Regulation of innate immunity through RNA structure and the protein kinase PKR

Nallagatla, Subba Rao; Toroney, Rebecca; Bevilacqua, Philip C.

doi:10.1016/j.sbi.2010.11.003

Cited by 121 publications

(124 citation statements)

References 62 publications

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“…The only pathway that is functional in mESCs seems to be the PKR pathway, which is activated by polyIC and responsible for polyIC-induced inhibition of translation and cell proliferation as we previously reported [7]. Although best characterized as dsRNAactivated kinase, recent studies have indicated that PKR can be also activated by ssRNA depending on certain structural features of ssRNA [44] and that there is a synergistic involvement of RIG-I and IFN [49]. The failure of ssRNA to activate PKR in mESCs can be explained by the inactive status of the RIG-I pathway and defected IFN expression mechanism in these cells.…”

Section: Discussionmentioning

confidence: 81%

“…It is noted that polyIC fails to induce type I IFN in D3 cells, but it can activate PKR and induce a strong inhibition of cell proliferation [7]. Since it has been reported that PKR can also be activated by ssRNA that have certain structural features [44], we analyzed the effect of 3p-EGFP mRNA on PKR activation in mESCs. As a positive control experiment, we show that transfection of D3 cells with polyIC significantly increased the level of phosphorylated eIF2a, indicating the activation of PKR (Fig.…”

Section: Short 3p-ssrna Induced Strong Antiviral Responses In Fibroblmentioning

confidence: 99%

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Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy

Wang

Teng

Spangler

et al. 2014

Stem Cells and Development

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We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFN) when exposed to viral infection and double-stranded RNA. In this study, we extended our investigation and demonstrated that single-stranded RNA and protein-encoding mRNA can induce strong IFN expression and cytotoxicity in fibroblasts and epithelial cells, but none of the effects associated with these antiviral responses were observed in mESCs. Our results provided additional data to support the conclusion that mESCs are intrinsically deficient in antiviral responses. While our findings represent a novel feature of mESCs that in itself is important for understanding innate immunity development, we exploited this property to develop a novel mRNA-mediated gene expression cell model. Direct introduction of synthetic mRNA to express desired genes has been shown as an effective alternative to DNA/viral vector-based gene expression. However, a major biological challenge is that a synthetic mRNA is detected as a viral RNA analog by the host cell, resulting in a series of adverse effects associated with antiviral responses. We demonstrate that the lack of antiviral responses in mESCs effectively avoids this problem. mESCs can tolerate repeated transfection and effectively express proteins from their synthetic mRNA with expected biological functions, as demonstrated by the expression of green fluorescent protein and the transcription factor Etv2. Therefore, mRNA-based gene expression could be developed into a novel ESC differentiation strategy that avoids safety concerns associated with viral/DNA-based vectors in regenerative medicine.

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Section: Discussionmentioning

confidence: 81%

Section: Short 3p-ssrna Induced Strong Antiviral Responses In Fibroblmentioning

confidence: 99%

Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy

Wang

Teng

Spangler

et al. 2014

Stem Cells and Development

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“…In support of this hypothesis, we find that exogenously supplied dsRNA can activate PKR and trigger necrosis without need for mRNA transcription. Activation of PKR by cellular RNAs is not without precedent: the 3′-untranslated regions (UTRs) of several cytoskeletal and cytokine mRNAs have been shown to activate this kinase in various physiological contexts (22)(23)(24)(25)(26). Indeed, such endogenous mRNAs can activate PKR even more potently than dsRNA (27).…”

Section: Discussionmentioning

confidence: 99%

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases

Thapa

Nogusa

Chen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

297

255

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Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinasemediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.necroptosis | apoptosis I nterferons (IFNs) are pleiotropic cytokines classified into two primary groups, type I (predominantly α/β) and type II (γ). Both classes of IFNs exert their effects via similar Janus kinase (JAK)-signal transducers and activators of transcription (STAT)-dependent signaling cascades to induce the expression of over 500 genes (1). Such IFN-stimulated genes (ISGs) have been reasonably well characterized in the context of antiviral or immune-modulatory signaling, but less is known about how they collaborate to mediate the cytotoxic and antiproliferative effects of IFNs.Recent studies have shed light on a new form of regulated cell death that is activated when caspase-dependent apoptotic pathways are inhibited. This mode of necrotic cell death, sometimes called "necroptosis," requires the serine-threonine kinases receptor-interacting protein 1 (RIP1) and RIP3, and results from overproduction of reactive oxygen species (ROS) and eventual mitochondrial dysfunction (2, 3). Strict negative control of the pronecrotic kinases RIP1 and RIP3 are essential for several aspects of mammalian development and homeostasis, including immune cell proliferation and progression through embryogenesis (4). The proteins FADD, caspase 8, and c-FLIP represent three such negative regulators; in the absence of any of these molecules, the RIP kinases trigger inopportune necrosis, often with severe consequences for the host (4). The core necrosis machinery is thus carefully regulated to execute cell death only in specific contexts, but how this regulation is achieved and which other upstream stimuli exploit RIP kinases to activate necrosis are still relatively poorly described.In the present study, we show that both IFN-γ and IFN-α/β t...

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“…PKR is synthesized in a latent state and is activated via autophosphorylation upon binding to duplex regions present in viral RNAs (Nallagatla et al 2011). Activated PKR then phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), leading to inhibition of protein synthesis in infected cells.…”

Section: Introductionmentioning

confidence: 99%

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Mayo

Wong

Lopez

et al. 2016

RNA

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Protein kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains a C-terminal kinase domain and two tandem dsRNA binding domains. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA of 30 bp is required for activation. However, short (∼15 bp) stem-loop RNAs containing flanking single-stranded tails (ss-dsRNAs) are capable of activating PKR. Activation was reported to require a 5 ′ -triphosphate. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15-nt single-stranded tails and a 15-bp stem and made systematic truncations of the tail and stem regions. Autophosphorylation assays and analytical ultracentrifugation experiments were used to correlate activation and binding affinity. PKR activation requires both 5 ′ -and 3 ′ -single-stranded tails but the triphosphate is dispensable. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. These results indicate that the single-stranded regions bind to PKR and support a model where ss-dsRNA induced dimerization is required but not sufficient to activate the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single-stranded regions may contribute to PKR activation in these cases.

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Regulation of innate immunity through RNA structure and the protein kinase PKR

Cited by 121 publications

References 62 publications

Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy

Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases

Activation of PKR by short stem–loop RNAs containing single-stranded arms

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