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`Author Manuscript
`Curr Opin Struct Biol. Author manuscript; available in PMC 2012 February 1.
`Published in final edited form as:
`Curr Opin Struct Biol. 2011 February ; 21(1): 119–127. doi:10.1016/j.sbi.2010.11.003.
`
`RNA structure and regulation of innate immunity through protein
`kinase PKR
`
`Subba Rao Nallagatlaa,c, Rebecca Toroneya,c, and Philip C. Bevilacquaa,b
`aDepartment of Chemistry, The Pennsylvania State University, 104 Chemistry Bldg, University
`Park, PA 16802, USA
`
`Abstract
`Molecular recognition of RNA structure is key to innate immunity. The protein kinase PKR
`differentiates self from non-self by recognition of molecular patterns in RNA. Certain biological
`RNAs induce autophosphorylation of PKR, activating it to phosphorylate eukaryotic initiation
`factor 2(cid:302) (eIF2(cid:302)), which leads to inhibition of translation. Additional biological RNAs inhibit
`PKR, while still others have no effect. The aim of this article is to develop a cohesive framework
`for understanding and predicting PKR function in the context of diverse RNA structure. We
`present effects of recently characterized viral and cellular RNAs on regulation of PKR, as well as
`siRNAs. A central conclusion is that assembly of accessible long double-stranded RNA (dsRNA)
`elements within the context of biological RNAs plays a key role in regulation of PKR kinase.
`Strategies for forming such elements in biology include RNA dimerization, formation of
`symmetrical helical defects, A-form dsRNA mimicry, and coaxial stacking of helices.
`
`Introduction
`Numerous remarkable roles for RNA in biology have been uncovered [1]. RNA is central to
`translation; it can function as an enzyme (ribozyme) and genetic switch (riboswitch); and
`small RNAs play key roles in regulating genes. Many of these discoveries have been
`transformative to our understanding of life processes [2].
`
`A central reason why RNA plays crucial roles in biology is that it embodies both diverse
`structural and decodable sequence information. The folding of RNA has been described as
`hierarchical [3], in which primary structure forms as the RNA is being transcribed, followed
`by folding of secondary structure, and then tertiary structure, as the nascent secondary
`structural elements assemble (Figure 1a).
`
`There is great diversity present in each element of the hierarchy: Primary structure embodies
`different sequence and length, as well as modifications at the ends and internally (Figure
`1b). Secondary structure has as its basis the A-form helix, but is highly diverse owing to
`assorted imperfections (defects) present in most helices such as bulges, hairpin loops, and
`
`© 2010 Elsevier Ltd. All rights reserved.
`bCorresponding author: Philip C. Bevilacqua (pcb5@psu.edu), tel: (814) 863-3812, fax: (814) 865-2927. sxn19@psu.edu,
`rut115@psu.edu.
`cThese authors contributed equally to this work.
`Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
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`For submission to Current Opinion in Structural Biology
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`internal loops (Figure 1c). Tertiary structures are compact and often (but not always)
`globular forms of RNA that bring together helices and are highly diverse (Figure 1d).
`Adding even further to this complexity, the fold and interactions of RNA are dynamic as
`well: RNA folds as it is being transcribed, and it interacts with ions, metabolites, proteins,
`and other RNAs (Figure 1e) [4].
`
`Innate immunity is the initial immune response to invasion by pathogens [5]. Many proteins
`are involved in this process, including toll-like receptors (TLRs), retinoic acid-inducible
`gene 1 (RIG-I), and the RNA-activated protein kinase (PKR). One key function of these
`proteins is distinguishing self from non-self through so-called pathogen-associated
`molecular patterns, or ‘PAMPs’ [6]. Given RNA's diversity in sequence and structure, it
`comes as no surprise to find that nature has chosen RNA for many key PAMPs. Specific
`sequences and structures present in pathogenic RNA allow the innate immune system to
`distinguish between cellular RNAs and RNAs from viruses and foreign organisms [7].
`
`This review focuses on the RNA-based activation of PKR and how RNAs can serve as
`PAMPs. The last few years have witnessed increased understanding of PKR interaction with
`RNAs of diverse structure. We begin with an overview of PKR structure and its well-known
`interaction with dsRNA. We then describe recent contributions within the context of the
`RNA folding hierarchy, proceeding from primary to tertiary structure and ending with
`siRNAs and a brief comparison to other RNA-based regulating proteins of innate immunity.
`Our central goal is to develop a cohesive framework for understanding and predicting PKR
`function in the context of RNA structure.
