Measurement of RNA-induced PKR Activation in vitro.
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ABSTRACT:
SUBMITTER: Yuen KC
PROVIDER: S-EPMC8376569 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protein Kinase R (PKR), the double-stranded RNA (dsRNA)-activated protein kinase, plays important roles in innate immunity. Previous studies have shown that PKR is activated by long stretches of dsRNA, RNA pseudoknots, and certain single-stranded RNAs; however, regulation of PKR by RNAs with globular tertiary structure has not been reported. In this study, the HDV ribozyme is used as a model of a mostly globular RNA. In addition to a catalytic core, the ribozyme contains a peripheral 13-bp pairing region (P4), which, upon shortening, affects neither the catalytic activity of the ribozyme nor its ability to crystallize. We report that the HDV ribozyme sequence alone can activate PKR. To elucidate the RNA structural basis for this, we prepared a number of HDV variants, including those with shortened or lengthened P4 pairing regions, with the anticipation that lengthening the P4 extension would yield a more potent activator since it would offer more base pairs of dsRNA. Surprisingly, the variant with a shortened P4 was the most potent activator. Through native gel mobility and enzymatic structure mapping experiments we implicate misfolded HDV ribozyme dimers as the PKR-activating species, and show that the shortened P4 leads to enhanced occupancy of the RNA dimer. These observations have implications for how RNA misfolding relates to innate immune response and human disease.
| S-EPMC3504668 | biostudies-literature
Project description:The double-stranded RNA (dsRNA)-activated protein kinase [protein kinase R (PKR)] plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15-bp dsRNA for one protein to bind and 30-bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eukaryotic initiation factor 2alpha, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15- to 30-bp limits: human immunodeficiency virus type 1 transactivation-responsive region (TAR) RNA, a 23-bp hairpin with three bulges that is known to dimerize. TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization to test whether RNA dimerization affects PKR dimerization and activation. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary-structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary-structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15- to 30-bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease.
| S-EPMC2763119 | biostudies-literature
Project description:NIPBL, a cohesin loader, has been implicated in transcriptional control and genome organization. Mutations in NIPBL, cohesin, and its deacetylase HDAC8 result in Cornelia de Lange syndrome. We report activation of the RNA-sensing kinase PKR in human lymphoblastoid cell lines carrying NIPBL or HDAC8 mutations, but not SMC1A or SMC3 mutations. PKR activation can be triggered by unmodified RNAs. Gene expression profiles in NIPBL-deficient lymphoblastoid cells and mouse embryonic stem cells reveal lower expression of genes involved in RNA processing and modification. NIPBL mutant lymphoblastoid cells show reduced proliferation and protein synthesis with increased apoptosis, all of which are partially reversed by a PKR inhibitor. Non-coding RNAs from an NIPBL mutant line had less m(6)A modification and activated PKR activity in vitro. This study provides insight into the molecular pathology of Cornelia de Lange syndrome by establishing a relationship between NIPBL and HDAC8 mutations and PKR activation.
| S-EPMC4904785 | biostudies-literature
Project description:Protein kinase R (PKR) functions in a plethora of cellular processes, including viral and cellular stress responses, by phosphorylating the translation initiation factor eIF2α. The minimum requirements for PKR function are homodimerization of its kinase and RNA-binding domains, and autophosphorylation at the residue Thr-446 in a flexible loop called the activation loop. We investigated the interdependence between dimerization and Thr-446 autophosphorylation using the yeast Saccharomyces cerevisiae model system. We showed that an engineered PKR that bypassed the need for Thr-446 autophosphorylation (PKR(T446∼P)-bypass mutant) could function without a key residue (Asp-266 or Tyr-323) that is essential for PKR dimerization, suggesting that dimerization precedes and stimulates activation loop autophosphorylation. We also showed that the PKR(T446∼P)-bypass mutant was able to phosphorylate eIF2α even without its RNA-binding domains. These two significant findings reveal that PKR dimerization and activation loop autophosphorylation are mutually exclusive yet interdependent processes. Also, we provide evidence that Thr-446 autophosphorylation during PKR activation occurs in a cis mechanism following dimerization.
