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Ronald T. Raines - One of the best experts on this subject based on the ideXlab platform.
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knockout of the Ribonuclease inhibitor gene leaves human cells vulnerable to secretory Ribonucleases
Biochemistry, 2016Co-Authors: Sydney P Thomas, Ronald T. RainesAbstract:Ribonuclease inhibitor (RNH1) is a cytosolic protein that binds with femtomolar affinity to human Ribonuclease 1 (RNase 1) and homologous secretory Ribonucleases. RNH1 contains 32 cysteine residues and has been implicated as an antioxidant. Here, we use CRISPR-Cas9 to knock out RNH1 in HeLa cells. We find that cellular RNH1 affords marked protection from the lethal ribonucleolytic activity of RNase 1 but not from oxidants. We conclude that RNH1 protects cytosolic RNA from invading Ribonucleases.
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human cancer antigen globo h is a cell surface ligand for human Ribonuclease 1
ACS central science, 2015Co-Authors: Chelcie H Eller, Tzuyuan Chao, Kiran Kumar Singarapu, Ouathek Ouerfelli, Guangbin Yang, John L Markley, Samuel J Danishefsky, Ronald T. RainesAbstract:Pancreatic-type Ribonucleases are secretory enzymes that catalyze the cleavage of RNA. Recent efforts have endowed the homologues from cow (RNase A) and human (RNase 1) with toxicity for cancer cells, leading to a clinical trial. The basis for the selective toxicity of Ribonuclease variants for cancerous versus noncancerous cells has, however, been unclear. A screen for RNase A ligands in an array of mammalian cell-surface glycans revealed strong affinity for a hexasaccharide, Globo H, that is a tumor-associated antigen and the basis for a vaccine in clinical trials. The affinity of RNase A and RNase 1 for immobilized Globo H is in the low micromolar-high nanomolar range. Moreover, reducing the display of Globo H on the surface of human breast adenocarcinoma cells with a small-molecule inhibitor of biosynthesis or a monoclonal antibody antagonist decreases the toxicity of an RNase 1 variant. Finally, heteronuclear single quantum coherence (HSQC) NMR spectroscopy showed that RNase 1 interacts with Globo H by using residues that are distal from the enzymic active site. The discovery that a systemic human Ribonuclease binds to a moiety displayed on human cancer cells links two clinical paradigms and suggests a mechanism for innate resistance to cancer.
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bovine pancreatic Ribonuclease fifty years of the first enzymatic reaction mechanism
Biochemistry, 2011Co-Authors: Claudi M Cuchillo, Victoria M Nogues, Ronald T. RainesAbstract:Fifty years ago, the group of Tony Mathias and Bob Rabin at University College London deduced the first mechanism for catalysis by an enzyme, Ribonuclease [Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., and Ross, C. A. (1961) Nature 190, 781–784]. Here, we celebrate this historic accomplishment by surveying knowledge of enzymology and protein science at that time, facts that led to the formulation of the mechanism, criticisms and alternative mechanisms, data that supported the proposed mechanism, and some of the refinements that have since provided a more precise picture of catalysis of RNA cleavage by Ribonucleases. The Mathias and Rabin mechanism has appeared in numerous textbooks, monographs, and reviews and continues to have a profound impact on biochemistry.
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variants of Ribonuclease inhibitor that resist oxidation
Protein Science, 2008Co-Authors: Wayne L Schultz, Ronald T. RainesAbstract:Human Ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type Ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic Ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328–Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type Ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein.
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evasion of Ribonuclease inhibitor as a determinant of Ribonuclease cytotoxicity
Current Pharmaceutical Biotechnology, 2008Co-Authors: Thomas J Rutkoski, Ronald T. RainesAbstract:Onconase® (ONC) is an amphibian member of the bovine pancreatic Ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma, and is poised to become the first chemotherapeutic agent based on a Ribonuclease. Investigations into the mechanism of Ribonuclease-based cytotoxicity have elucidated several important determinants for cytotoxicity, including efficient deliverance of ribonucleolytic activity to the cytosol and preservation of conformation stability. Nevertheless, the most-striking similarity between ONC and bovine seminal Ribonuclease, another naturally cytotoxic Ribonuclease, is their insensitivity to inhibition by the potent cytosolic Ribonuclease inhibitor protein (RI). RI typically binds to its Ribonuclease ligands with femtomolar affinity—an extraordinary feat considering the lack of sequence identity among the bound Ribonucleases. Mammalian Ribonucleases such as RNase A or its human homologue, RNase 1, have the potential to be more desirable chemotherapeutic agents than ONC owing to their higher catalytic activity, low potential for immunogenicity, favorable tissue distribution, and high therapeutic index, but are limited by their sensitivity to RI. These non-toxic mammalian Ribonucleases can be transformed into potent cytotoxins by engendering them with RI-evasion using protein engineering strategies such as site-directed mutagenesis, multimerization, fusion to a targeting moiety, and chemical modification. In several instances, these engineered Ribonucleases exhibit greater cytotoxicity in vitro than does ONC. Herein, we review the biochemical characteristics of RI·Ribonuclease complexes and progress towards the development of mammalian Ribonuclease-based chemotherapeutics through the elicitation of RI-evasion.
