Double-stranded RNA (dsRNA), a hallmark viral material that activates antiviral interferon (IFN) responses, can appear in human cells also in the absence of viruses. We identify phosphorothioate DNAs (PS DNAs) as triggers of such endogenous dsRNAs (endo-dsRNAs). PS DNAs inhibit decay of nuclear RNAs and induce endo-dsRNA via accumulation of Intronic and Intergenic Inverted Retroelements (IIIR). IIIRs activate responses distinct from the antiviral response and do not turn on transcriptional RIG-I/MDA5/IFN signaling, but trigger the dsRNA-sensing pathways of OAS3/RNase L and PKR. The RNase L pathway acts both as a sensor and a cleavage system for these classes of RNAs. Thus, our data suggest that nuclear RNA decay and nuclear-cytosolic RNA sorting provide a checkpoint controlling innate immune response to human endo-dsRNAs. OAS3/RNase L and PKR form a specialized arm of the innate immune system, distinct from antiviral IFN signaling, which monitors quality of RNA sorting by sensing escaping IIIRs.

ADAR1 isoforms are adenosine deaminases that edit and destabilize double-stranded RNA reducing its immunostimulatory activities. Mutation of ADAR1 leads to a severe neurodevelopmental and inflammatory disease of children, Aicardi-Goutiéres syndrome. In mice, Adar1 mutations are embryonic lethal but are rescued by mutation of the Mda5 or Mavs genes, which function in IFN induction. However, the specific IFN regulated proteins responsible for the pathogenic effects of ADAR1 mutation are unknown. We show that the cell-lethal phenotype of ADAR1 deletion in human lung adenocarcinoma A549 cells is rescued by CRISPR/Cas9 mutagenesis of the RNASEL gene or by expression of the RNase L antagonist, murine coronavirus NS2 accessory protein. Our result demonstrate that ablation of RNase L activity promotes survival of ADAR1 deficient cells even in the presence of MDA5 and MAVS, suggesting that the RNase L system is the primary sensor pathway for endogenous dsRNA that leads to cell death.

The mammalian innate immune system uses several sensors of double-stranded RNA (dsRNA) to develop the interferon response. Among these sensors are dsRNA-activated oligoadenylate synthetases (OAS), which produce signaling 2',5'-linked RNA molecules (2-5A) that activate regulated RNA decay in mammalian tissues. Different receptors from the OAS family contain one, two, or three copies of the 2-5A synthetase domain, which in several instances evolved into pseudoenzymes. The structures of the pseudoenzymatic domains and their roles in sensing dsRNA are unknown. Here we present the crystal structure of the first catalytically inactive domain of human OAS3 (hOAS3.DI) in complex with a 19-bp dsRNA, determined at 2.0-Å resolution. The conformation of hOAS3.DI is different from the apo- and the dsRNA-bound states of the catalytically active homolog, OAS1, reported previously. The unique conformation of hOAS3.DI disables 2-5A synthesis by placing the active site residues nonproductively, but favors the binding of dsRNA. Biochemical data show that hOAS3.DI is essential for activation of hOAS3 and serves as a dsRNA-binding module, whereas the C-terminal domain DIII carries out catalysis. The location of the dsRNA-binding domain (DI) and the catalytic domain (DIII) at the opposite protein termini makes hOAS3 selective for long dsRNA. This mechanism relies on the catalytic inactivity of domain DI, revealing a surprising role of pseudoenzyme evolution in dsRNA surveillance.

Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.

Double-stranded RNA (dsRNA) activates the innate immune system of mammalian cells and triggers intracellular RNA decay by the pseudokinase and endoribonuclease RNase L. RNase L protects from pathogens and regulates cell growth and differentiation by destabilizing largely unknown mammalian RNA targets. We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and nontargets. We show that this RNase L-dependent decay selectively affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 and other miRs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these miRs and acts as a suppressor of proliferation and adhesion in mammalian cells. Our data suggest that RNase L-dependent decay serves to establish an antiproliferative state via destabilization of the miR-regulated transcriptome.

Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.

One of the hallmark mechanisms activated by type I interferons (IFNs) in human tissues involves cleavage of intracellular RNA by the kinase homology endoribonuclease RNase L. We report 2.8 and 2.1 angstrom crystal structures of human RNase L in complexes with synthetic and natural ligands and a fragment of an RNA substrate. RNase L forms a crossed homodimer stabilized by ankyrin (ANK) and kinase homology (KH) domains, which positions two kinase extension nuclease (KEN) domains for asymmetric RNA recognition. One KEN protomer recognizes an identity nucleotide (U), whereas the other protomer cleaves RNA between nucleotides +1 and +2. The coordinated action of the ANK, KH, and KEN domains thereby provides regulated, sequence-specific cleavage of viral and host RNA targets by RNase L.