`
`Structure and function of PKR
`The structural biology of PKR is best viewed as a work in progress. PKR is a 551 amino
`acid protein that consists of two functional domains: an N-terminal dsRNA binding domain
`(dsRBD) that comprises two dsRNA binding motifs (dsRBMs) spaced by a flexible 20
`amino acid linker,1 and a C-terminal kinase domain that contains the major sites for
`phosphorylation (Figure 2a) [8,9]. The dsRBM is a common motif that occurs in all
`kingdoms of life and is present in a number of notable proteins beyond PKR, including
`dicer, drosha, and adenosine deaminases that act on RNA (ADARs) [10]. The dsRBM
`typically recognizes dsRNA non-sequence specifically via minor groove interactions, and
`several reports indicate interactions with the bases [11,12]. Available structural biology of
`PKR includes an NMR structure of the dsRBD solved without RNA present [13], and a
`crystal structure for the kinase domain complexed with eIF2(cid:302) substrate [14]. The NMR
`structure reveals the typical (cid:302)(cid:533)(cid:533)(cid:533)(cid:302) architecture for each dsRBM [13], while the X-ray
`structure indicates a smaller, mostly (cid:533)-sheet N-terminal lobe (N-lobe) with a larger, stable,
`largely helical C-terminal lobe (C-lobe) (Figure 2a). The N-lobe of the kinase domain is
`involved in dimerization of PKR, whereas the (cid:302)G helix from the C-lobe acts as a substrate-
`docking motif [14]. Low-resolution structural models of full length latent (inactive) PKR
`have been constructed by small angle X-ray scattering (SAXS) and reveal that PKR has
`intrinsically disordered regions, which may become ordered upon RNA binding;
`interestingly, data from this method are not fully consistent with the autoinhibition model
`previously proposed for PKR (described below) in which the latent protein is locked into
`closed conformation, as described below [15]*.
`
`At present there are no RNA-bound structures of PKR, probably because the non-sequence
`specific nature of RNA binding and the disordered region between the dsRBD and the
`
`1This nomenclature is the convention used in the PKR field. However, more generally speaking, ‘dsRBM’ refers to the sequence
`motif, while ‘dsRBD’ refers to an independently folding domain.
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`kinase domain leads to heterogeneous states. However, a few structures of other dsRBMs
`bound to dsRNA have been solved; see for example [16,17]. In general, the dsRBM binds
`into the wide accessible minor groove of dsRNA, and multiple dsRBMs can pack along the
`length of the helix. Shown in Figure 2b is packing of two dsRBMs on ~20 bp of dsRNA.
`Packing of four dsRBMs on 33 bp of dsRNA, which is the minimum activating length, can
`be modeled similarly.
`
`The function of PKR in biology is quite diverse. A number of excellent reviews of PKR
`function are available [8,18-20], and only a very brief overview is presented here. In
`general, activation of latent PKR requires dimerization and autophosphorylation, which
`occurs upon recognition of sufficiently long dsRNA, such as from intermediates generated
`during viral replication. In general, 33 bp are needed for minimal activation, with longer
`dsRNAs activating to a greater extent. Shorter dsRNA, 15-30 bp, inhibits PKR through
`competitive binding [21,22]. The protein activator PACT and the polyanion heparin can also
`activate PKR [20], and PKR can even autophosphorylate in the absence of activator if its
`concentration is high enough [23]. The activated dimer of PKR goes on to phosphorylate its
`cellular substrate eIF2(cid:302) on Ser51 leading to translational arrest [8,19]. This process provides
`essential antiviral and antiproliferative capabilities for the host cell. More recently it was
`found that phosphorylation of three tyrosine residues on PKR, in addition to multiple serine/
`threonine phosphorylating sites, is required for full-scale activation of the kinase [24].
`
`In addition to the antiviral functions, PKR has been implicated in modulating cell-signalling
`pathways to alter numerous cellular responses [19]. In addition, several diseases, such as
`Huntington, Parkinson and Alzheimer's, have been linked to PKR regulation [20]. A recent
`report suggested that p53-mediated tumor suppression can be attributed to p53's induction of
`PKR under genotoxic conditions [25], while another recent study indicated that PKR
`regulates insulin action and metabolism in response to nutrient signals and endoplasmic
`reticulum stress [26].
`
`RNA primary structure-based regulation of PKR
`As presented in the Introduction, the folding of RNA is largely hierarchical (Figure 1), and
`high information content exists at each level in the folding hierarchy. The next three sections
`consider the three levels of RNA folding. The interplay of these RNA elements with
`regulation of PKR function is summarized in Figure 3.