Helene F Rosenberg - One of the best experts on this subject based on the ideXlab platform.
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eosinophil associated Ribonuclease 11 is a macrophage chemoattractant
Journal of Biological Chemistry, 2015Co-Authors: Kelsey J Yamada, Kimberly D Dyer, James J Lee, Tolga Barker, Tyler A Rice, Caroline M Percopo, Katia E Garciacrespo, Soochin Cho, Kirk M Druey, Helene F RosenbergAbstract:RNase A is the prototype of an extensive family of divergent proteins whose members share a unique disulfide-bonded tertiary structure, conserved catalytic motifs, and the ability to hydrolyze polymeric RNA. Several members of this family maintain independent roles as Ribonucleases and modulators of innate immunity. Here we characterize mouse eosinophil-associated RNase (Ear) 11, a divergent member of the eosinophil Ribonuclease cluster, and the only known RNase A Ribonuclease expressed specifically in response to Th2 cytokine stimulation. Mouse Ear 11 is differentially expressed in somatic tissues at baseline (brain ≪ liver < lung < spleen); systemic stimulation with IL-33 results in 10-5000-fold increased expression in lung and spleen, respectively. Ear 11 is also expressed in response to protective priming of the respiratory mucosa with Lactobacillus plantarum; transcripts are detected both locally in lung as well as systemically in bone marrow and spleen. Mouse Ear 11 is enzymatically active, although substantially less so than mEar 1 and mEar 2; the relative catalytic efficiency (kcat/Km) of mEar 11 is diminished ∼1000-1500-fold. However, in contrast to RNase 2/EDN and mEar 2, which have been characterized as selective chemoattractants for CD11c(+) dendritic cells, mEar 11 has prominent chemoattractant activity for F4/80(+)CD11c(-) tissue macrophages. Chemoattractant activity is not dependent on full enzymatic activity, and requires no interaction with the pattern recognition receptor, Toll-like receptor 2 (TLR2). Taken together, this work characterizes a divergent RNase A Ribonuclease with a unique expression pattern and function, and highlights the versatility of this family in promoting innate immunity.
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evolution and function of leukocyte rnase a Ribonucleases of the avian species gallus gallus
Journal of Biological Chemistry, 2006Co-Authors: Takeaki Nitto, Kimberly D Dyer, Meggan Czapiga, Helene F RosenbergAbstract:Abstract In this study, we explore the evolution and function of two closely related RNase A Ribonucleases from the chicken, Gallus gallus. Separated by ∼10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN > dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both ∼16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s-1, respectively), and surprisingly, both interact with human placental Ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of Ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the Ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.
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characterization of a Ribonuclease gene and encoded protein from the reptile iguana iguana
Gene, 2005Co-Authors: Takeaki Nitto, Kimberly D Dyer, Robert Wagner, Helene F RosenbergAbstract:Abstract In this work we identify an intronless open reading frame encoding an RNase A Ribonuclease from genomic DNA from the Iguana iguana IgH2 cell line. The iguana RNase is expressed primarily in pancreas, and represents the majority of the specific enzymatic activity in this tissue. The encoded sequence shares many features with its better-known mammalian counterparts including the crucial His 12 , Lys 40 and His 114 catalytic residues and efficient hydrolytic activity against yeast tRNA substrate ( k cat / K m = 6 × 10 4 M −1 s −1 ), albeit at a reduced pH optimum (pH 6.0). Although the catalytic activity of the iguana RNase is not diminished by human placental RI, iguana RNase is not bactericidal nor is it cytotoxic even at micromolar concentrations. Phylogenetic analysis indicates moderate (46%) amino acid sequence similarity to a pancreatic RNase isolated from Chelydra serpentina (snapping turtle) although no specific relationship could be determined between these RNases and the pancreatic Ribonucleases characterized among mammalian species. Further analysis of Ribonucleases from non-mammalian vertebrate species is needed in order to define relationships and lineages within the larger RNase A gene superfamily.