The human sensor of double-stranded RNA (dsRNA) oligoadenylate synthetase 1 (hOAS1) polymerizes ATP into 2',5'-linked iso-RNA (2-5A) involved in innate immunity, cell cycle, and differentiation. We report the crystal structure of hOAS1 in complex with dsRNA and 2'-deoxy ATP at 2.7 Å resolution, which reveals the mechanism of cytoplasmic dsRNA recognition and activation of oligoadenylate synthetases. Human OAS1 recognizes dsRNA using a previously uncharacterized protein/RNA interface that forms via a conformational change induced by binding of dsRNA. The protein/RNA interface involves two minor grooves and has no sequence-specific contacts, with the exception of a single hydrogen bond between the -NH(2) group of nucleobase G17 and the carbonyl oxygen of serine 56. Using a biochemical readout, we show that hOAS1 undergoes more than 20,000-fold activation upon dsRNA binding and that canonical or GU-wobble substitutions produce dsRNA mutants that retain either full or partial activity, in agreement with the crystal structure. Ultimately, the binding of dsRNA promotes an elaborate conformational rearrangement in the N-terminal lobe of hOAS1, which brings residues D75, D77, and D148 into proximity and creates coordination geometry for binding of two catalytic Mg(2+) ions and ATP. The assembly of this critical active-site structure provides the gate that couples binding of dsRNA to the production and downstream functions of 2-5A.

BACKGROUND: Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity. RESULTS: Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+ and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors. CONCLUSIONS: Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer.

BACKGROUND: The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. RESULTS: This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. CONCLUSIONS: Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein-RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes.

Accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). Ire1, an ER-resident transmembrane kinase/RNase, senses the protein folding status inside the ER. When activated, Ire1 oligomerizes and trans-autophosphorylates, activating its RNase and initiating a nonconventional mRNA splicing reaction. Splicing results in production of the transcription factor Hac1 that induces UPR target genes; expression of these genes restores ER homeostasis by increasing its protein folding capacity and allows abatement of UPR signaling. Here, we uncouple Ire1's RNase from its kinase activity and find that cells expressing kinase-inactive Ire1 can regulate Ire1's RNase, splice HAC1 mRNA, produce Hac1 protein, and induce UPR target genes. Unlike wild-type IRE1, kinase-inactive Ire1 cells display defects in Ire1 deactivation. Failure to properly inactivate Ire1 causes chronic ER stress and reduces cell survival under UPR-inducing conditions. Thus, Ire1-catalyzed phosphoryl-transfer aids disassembly of Ire1 signaling complexes and is a critical component of the UPR homeostatic feedback loop.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), an intracellular signaling pathway that adjusts the protein folding capacity of the ER according to need. If homeostasis in the ER protein folding environment cannot be reestablished, cells commit to apoptosis. The ER-resident transmembrane kinase-endoribonuclease inositol-requiring enzyme 1 (IRE1) is the best characterized UPR signal transduction molecule. In yeast, Ire1 oligomerizes upon activation in response to an accumulation of misfolded proteins in the ER. Here we show that the salient mechanistic features of IRE1 activation are conserved: mammalian IRE1 oligomerizes in the ER membrane and oligomerization correlates with the onset of IRE1 phosphorylation and RNase activity. Moreover, the kinase/RNase module of human IRE1 activates cooperatively in vitro, indicating that formation of oligomers larger than four IRE1 molecules takes place upon activation. High-order IRE1 oligomerization thus emerges as a conserved mechanism of IRE1 signaling. IRE1 signaling attenuates after prolonged ER stress. IRE1 then enters a refractive state even if ER stress remains unmitigated. Attenuation includes dissolution of IRE1 clusters, IRE1 dephosphorylation, and decline in endoribonuclease activity. Thus IRE1 activity is governed by a timer that may be important in switching the UPR from the initially cytoprotective phase to the apoptotic mode.

Deficiencies in the protein-folding capacity of the endoplasmic reticulum (ER) in all eukaryotic cells lead to ER stress and trigger the unfolded protein response (UPR). ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the messenger RNA encoding a key transcription activator, Hac1 in yeast or XBP1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base-pairing interaction between the HAC1 intron and the HAC1 5' untranslated region. After excision of the intron, transfer RNA ligase joins the severed exons, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue. Hac1 in turn drives the UPR gene expression program comprising 7-8% of the yeast genome to counteract ER stress. Here we show that, on activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3' untranslated region. Disruption of either Ire1 clustering or HAC1 mRNA recruitment impairs UPR signalling. The HAC1 3' untranslated region element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfils this requirement for HAC1 mRNA. Recruitment of mRNA to signalling centres provides a new paradigm for the control of eukaryotic gene expression.

Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-A crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors.