`
`Early studies used perfect dsRNAs such as poly I:C and T7 transcribed dsRNAs of various
`lengths to characterize PKR activation [19]. More recent studies reveal additional activation
`by RNAs that are non-perfectly double stranded [27,28]. At the primary RNA sequence
`level, ssRNAs with a small stem-loop and an imperfect 16 base-paired dsRNA with 10-15 nt
`single-strand tails (so-called ‘ss-dsRNA’) have been shown to activate PKR in a 5’-
`triphosphate dependent manner [27,28]. This 5’-triphosphate functional group of ssRNA is
`key in PKR activation, as 5’-diphosphate, -monophosphte, -hydroxyl and 7mG cap-
`containing ssRNAs do not activate PKR [28]. Most endogenous cytoplasmic RNAs contain
`5’-monophophate or 7mG cap, generated through RNA processing, whereas bacterial and
`some viral RNAs contain 5’-triphosphate; the 5’-triphosphate functionality thus constitutes a
`PAMP for PKR. In contrast, activation of PKR by dsRNA does not require a 5’-
`triphosphate, indicating that PKR uses different strategies for recognition of ssRNA and
`dsRNA. The 5’-triphosphate serves as a PAMP for PKR in recognition of the viral RNA
`from influenza B virus as well [29]**. Additional experiments have demonstrated that
`internal nucleoside modifications in 5’-triphosphate ssRNA abrogate PKR activation [30]*,
`indicating that these may also serve in distinguishing self from non-self.
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`RNA secondary structure-based regulation of PKR
`Long stretches of double-stranded RNA ((cid:5795)33 bp) activate PKR potently, and have been
`proposed as the major activators of PKR in vivo. The molecular mechanism behind dsRNA-
`based activation of PKR has been studied extensively. Several models have been advanced,
`including an autoinhibition model, in which dsRNA binding to the dsRBD releases PKR
`from an inactive conformation, and a dimerization model, in which dsRNA binding serves to
`promote kinase dimerization [8]. Recently, analytical ultracentrifugation (AUC) has been
`employed to investigate the length dependence and stoichiometry of PKR binding to dsRNA
`[22,31]. These studies have demonstrated that dsRBM1 functions primarily in recognition of
`shorter dsRNA sequences (<20 bp), while both dsRBMs participate in recognition of longer
`dsRNAs, which are capable of activating PKR. Additionally, AUC studies have shown that
`the minimum requirement of ~33 bp for activation of PKR correlates with the ability to bind
`two PKR monomers. These data are consistent with a model in which long dsRNA functions
`to bring two PKR monomers into close proximity, which promotes dimerization and thus
`activation of the kinase domains.
`
`In addition to dsRNA, PKR has been found to be activated by a variety of viral and cellular
`RNAs, which typically contain various secondary structure imperfections. One such viral
`RNA is the human immunodeficiency virus transactivation-response region (HIV TAR), a
`23 bp hairpin RNA interrupted by three bulges that can exist as a dimer (Figure 4a) [32].
`There has been longstanding discrepancy about the role of HIV TAR RNA in regulation of
`PKR; recent evidence, however, strongly supports that a dimeric form of TAR activates
`PKR [33,34]. In this study, monomers and dimers of TAR were isolated by native gel
`electrophoresis and studied both structurally and functionally. In particular, it was found that
`two TAR hairpin monomers refold to form an extended duplex with two asymmetric bulges,
`which effectively doubles the number of base pairs from ~23 bp in TAR monomer to ~46 bp
`in TAR dimer. It was found that monomer inhibited PKR, while dimer activated it,
`consistent with the known dependence of PKR function on dsRNA length. Thus, in this
`case, RNA dimerization promoted PKR dimerization and activation. In addition, this study
`showed that RNA dimers with fewer asymmetrical secondary structure defects were more
`potent activators of PKR, suggesting that such defects function as antideterminants of PKR
`binding.
`
`The IRES of HCV has been reported to regulate PKR [35-37]. A strategy by which dsRNAs
`with imperfections can activate PKR is through structural mimicry of perfect A-form
`dsRNA, as recently demonstrated in activation of PKR by domain II of hepatitis C virus
`internal ribosome entry site (HCV IRES) RNA [36]*. The IRES element of HCV has a
`complex secondary structure with four distinct structural domains containing multiple
`symmetric and asymmetric bulges, internal loops, and a pseudoknot (Figure 4b). Despite
`these complicated structural elements, several domains of HCV IRES RNA have been
`reported as activators of PKR, including domains III-IV, which contains several multi-helix
`junctions and a pseudoknot, and domain II, a shorter hairpin with several internal loops and
`bulges [36-38]. Given both the presence of imperfections and the limited number of
`canonical base pairs (< 33 bp), activation of PKR by domain II in particular is surprising.
