast cancer cells, we discovered not only canonical endomotif-containing RIDD substrates, but also many targets lacking recognizable motifs—degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displayed two basic endoribonuclease modalities: endomotif-specific cleavage, minimally requiring dimers; and endomotif-independent promiscuous processing, requiring phospho-oligomers. An oligomer-deficient mutant that did not support RIDDLE failed to rescue cancer-cell viability. These results link IRE1α oligomers, RIDDLE, and cell survival, advancing mechanistic understanding of the UPR.
Project description:Eukaryotic IRE1 mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, mammalian RIDD seemingly targets only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human breast cancer cells, we discovered not only canonical endomotif-containing RIDD substrates, but also many targets lacking recognizable motifs—degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displayed two basic endoribonuclease modalities: endomotif-specific cleavage, minimally requiring dimers; and endomotif-independent promiscuous processing, requiring phospho-oligomers. An oligomer-deficient mutant that did not support RIDDLE failed to rescue cancer-cell viability. These results link IRE1α oligomers, RIDDLE, and cell survival, advancing mechanistic understanding of the UPR.
Project description:We measured steady-state mRNA levels by microarray hybridization, comparing WT, (delta)ire1, (delta)gcn4, and (delta)gcn2 cells treated with 2 mM DTT for 30 min (by which time the UPR is qualitatively complete) to untreated samples of the same genotype. WT cells were taken as a positive control for UPR induction, and (delta)ire1 cells as a negative control. Fold change in expression of a given gene was computed as the ratio of mRNA level in the treated sample to the level in an untreated sample of the same genotype. Values reported here are the log2 fold change. Keywords = unfolded protein response Keywords = UPR Keywords = ire1 Keywords = gcn4 Keywords = gcn2