Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The unfolded protein response is an intracellular signaling pathway that, in response to accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER), upregulates transcription of ER resident chaperones. A key step in this pathway is the non-conventional, regulated splicing of the mRNA encoding the positive transcriptional regulator Hac1p. In the yeast Saccharomyces cerevisiae, the bifunctional transmembrane kinase/endoribonuclease Ire1p cleaves HAC1 mRNA at both splice junctions and tRNA ligase joins the two exons together. We have reconstituted HAC1 mRNA splicing in an efficient in vitro reaction and show that, in many ways, the mechanism of HAC1 mRNA splicing resembles that of pre-tRNA splicing. In particular, Ire1p endonucleolytic cleavage leaves 2', 3'-cyclic phosphates, the excised exons remain associated by base pairing, and exon ligation by tRNA ligase follows the same chemical steps as for pre-tRNA splicing. To date, this mechanism of RNA processing is unprecedented for a messenger RNA. In contrast to the striking similarities to tRNA splicing, the structural features of the splice junctions recognized by Ire1p differ from those recognized by tRNA endonuclease. We show that small stem-loop structures predicted to form at both splice junctions of HAC1 mRNA are required and sufficient for Ire1p cleavage.
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PMID:Mechanism of non-spliceosomal mRNA splicing in the unfolded protein response pathway. 1035 23

Genetic analysis of the cellular adaptation to malfolded proteins in the endoplasmic reticulum (the unfolded protein response - UPR) has revealed a novel signaling pathway initiated by activation of IRE1, an ER-resident protein kinase and endonuclease. In yeast, Ire1p activates gene expression by promoting a non-conventional splicing event that converts the mRNA encoding the Hac1p transcription factor from an inefficiently translated inactive mRNA to an actively translated one. Hac1p binds to the promoters of genes encoding chaperones and other targets of the UPR and activates them. Recently, mammalian IRE1 homologues have been identified and their response to ER stress is regulated by binding to the ER chaperone BiP. The mechanisms by which mammalian IRE1 activates gene expression have not been completely characterized and mammalian HAC1 homologues have not been identified. Surprisingly, mammalian IRE1s are able to activate both JUN N-terminal kinases and an alternative ER-stress signaling pathway mediated by the transcription factor ATF6. This indicates that the mammalian UPR is more complex than that found in yeast.
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PMID:IRE1 and efferent signaling from the endoplasmic reticulum. 1103 98

The global gene expression program that accompanies the adaptation of Saccharomyces cerevisiae to an abrupt transfer from a fermentable to a nonfermentable carbon source was characterized by using a cDNA microarray to monitor the relative abundances and polysomal distributions of mRNAs. Features of the program included a transient reduction in global translational activity and a severe decrease in polysome size of transcripts encoding ribosomal proteins. While the overall translation initiation of newly synthesized and preexisting mRNAs was generally repressed after the carbon source shift, the mRNA encoded by YPL250C was an exception in that it selectively mobilized into polysomes, although its relative abundance remained unchanged. In addition, splicing of HAC1 transcripts, which has previously been reported to occur during accumulation of unfolded proteins in the endoplasmic reticulum, was observed after the carbon shift. This finding suggests that the nonconventional splicing complex, composed of the kinase-endonuclease Ire1p and the tRNA ligase Rlg1p, was activated. While spliced HAC1 transcripts mobilized into polysomes, the vast majority of unspliced HAC1 RNA accumulated in nonpolysomal fractions before and after the carbon source shift, indicating that translation of unspliced HAC1 RNA is blocked at the translation initiation step, in addition to the previously reported elongation step. These findings reveal that S. cerevisiae reacts to the carbon source shift with a remarkable variety of responses, including translational regulation of specific mRNAs and activation of specific enzymes involved in a nonconventional splicing mechanism.
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PMID:Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source. 1115 78

IRE1 proteins mediate cellular responses to accumulation of malfolded proteins in the endoplasmic reticulum in the yeast and mammalian unfolded protein responses. A sensitive in vivo u.v. crosslinking assay showed that IRE1 proteins are intimately associated with RNA in mammalian cells. The IRE1-associated RNA fragments recovered by this assay were different in stressed and unstressed cells. The amount of RNA associated with IRE1 that could be revealed by end-labeling with T4 kinase was greater in IRE1-containing complexes isolated from stressed cells. Furthermore, the RNA fragments recovered from complexes found in stressed cells were shorter than those from unstressed cells, revealing a dynamic change in the IRE1-RNA complex during the UPR. Formation of the complex between IRE1 and RNA was dependent on both the kinase and endonuclease domains of IRE1, and involved pre-existing RNA species. When viewed in the context of the known importance of Ire1p-HAC1 mRNA interactions to the yeast unfolded protein response, these findings suggest that full-length mammalian IRE1s also engage RNA molecules as downstream effectors.
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PMID:Alterations in an IRE1-RNA complex in the mammalian unfolded protein response. 1159 Feb 47

Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR). How the UPR is downregulated is not well understood. Inositol requirement 1 (Ire1) is an endoplasmic reticulum transmembrane UPR sensor in Saccharomyces cerevisiae. When the UPR is triggered, Ire1 is autophosphorylated, on Ser 840 and Ser 841, inducing the cytosolic endonuclease activity of Ire1, thereby initiating the splicing and translational de-repression of HAC1 mRNA. Homologous to Atf/Creb1 (Hac1) activates UPR transcription. Here, we report that the dose-dependent cell-cycle regulator 2 (Dcr2) phosphatase functionally and physically interacts with Ire1. We identified genetic interactions between DCR2 and genes, including IRE1, which are involved in secretory processes. Overexpression of DCR2, but not of a catalytically inactive DCR2 allele, significantly delays HAC1 splicing and sensitizes cells to the UPR. Furthermore, Dcr2 physically interacts in vivo with Ire1-S840E,S841E, which mimics phosphorylated Ire1, and Dcr2 de-phosphorylates Ire1 in vitro. Our results are consistent with de-phosphorylation of Ire1 being a mechanism for antagonizing UPR signalling.
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PMID:Dcr2 targets Ire1 and downregulates the unfolded protein response in Saccharomyces cerevisiae. 1699 Aug 50

The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin alpha (Kap60p) and a novel betaNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin beta homologues. Kinetic binding data suggest that binding to importin beta proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling.
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PMID:The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins. 1703 34

In the yeast Saccharomyces cerevisiae, the unfolded protein response (UPR) involves the unconventional splicing of HAC1 mRNA, which is mediated by the activated Ire1p transmembrane kinase/endonuclease. In this study, we isolated and characterized a Yarrowia lipolytica HAC1 (YlHAC1) encoding a basic-leucine zipper transcription factor. The null mutant strain of YlHAC1 (DeltaYlhac1) displayed a significantly increased sensitivity to dithiothreitol (DTT) and tunicamycin (TM), along with a defect in hyphal growth, suggesting the essential function of YlHAC1 in UPR. The unconventional splicing of YlHAC1 mRNA occurred under the UPR conditions induced by DTT or TM treatment. Unlike S. cerevisiae HAC1 mRNA with an intron of 252 nt, YlHAC1 mRNA was shown to harbour a short intron of length 29 nt. The YlHAC1 mRNA harboured the nucleotides CAG, conserved at the intron borders in the filamentous fungi hac1/hacA and mammalian XBP1, as well as a conserved bipartite element within the 3' untranslated region. The expression of the spliced form of YlHAC1 mRNA in the wild-type andDeltaYlhac1 strains resulted in an increased resistance to DTT, thereby indicating that the spliced form is translated into a functional YlHac1p.
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PMID:Functional characterization of the unconventional splicing of Yarrowia lipolytica HAC1 mRNA induced by unfolded protein response. 2016 30

Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1's RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide-binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1's kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence of enhanced dimer formation in quercetin's mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.
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PMID:Flavonol activation defines an unanticipated ligand-binding site in the kinase-RNase domain of IRE1. 2041 96

The monoterpene carvacrol, the major component of oregano and thyme oils, is known to exert potent antifungal activity against the pathogenic yeast Candida albicans. This monoterpene has been the subject of a considerable number of investigations that uncovered extensive pharmacological properties, including antifungal and antibacterial effects. However, its mechanism of action remains elusive. Here, we used integrative chemogenomic approaches, including genome-scale chemical-genetic and transcriptional profiling, to uncover the mechanism of action of carvacrol associated with its antifungal property. Our results clearly demonstrated that fungal cells require the unfolded protein response (UPR) signaling pathway to resist carvacrol. The mutants most sensitive to carvacrol in our genome-wide competitive fitness assay in the yeast Saccharomyces cerevisiae expressed mutations of the transcription factor Hac1 and the endonuclease Ire1, which is required for Hac1 activation by removing a nonconventional intron from the 3' region of HAC1 mRNA. Confocal fluorescence live-cell imaging revealed that carvacrol affects the morphology and the integrity of the endoplasmic reticulum (ER). Transcriptional profiling of pathogenic yeast C. albicans cells treated with carvacrol demonstrated a bona fide UPR transcriptional signature. Ire1 activity detected by the splicing of HAC1 mRNA in C. albicans was activated by carvacrol. Furthermore, carvacrol was found to potentiate antifungal activity of the echinocandin antifungal caspofungin and UPR inducers dithiothreitol and tunicamycin against C. albicans. This comprehensive chemogenomic investigation demonstrated that carvacrol exerts its antifungal activity by altering ER integrity, leading to ER stress and the activation of the UPR to restore protein-folding homeostasis.
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PMID:The Monoterpene Carvacrol Generates Endoplasmic Reticulum Stress in the Pathogenic Fungus Candida albicans. 2601 32

The Hac1 transcription factor in yeast up-regulates a collection of genes that control protein homeostasis. Base-pairing interactions between sequences in the intron and the 5'-untranslated region (5' UTR) of the HAC1 mRNA represses Hac1 protein production under basal conditions, whereas cytoplasmic splicing of the intron by the Ire1 kinase-endonuclease, activated under endoplasmic reticulum stress conditions, relieves the inhibition and enables Hac1 synthesis. Using a random mutational screen as well as site-directed mutagenesis, we identify point mutations within the 5' UTR-intron interaction site that derepress translation of the unspliced HAC1 mRNA. We also show that insertion of an in-frame AUG start codon upstream of the interaction site releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhances production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. These results suggest that the major block of translation occurs at the initiation stage. Supporting this interpretation, the point mutations that enhanced Hac1 production resulted in an increased percentage of the HAC1 mRNA associating with polysomes versus free ribosomal subunits. Thus, our results provide evidence that the 5' UTR-intron interaction represses translation initiation on the unspliced HAC1 mRNA.
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PMID:Evidence That Base-pairing Interaction between Intron and mRNA Leader Sequences Inhibits Initiation of HAC1 mRNA Translation in Yeast. 2617 53


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