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diminished expression of an antiviral Ribonuclease in response to pneumovirus infection in vivo
Antiviral Research, 2003Co-Authors: Joanne M Moreau, Takeaki Nitto, Kimberly D Dyer, Joseph B Domachowske, Cynthia A Bonville, Nora L Vasquez, Andrew J Easton, Helene F RosenbergAbstract:Abstract The mouse eosinophil-associated Ribonucleases (mEars) are species specific, divergent orthologs of the human antiviral RNase A Ribonucleases, eosinophil-derived neurotoxin (RNase 2) and eosinophil cationic protein (RNase 3). We show here that mEar 2 is also an antiviral Ribonuclease, as micromolar concentrations promote a ∼sixfold reduction in the infectivity of pneumonia virus of mice (PVM) for target respiratory epithelial cells in vitro. Although initially identified as a component of eosinophilic leukocytes, mEar 2 mRNA and protein were also detected in lung tissue accompanied by enzymatically active mEar 2 in bronchoalveolar lavage fluid (BALF). At t =3 days post-inoculation with PVM (strain J3666), we observed the characteristic inflammatory response accompanied by diminished expression of total mEar mRNA and protein in lung tissue and a corresponding fivefold drop in Ribonuclease activity in BALF. No change in mEar expression was observed in response to infection with PVM strain 15, a replication-competent strain of PVM that does not elicit a cellular inflammatory response. However, mEar expression is not directly dependent on inflammation per se, as diminished expression of mEar mRNA and BAL Ribonuclease activity were also observed in PVM-infected, inflammation-deficient, MIP-1α −/− mice. We propose that this mechanism may represent a novel virus-mediated evasion strategy, with a mechanism that is linked in some fashion to virus-specific pathogenicity.
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eosinophils Ribonucleases and host defense solving the puzzle
Immunologic Research, 1999Co-Authors: Helene F Rosenberg, Joseph B DomachowskeAbstract:The eosinophil Ribonucleases eosinophil-derived neurotoxin (EDN/ RNase 2) and eosinophil cationic protein (ECP/RNase 3) are among the major secretory effector proteins of human eosinophilic leukocytes, cells whose role in host defense remains controversial and poorly understood. We have recently described the unusual manner in which this Ribonuclease lineage has evolved, with extraordinary diversification observed in primate as well as in rodent EDNs and ECPs. The results of our evolutionary studies suggest that the EDN/ ECP Ribonucleases are in the process of being tailored for a specific, Ribonuclease-related goal. With this in mind, we have begun to look carefully at some of the intriguing associations that link eosinophils and their Ribonucleases to disease caused by the single-stranded RNA viral pathogen, respiratory syncytial virus (RSV). Recent work in our laboratory has demonstrated that eosinophils can mediate a direct, Ribonuclease-dependent reduction in infectivity of RSV in vitro, and that EDN can function alone as an independent antiviral agent. The results of this work have led us to consider the possibility that the EDN/ECP Ribonucleases represent a heretofore unrecognized element of innate and specific antiviral host defense.
Johann Deisenhofer - One of the best experts on this subject based on the ideXlab platform.
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mechanism of Ribonuclease inhibition by Ribonuclease inhibitor protein based on the crystal structure of its complex with Ribonuclease a
Journal of Molecular Biology, 1996Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:We describe the mechanism of Ribonuclease inhibition by Ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and Ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the Ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of Ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse Ribonucleases. However, the inhibition is lost with amphibian Ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal Ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited
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crystal structure of porcine Ribonuclease inhibitor a protein with leucine rich repeats
Nature, 1993Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:Ribonuclease inhibitor is a cytoplasmic protein that tightly binds and inhibits Ribonucleases of the pancreatic Ribonuclease super-family1. The primary sequence of this inhibitor contains leucine-rich repeats2 (LRRs); these motifs are present in many proteins that participate in protein–protein interactions and have different functions and cellular locations. In vivo, Ribonuclease inhibitor may have a role in the regulation of RNA turnover in mammalian cells3 and in angiogenesis4. To define the structural features of LRR proteins and to understand better the nature of the tight interaction of Ribonuclease inhibitor with Ribonucleases, we have determined the crystal structure of the porcine inhibitor. To our knowledge, this is the first three-dimensional structure of a protein containing LRRs and represents a new class of α/β protein fold. Individual repeats constitute β–α structural units that probably also occur in other proteins containing LRRs. The non-globular shape of the structure and the exposed face of the parallel β-sheet may explain why LRRs are used to achieve strong protein–protein interactions. A possible Ribonuclease-binding region incorporates the surface formed by the parallel β-sheet and the βα loops.