`Footprinting and mutational analysis suggest that PKR binds and is potently activated by
`domain II RNA because the overall topology of its symmetrical loop regions is primarily A-
`form [36]*. Non-Watson-Crick interactions in the loops of domain II maintain an overall A-
`form helical backbone geometry and contribute to an activating total of ~33 bp. Mimicry of
`A-form dsRNA by symmetrical loops may serve as a general mechanism for PKR activation
`by RNAs with multiple helical imperfections.
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`Regulation of PKR by RNA secondary structure is also typified by abrogation of PKR
`dimerization and activation through binding of inhibitory RNAs, such as those encoded by
`adenovirus (VAI) and Epstein-Barr virus (EBERI). Both RNAs bind PKR with similar
`affinity as activating RNAs, but prevent PKR dimerization and subsequent
`autophosphorylation [39]. VAI and EBERI have roughly similar structures with three
`distinct domains: an apical stem-loop, a central domain, and a terminal stem. In the case of
`VAI, the apical stem-loop has been identified as the PKR dsRBD binding site, and the three-
`way junction within the central domain is the determinant for PKR inhibition [40]*; this
`domain includes elements of tertiary structure, which will be discussed in the next section.
`The terminal stem is completely dispensable for inhibition [41]*. The VAI apical stem-loop
`consists of ~18 canonical and non-canonical base pairs, which is sufficient for binding one
`PKR monomer but not long enough to promote PKR dimerization. Interestingly, the apical
`stem-loop domain of VAI exists as a population of two conformations, one of which
`potently inhibits PKR, and the other of which displays markedly decreased inhibition
`activity [42]*. Possible benefits of these functionally distinct structures for either the virus
`or the host have yet to be determined.
`
`Although the function of PKR is typically to serve as a sensor of non-self RNA, certain
`cellular RNAs activate PKR. Previous work by Davis et al. and Nussbaum et al. identified
`the 3’-UTRs (untranslated region) of several highly structured cytoskeletal mRNAs as
`activators of PKR [43,44]. Interestingly, PKR activation by cytoskeletal 3’-UTRs is
`predicted to play a role in the tumor-related activities of these sequences. Similar to
`previously discussed viral RNAs, these cellular RNAs contain long helical stretches
`interrupted by bulges, internal loops, and branch points. Also, an element of the 3’-UTR of
`tumor necrosis factor (cid:302) mRNA (TNF-(cid:302)) has also been shown to activate PKR [45]. Control
`of exogenous gene expression by PKR is attenuated by full-length ADAR1 as well as its
`dsRBMs alone, suggesting that PKR and ADAR1 compete for binding to the same RNAs
`[46,47]. Whether this effect carries over to cellular RNAs is unclear at present [48].
`
`RNA tertiary structure-based regulation of PKR
`Tertiary structure has the potential to activate or inhibit PKR. Given PKR's penchant for
`dsRNA, one simple idea is that if the tertiary structure is globular, activation is unlikely, but
`that if it is extended, activation is possible. The 5’-UTR of the cellular mRNA for interferon-
`gamma (IFN-(cid:534)) fits this model (Figure 4c). As part of the interferon-mediated antiviral
`response, PKR participates in a negative feedback loop whereby IFN-(cid:534) regulates its own
`translation via competition between the ribosome and PKR for binding to IFN-(cid:534) mRNA
`[49,50]. If the level of PKR in the cell is low, the ribosome binds to IFN-(cid:534) mRNA to
`promote interferon synthesis. Upon clearing the ribosome, the 5’-UTR refolds to generate an
`RNA structure containing a pseudoknot, which is capable of activating PKR. Four adjoining
`short helices within IFN-(cid:534) mRNA coaxially stack within the pseudoknot to cumulate to an
`activating total of ~33 bp. Thus, in addition to RNA oligomerization by HIV TAR and A-
`form structural mimicry by HCV domain II, the amalgamation of secondary and tertiary
`features in IFN-(cid:534) mRNA demonstrates another means by which the hierarchical nature of
`RNA folding can generate RNA structures capable of activating PKR.