Restrictocin, a member of the alpha-sarcin family of site-specific endoribonucleases, uses electrostatic interactions to bind to the ribosome and to RNA oligonucleotides, including the minimal specific substrate, the sarcin/ricin loop (SRL) of 23S-28S rRNA. Restrictocin binds to the SRL by forming a ground-state E:S complex that is stabilized predominantly by Coulomb interactions and depends on neither the sequence nor structure of the RNA, suggesting a nonspecific complex. The 22 cationic residues of restrictocin are dispersed throughout this protein surface, complicating a priori identification of a Coulomb interacting surface. Structural studies have identified an enzyme-substrate interface, which is expected to overlap with the electrostatic E:S interface. Here, we identified restrictocin residues that contribute to binding in the E:S complex by determining the salt dependence [partial differential log(k 2/ K 1/2)/ partial differential log[KCl]] of cleavage of the minimal SRL substrate for eight point mutants within the protein designed to disrupt contacts in the crystallographically defined interface. Relative to the wild-type salt dependence of -4.1, a subset of the mutants clustering near the active site shows significant changes in salt dependence, with differences of magnitude being >or=0.4. This same subset was identified using calculated salt dependencies for each mutant derived from solutions to the nonlinear Poisson-Boltzmann equation. Our findings support a mechanism in which specific residues on the active site face of restrictocin (primarily K110, K111, and K113) contribute to formation of the E:S complex, thereby positioning the SRL substrate for site-specific cleavage. The same restrictocin residues are expected to facilitate targeting of the SRL on the surface of the ribosome.

Restrictocin is a site-specific endoribonuclease that inactivates ribosomes by cleaving the sarcin/ricin loop (SRL) of 23S-28S rRNA. Here we present a kinetic and thermodynamic analysis of the SRL cleavage reaction based on monitoring the cleavage of RNA oligonucleotides (2-27-mers). Restrictocin binds to a 27-mer SRL model substrate (designated wild-type SRL) via electrostatic interactions to form a nonspecific ground state complex E:S. At pH 6.7, physical steps govern the reaction rate: the wild-type substrate reacts at a partially diffusion-limited rate, and a faster-reacting SRL, containing a 3'-sulfur atom at the scissile phosphate, reacts at a fully diffusion-limited rate (k2/K1/2 = 1.1 x 10(9) M-1 s-1). At pH 7.4, the chemical step apparently limits the SRL cleavage rate. After the nonspecific binding step, restrictocin recognizes the SRL structure, which imparts 4.3 kcal/mol transition state stabilization relative to a single-stranded RNA. The two conserved SRL modules, bulged-G motif and GAGA tetraloop, contribute at least 2.4 and 1.9 kcal/mol, respectively, to the recognition. These findings suggest a model of SRL recognition in which restrictocin contacts the GAGA tetraloop and the bulged guanosine of the bulged-G motif to progress from the nonspecific ground state complex (E:S) to the higher-energy-specific complex (E.S) en route to the chemical transition state. Comparison of restrictocin with other ribonucleases revealed that restrictocin exhibits a 10(3)-10(6)-fold smaller ribonuclease activity against single-stranded RNA than do the restrictocin homologues, non-structure-specific ribonucleases T1 and U2. Together, these findings show how structural features of the SRL substrate facilitate catalysis and provide a mechanism for distinguishing between cognate and noncognate RNA.

Alpha-sarcin and ricin represent two structurally and mechanistically distinct families of site-specific enzymes that block translation by irreversibly modifying the sarcin/ricin loop (SRL) of 23S-28S rRNA. alpha-Sarcin family enzymes are designated as ribotoxins and act as endonucleases. Ricin family enzymes are designated as ribosome inactivating proteins (RIP) and act as N-glycosidases. Recently, we demonstrated that basic surface residues of the ribotoxin restrictocin promote rapid and specific ribosome targeting by this endonuclease. Here, we report that three RIP: ricin A, saporin, and gypsophilin depurinate the ribosome with strong salt sensitivity and achieve unusually fast kcat/Km approximately 10(9)-10(10) M(-1) s(-1), implying that RIP share with ribotoxins a common mechanism of electrostatically facilitated ribosome targeting. Bioinformatics analysis of RIP revealed that surface charge properties correlate with the presence of the transport chain in the RIP molecule, suggesting a second role for the surface charge in RIP transport. These findings put forward surface electrostatics as an important determinant of RIP activity.

Alpha-sarcin ribotoxins comprise a unique family of ribonucleases that cripple the ribosome by catalyzing endoribonucleolytic cleavage of ribosomal RNA at a specific location in the sarcin/ricin loop (SRL). The SRL structure alone is cleaved site-specifically by the ribotoxin, but the ribosomal context enhances the reaction rate by several orders of magnitude. We show that, for the alpha-sarcin-like ribotoxin restrictocin, this catalytic advantage arises from favorable electrostatic interactions with the ribosome. Restrictocin binds at many sites on the ribosomal surface and under certain conditions cleaves the SRL with a second-order rate constant of 1.7 x 10(10) M(-1) s(-1), a value that matches the predicted frequency of random restrictocin-ribosome encounters. The results suggest a mechanism of target location whereby restrictocin encounters ribosomes randomly and diffuses within the ribosomal electrostatic field to the SRL. These studies show a role for electrostatics in protein-ribosome recognition.