Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

Toroney, Rebecca; Hull, Chelsea M.; Sokoloski, Joshua E.; Bevilacqua, Philip C.

doi:10.1261/rna.034520.112

Cited by 17 publications

(31 citation statements)

References 43 publications

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“…Here, we find that both tails are required for activity and symmetric deletion to 10-nt 5 ′ and 3 ′ tails blocks activation. It has been reported that a 5 ′ -ppp is crucial for activation of PKR by the canonical ss-dsRNAs as well as ssRNAs containing short duplexes (Nallagatla et al 2007;Toroney et al 2012). In contrast, we find that this moiety does not significantly contribute to PKR activation and only slightly affects binding affinity.…”

Section: Discussioncontrasting

confidence: 70%

“…It has been reported that a 5 ′ -ppp is crucial for activation of PKR by the canonical ss-dsRNAs as well as ssRNAs containing short duplexes (Nallagatla et al 2007;Toroney et al 2012). However, dephosphorylation of ppp-15-15-15 and ppp-5-15-10 by treatment with calf intestinal alkaline phosphatase (CIP) has no significant effect on the extent of PKR activation (Fig.…”

Section: ′ -Triphosphatementioning

confidence: 99%

“…The 5 ′ -ppp binding site lies within a positively charged pocket at the C-terminus of RIG-I (Cui et al 2008;Takahasi et al 2008;Lu et al 2010;Luo et al 2012). However, sites for ssRNA or triphosphate binding have not been identified in PKR (Toroney et al 2012). The duplex regions in all of these RNAs are too short to mediate PKR activation alone and the mechanism by which the 5 ′ -ppp and ssRNA regions elicit activation is not known.…”

Section: Introductionmentioning

confidence: 99%

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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: Discussioncontrasting

confidence: 70%

Section: ′ -Triphosphatementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Mayo

Wong

Lopez

et al. 2016

RNA

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“…Possibly a 5′-triphosphate increases the binding affinity of some snoRNAs to PKR. Consistent with this hypothesis, PKR contains a potential triphosphate-binding site (31). Most snoRNAs are encoded in introns of host genes.…”

Section: Discussionmentioning

confidence: 69%

Potential role for snoRNAs in PKR activation during metabolic stress

Youssef

Safran

Nakamura

et al. 2015

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

105

103

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Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKR WT ) or PKR with a point mutation in each dsRNA-binding motif (PKR RM ). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV-cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKR WT samples after PA treatment, but not in the PKR RM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5′-triphosphate enhanced PKR activity compared with the activity with a 5′-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress.PKR | snoRNA | metabolic stress | phosphorylation | RNA-binding protein

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“…First, AAV vectors cannot be used in patients with preexisting antibodies 96 or lose their efficacy on development of such antibodies in treated patients. Second, even subtle structural differences between synthetic or recombinant small RNA molecules and endogenous counterparts, for example, miRs, may activate the innate immune system through both toll-like receptor-dependent and toll-like receptor-independent mechanisms, 97,98 and considerable efforts have been made to define structural requirements to avoid this problem. Only in noncardiovascular diseases, for example, cancer, immunostimulation by ncRNAs may be an acceptable or even useful side effect.…”

Section: Side Effectsmentioning

confidence: 99%

Cardiovascular RNA Interference Therapy

Poller

Tank

Skurk

et al. 2013

Circulation Research

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Abstract:Understanding of the roles of noncoding RNAs (ncRNAs) within complex organisms has fundamentally changed. It is increasingly possible to use ncRNAs as diagnostic and therapeutic tools in medicine. Regarding disease pathogenesis, it has become evident that confinement to the analysis of protein-coding regions of the human genome is insufficient because ncRNA variants have been associated with important human diseases. Thus, inclusion of noncoding genomic elements in pathogenetic studies and their consideration as therapeutic targets is warranted. We consider aspects of the evolutionary and discovery history of ncRNAs, as far as they are relevant for the identification and selection of ncRNAs with likely therapeutic potential. Novel therapeutic strategies are based on ncRNAs, and we discuss here RNA interference as a highly versatile tool for gene silencing. RNA interference-mediating RNAs are small, but only parts of a far larger spectrum encompassing ncRNAs up to many kilobasepairs in size. We discuss therapeutic options in cardiovascular medicine offered by ncRNAs and key issues to be solved before clinical translation. Convergence of multiple technical advances is highlighted as a prerequisite for the translational progress achieved in recent years. Regarding safety, we review properties of RNA therapeutics, which may immunologically distinguish them from their endogenous counterparts, all of which underwent sophisticated evolutionary adaptation to specific biological contexts.

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Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

Cited by 17 publications

References 43 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

Potential role for snoRNAs in PKR activation during metabolic stress

Cardiovascular RNA Interference Therapy

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