`
`Finally, a role for RNA tertiary structure in PKR activation lies in the VAI viral RNA. It was
`recently determined that Mg2+, which is often required for stabilization of RNA tertiary
`structure, is required for correct folding of the VAI central domain and leads to binding of
`just one PKR. This helps explain the well-established inhibitory role of this RNA [51]*.
`Melting profiles and compensatory base pair modifications suggested a possible role of
`RNA tertiary structure in PKR inhibition by VAI RNA [40]. It has been suggested that,
`while the terminal stem of VAI may function to stabilize this tertiary structure, in the
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`absence of the terminal stem, PKR binding to VAI may stabilize tertiary structure [41],
`although the exact nature of this tertiary structure has not been fully characterized [52].
`
`siRNA-based regulation of PKR
`There exist conflicting results on the activation of PKR by small interfering RNAs (siRNA).
`siRNA are short, 19-27 bp, dsRNAs that mediate RNA interference. Several groups have
`reported that siRNAs of 19-21 bp do not activate PKR, supporting the aforementioned
`requirement of 33 bp dsRNA for PKR activation [28,53]. In particular, Kim et al. [53]
`designed long siRNAs of 27 bp to enhance RNAi potency and efficiency and showed that
`they do not activate PKR, while we found that 21 bp double-stranded siRNAs do not
`activate PKR [27,28].
`
`In contrast to these observations, activation of PKR by siRNA containing just 19 to 21 bp
`has been reported [54,55]. A proposed model for PKR activation by these shorter dsRNAs
`suggested that a PKR dimer assembled on one siRNA to phosphorylate a PKR dimer bound
`to a different siRNA [55].
`
`Activation of PKR has a strong dependence on ionic strength, and lower salt conditions
`favor short RNA binding [22]. Also blunt end siRNAs activated PKR less potently than
`sticky ends [55]. Thus, experimental conditions, sequence, and helix termini of siRNA may
`play crucial roles in determining which siRNAs activate PKR [55]. Lastly, other studies
`indicate that activation of PKR by siRNAs is more efficient in vitro than in vivo [56], while
`others indicated that in vivo effects may be indirect [57]. More work is needed to sort out
`these discrepancies.
`
`Comparison of PKR to other RNA-based regulating proteins in innate
`immunity
`
`RIG-I and Toll-like receptors (TLR 3, 7 and 8) are additional sensors in innate immunity
`that recognize patterns associated with non-self RNAs. Indeed, dsRNA and 5’-triphosphate
`groups, which PKR recognizes as mentioned, can also be recognized by RIG-I [28,58,59].
`Moreover, several natural nucleoside modifications in RNA can negate the 5’-triphosphate
`and dsRNA dependent activation of PKR and RIG-I [30,58,60]. Indeed, in vitro transcribed
`pseudouridine-containing mRNA translates better than unmodified mRNA owing to
`diminishing PKR activation [61]*. Regarding TLRs, they are similarly affected: TLR3 is
`regulated by similar nucleoside modifications in dsRNA [62], while TLR 7 and 8 are
`regulated by such modifications in ssRNA. Remarkably, PKR, RIG-I, and TLRs are not
`sequence homologues, supporting unique molecular recognition strategies by each and
`suggesting convergent evolution.
`
`Conclusions and outlook
`The RNA-activated protein kinase PKR is activated by much more than long perfect RNA
`helices. Recent studies indicate that biological RNAs activate PKR by diverse strategies and
`to varying extents: dimerization of RNA, inclusion of symmetrical defects, mimicry of A-
`form dsRNA, and coaxial stacking of helices. A common theme is assembly of accessible
`double-stranded elements that reach the activating length of ~33 bp. Covalent modifications
`to the 5’–end and internal regions of RNA can either activate or inhibit the kinase. Much
`remains to be understood about the link between RNA structure and extent of PKR
`activation, including roles of RNA tertiary structure, RNA aggregation, and co-
`transcriptional folding. Additional cellular and viral RNAs that regulate PKR surely await
`discovery, and high-resolution structures of PKR bound to dsRNA and complex biological
`RNAs are needed. Moreover, ways in which various RNAs alter the fraction and extent of
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`PKR phosphorylation are unknown. Such future advances would help to further define the
`RNA features that allow PKR to perform its essential functions in innate immunity.
`
`Acknowledgments
`We thank Pete Beal, Jim Cole, Rick Russell and Scott Showalter for helpful comments on the manuscript, and
`National Institutes of Health grant R01-58709 for support.
`
`References and recommended reading
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`• of special interest
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`• • of outstanding interest
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`Curr Opin Struct Biol. Author manuscript; available in PMC 2012 February 1.
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