Bestjan Kobe - One of the best experts on this subject based on the ideXlab platform.
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mechanism of Ribonuclease inhibition by Ribonuclease inhibitor protein based on the crystal structure of its complex with Ribonuclease a
Journal of Molecular Biology, 1996Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:We describe the mechanism of Ribonuclease inhibition by Ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and Ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the Ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of Ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse Ribonucleases. However, the inhibition is lost with amphibian Ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal Ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited
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crystal structure of porcine Ribonuclease inhibitor a protein with leucine rich repeats
Nature, 1993Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:Ribonuclease inhibitor is a cytoplasmic protein that tightly binds and inhibits Ribonucleases of the pancreatic Ribonuclease super-family1. The primary sequence of this inhibitor contains leucine-rich repeats2 (LRRs); these motifs are present in many proteins that participate in protein–protein interactions and have different functions and cellular locations. In vivo, Ribonuclease inhibitor may have a role in the regulation of RNA turnover in mammalian cells3 and in angiogenesis4. To define the structural features of LRR proteins and to understand better the nature of the tight interaction of Ribonuclease inhibitor with Ribonucleases, we have determined the crystal structure of the porcine inhibitor. To our knowledge, this is the first three-dimensional structure of a protein containing LRRs and represents a new class of α/β protein fold. Individual repeats constitute β–α structural units that probably also occur in other proteins containing LRRs. The non-globular shape of the structure and the exposed face of the parallel β-sheet may explain why LRRs are used to achieve strong protein–protein interactions. A possible Ribonuclease-binding region incorporates the surface formed by the parallel β-sheet and the βα loops.
Quentin Tavernier - One of the best experts on this subject based on the ideXlab platform.
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regulation of ire1 rnase activity by the Ribonuclease inhibitor 1 rnh1
Cell Cycle, 2018Co-Authors: Quentin Tavernier, Evangeline Bennana, Nicolas Pietrancosta, Virginie Poindessous, Celine Schaeffer, Luca Rampoldi, Nicolas PalletAbstract:ABSTRACTAdaptation to endoplasmic reticulum (ER) stress depends on the activation of the sensor inositol-requiring enzyme 1α (IRE1), an endoRibonuclease that splices the mRNA of the transcription factor XBP1 (X-box-binding protein 1). To better understand the protein network that regulates the activity of the IRE1 pathway, we systematically screened the proteins that interact with IRE1 and identified a Ribonuclease inhibitor called Ribonuclease/angiogenin inhibitor 1 (RNH1). RNH1 is a leucine-rich repeat domains-containing protein that binds to and inhibits Ribonucleases. Immunoprecipitation experiments confirmed this interaction. Docking experiments indicated that RNH1 physically interacts with IRE1 through its cytosolic RNase domain. Upon ER stress, the interaction of RNH1 with IRE1 in the ER increased at the expense of the nuclear pool of RNH1. Inhibition of RNH1 expression using siRNA mediated RNA interference upon ER stress led to an increased splicing activity of XBP1. Modulation of IRE1 RNase activ...
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Regulation of IRE1 RNase activity by the Ribonuclease inhibitor 1 (RNH1)
2018Co-Authors: Quentin Tavernier, Evangeline Bennana, Nicolas Pietrancosta, Virginie Poindessous, Celine Schaeffer, Luca Rampoldi, Nicolas PalletAbstract:Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the sensor inositol-requiring enzyme 1α (IRE1), an endoRibonuclease that splices the mRNA of the transcription factor XBP1 (X-box-binding protein 1). To better understand the protein network that regulates the activity of the IRE1 pathway, we systematically screened the proteins that interact with IRE1 and identified a Ribonuclease inhibitor called Ribonuclease/angiogenin inhibitor 1 (RNH1). RNH1 is a leucine-rich repeat domains-containing protein that binds to and inhibits Ribonucleases. Immunoprecipitation experiments confirmed this interaction. Docking experiments indicated that RNH1 physically interacts with IRE1 through its cytosolic RNase domain. Upon ER stress, the interaction of RNH1 with IRE1 in the ER increased at the expense of the nuclear pool of RNH1. Inhibition of RNH1 expression using siRNA mediated RNA interference upon ER stress led to an increased splicing activity of XBP1. Modulation of IRE1 RNase activity by RNH1 was recapitulated in a cell-free system, suggesting direct regulation of IRE1 by RNH. We conclude that RNH1 attenuates the activity of IRE1 by interacting with its Ribonuclease domain. These findings have implications for understanding the molecular mechanism by which IRE1 signaling is attenuated upon ER stress.
