Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

Heinicke, Laurie A.; Bevilacqua, Philip C.

doi:10.1261/rna.034744.112

Cited by 26 publications

(40 citation statements)

References 37 publications

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“…Thus, the tails do not adopt unwanted secondary structure. Self-complementary RNA hairpins have the potential to dimerize, which can complicate analysis of their interactions with PKR (Heinicke and Bevilacqua 2012;Heinicke et al 2009). Sedimentation velocity analysis indicates that all of the ssdsRNAs are homogeneous and monomeric (Supplemental Table S2).…”

Section: Characterization Of Ss-dsrnasmentioning

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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Section: Characterization Of Ss-dsrnasmentioning

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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“…Several studies support an activation of interferon signalling pathways by HDV: in vitro, HDV replication was first shown to be associated with increased expression of IFNβ (191); later the expression of HDAg was associated with an induction of MxA (an Interferon Stimulated Gene -ISG) (192) and recently misfolded HDV RNA was shown to directly activate PKR (193). In vivo HDV was also associated with an increased expression of ISGs, both in the hNTCP transgenic mouse and in the humanized mice model (68,194).…”

Section: Natural History and Pathogenesismentioning

confidence: 99%

Hepatitis delta virus: From biological and medical aspects to current and investigational therapeutic options

2015

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“…Activation of PKR is well known to be promoted by long stretches of dsRNA (.33 base pairs), which may arise as intermediates during viral replication. RNA species of this length are long enough to accommodate a PKR dimer [10][11][12]. Two models of RNA-mediated activation of PKR have been offered: 1) a model in which PKR has intrinsically disordered regions that become ordered upon RNA binding, and 2) an autoinhibition model in which the latent protein is locked into a closed conformation that is relieved upon binding to dsRNA of sufficient length [3,13].…”

Section: Introductionmentioning

confidence: 99%

Native Tertiary Structure and Nucleoside Modifications Suppress tRNA’s Intrinsic Ability to Activate the Innate Immune Sensor PKR

et al. 2013

Self Cite

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Interferon inducible protein kinase PKR is an essential component of innate immunity. It is activated by long stretches of dsRNA and provides the first line of host defense against pathogens by inhibiting translation initiation in the infected cell. Many cellular and viral transcripts contain nucleoside modifications and/or tertiary structure that could affect PKR activation. We have previously demonstrated that a 5′-end triphosphate–a signature of certain viral and bacterial transcripts–confers the ability of relatively unstructured model RNA transcripts to activate PKR to inhibit translation, and that this activation is abrogated by certain modifications present in cellular RNAs. In order to understand the biological implications of native RNA tertiary structure and nucleoside modifications on PKR activation, we study here the heavily modified cellular tRNAs and the unmodified or the lightly modified mitochondrial tRNAs (mt-tRNA). We find that both a T7 transcript of yeast tRNAPhe and natively extracted total bovine liver mt-tRNA activate PKR in vitro, whereas native E. coli, bovine liver, yeast, and wheat tRNAPhe do not, nor do a variety of base- or sugar-modified T7 transcripts. These results are further supported by activation of PKR by a natively folded T7 transcript of tRNAPhe in vivo supporting the importance of tRNA modification in suppressing PKR activation in cells. We also examine PKR activation by a T7 transcript of the A14G pathogenic mutant of mt-tRNALeu, which is known to dimerize, and find that the misfolded dimeric form activates PKR in vitro while the monomeric form does not. Overall, the in vitro and in vivo findings herein indicate that tRNAs have an intrinsic ability to activate PKR and that nucleoside modifications and native RNA tertiary folding may function, at least in part, to suppress such activation, thus serving to distinguish self and non-self tRNA in innate immunity.

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Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

Cited by 26 publications

References 37 publications

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

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

Hepatitis delta virus: From biological and medical aspects to current and investigational therapeutic options

Native Tertiary Structure and Nucleoside Modifications Suppress tRNA’s Intrinsic Ability to Activate the Innate Immune Sensor PKR

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