Project description:IRE1a is a critical modulator of the unfolded protein response. Its RNAse activity generates the mature transcript for the XBP1 transcription factor and also degrades other ER associated mRNAs in a process termed Regulated IRE1a Dependent mRNA Decay or RIDD. To determine if IRE1a is critical in the response to oncogenic Ras we used ShRNA to knockdown Ire1a or Xbp1 in primary mouse epidermal keratinocytes transduced with a v-HRAS retrovirus.
Project description:Ribosome profiling (Ribo-Seq) and RNA-Seq analysis of Saccharomyces cerevisiae with and without 10 minute glucose starvation to determine effects on translation.
Project description:IRE1a and XBP1 are key regulators of the unfolded protein response (UPR). XBP1 ablation causes profound hypolipidemia in mice, and triggers feedback activation of its upstream enzyme IRE1a, instigating regulated IRE1-dependent decay (RIDD), an mRNA degradation mechanism dependent on IRE1a's endoribonuclease activity. Comprehensive microarray analysis of XBP1 and/or IRE1a deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1a. To identify RIDD substrate mRNAs and direct XBP1 targets in the liver, we performed a comprehensive comparative microarray analysis of three groups of RNA samples: WT and XBP1 deficient mice, WT and IRE1a deficient mice untreated or injected with tunicamycin, and XBP1 deficient mice injected with luciferase or IRE1a siRNA.
Project description:The ER-resident protein kinase/endoribonuclease IRE1 is activated through trans-autophosphorylation in response to protein folding overload in the ER lumen and maintains ER homeostasis by triggering a key branch of the unfolded protein response. Here we show that mammalian IRE1a in liver cells is also phosphorylated by a kinase other than itself in response to metabolic stimuli. Glucagon stimulated protein kinase PKA, which in turn phosphorylated IRE1a at Ser724, a highly conserved site within the kinase activation domain. Blocking Ser724 phosphorylation impaired the ability of IRE1a to augment the upregulation by glucagon signaling of the expression of gluconeogenic genes. Moreover, hepatic IRE1a was highly phosphorylated at Ser724 by PKA in mice with obesity, and silencing hepatic IRE1a markedly reduced hyperglycemia and glucose intolerance. Hence, these results suggest that IRE1a integrates signals from both the ER lumen and the cytoplasm in the liver and is coupled to the glucagon signaling in the regulation of glucose metabolism. We used DNA microarray to analyze the transcriptomic change upon IRE1a overexpression or IRE1a depletion in primary hepatocytes, to study the changes related to IRE1a Primary hepatocytes were infected with the desired adenoviruses (Ad-EGFP, Ad-WT, Ad-S724A, Ad-shCON, Ad-shIRE1a#2) or treated with glucagon. Total cellular RNA was isolated with TRIzol (Invitrogen) and subjected to analysis by Affymetrix Mouse Genome 430 2.0 Arrays. Three experiments were independently conducted.
Project description:The ER-resident protein kinase/endoribonuclease IRE1 is activated through trans-autophosphorylation in response to protein folding overload in the ER lumen and maintains ER homeostasis by triggering a key branch of the unfolded protein response. Here we show that mammalian IRE1a in liver cells is also phosphorylated by a kinase other than itself in response to metabolic stimuli. Glucagon stimulated protein kinase PKA, which in turn phosphorylated IRE1a at Ser724, a highly conserved site within the kinase activation domain. Blocking Ser724 phosphorylation impaired the ability of IRE1a to augment the upregulation by glucagon signaling of the expression of gluconeogenic genes. Moreover, hepatic IRE1a was highly phosphorylated at Ser724 by PKA in mice with obesity, and silencing hepatic IRE1a markedly reduced hyperglycemia and glucose intolerance. Hence, these results suggest that IRE1a integrates signals from both the ER lumen and the cytoplasm in the liver and is coupled to the glucagon signaling in the regulation of glucose metabolism. We used DNA microarray to analyze the transcriptomic change upon IRE1a overexpression or IRE1a depletion in primary hepatocytes, to study the changes related to IRE1a
Project description:IRE1a and XBP1 are key regulators of the unfolded protein response (UPR). XBP1 ablation causes profound hypolipidemia in mice, and triggers feedback activation of its upstream enzyme IRE1a, instigating regulated IRE1-dependent decay (RIDD), an mRNA degradation mechanism dependent on IRE1a's endoribonuclease activity. Comprehensive microarray analysis of XBP1 and/or IRE1a deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1a.
Project description:The purpose of the study was to examine the role of the IRE1a-XBP1 pathway during Th2 lymphocyte activation and differentiation. In vitro Th2 cells were treated with 4μ8c, a drug that specifically inhibits IRE1a endonuclease activity, and transcriptomes were compared.
Project description:Arabidopsis seedlings of wildtype or ire1a ire1b double mutant were treated with or without tunicamycine in the presence of actinomycin D (ActD).