| S-EPMC3937647 | biostudies-literature
Project description:The protein kinase PKR (protein kinase R) is a sensor in innate immunity. PKR autophosphorylates in the presence of double-stranded RNA enabling it to phosphorylate its substrate, eIF2α (eukaryotic initiation factor 2α), halting cellular translation. Classical activators of PKR are long viral double-stranded RNAs, but recently, PKR has been found to be activated by bacterial RNA. However, the features of bacterial RNA that activate PKR are unknown. We studied the Bacillus subtilis trp 5'-UTR (untranslated region), which is an indirect riboswitch with secondary and tertiary RNA structures that regulate gene function. Additionally, the trp 5'-UTR binds a protein, TRAP (tryptophan RNA-binding attenuation protein), which recognizes l-tryptophan. We present the first evidence that multiple structural features in this RNA, which are typical of bacterial RNAs, activate PKR in TRAP-free and TRAP/l-Trp-bound forms. Segments from the 5'-UTR, including the terminator 5'-stem-loop and Shine-Dalgarno blocking hairpins, demonstrated 5'-triphosphate and flanking RNA tail dependence on PKR activation. Disruption of long-distance tertiary interactions in the 5'-UTR led to partial loss in activation, consistent with highly base-paired regions in bacterial RNA activating PKR. One physiological change a bacterial RNA would face in a human cell is a decrease in the concentration of free magnesium. Upon lowering the magnesium concentration to human physiological conditions of 0.5mM, the trp 5'-UTR continued to activate PKR potently. Moreover, total RNA from Escherichia coli, depleted of rRNA, also activated PKR under these ionic conditions. This study demonstrates that PKR can signal the presence of bacterial RNAs under physiological ionic conditions and offers a potential explanation for the apparent absence of riboswitches in the human genome.
| S-EPMC4624505 | biostudies-literature
Project description:Protein kinase R (PKR) is a key pattern recognition receptor of the innate immune pathway. PKR is activated by double-stranded RNA (dsRNA) that is often produced during viral genome replication and transcription. PKR contains two tandem double-stranded RNA binding domains at the N-terminus, dsRBD1 and dsRBD2, and a C-terminal kinase domain. In the canonical model for activation, RNAs that bind multiple PKRs induce dimerization of the kinase domain that promotes an active conformation. However, there is evidence that dimerization of the kinase domain is not sufficient to mediate activation and PKR activation is modulated by the RNA-binding mode. dsRBD2 lacks most of the consensus RNA-binding residues, and it has been suggested to function as a modulator of PKR activation. Here, we demonstrate that dsRBD2 regulates PKR activation and identify the N-terminal helix as a critical region for modulating kinase activity. Mutations in dsRBD2 that have minor effects on overall dsRNA-binding affinity strongly inhibit the activation of PKR by dsRNA. These mutations also inhibit RNA-independent PKR activation. These data support a model where dsRBD2 has evolved to function as a regulator of the kinase.
| S-EPMC8153938 | biostudies-literature
Project description:Protein kinase R (PKR) is a central component of the interferon antiviral defense pathway. Upon binding to dsRNA, PKR undergoes autophosphorylation reactions that activate the kinase, resulting in the inhibition of protein synthesis in virally-infected cells. We have used analytical ultracentrifugation and related biophysical methods to quantitatively characterize the stoichiometries, affinities, and free energy couplings that govern the assembly of the macromolecular complexes in the PKR activation pathway. These studies demonstrate that PKR dimerization play a key role in enzymatic activation and support a model where the role of dsRNA is to bring two or more PKR monomers in close proximity to enhance dimerization.
| S-EPMC2926283 | biostudies-literature
Project description:Although the antiviral kinase PKR was originally characterized as a double-stranded RNA activated enzyme it can be stimulated by RNAs containing limited secondary structure. Single-stranded regions in such RNAs contribute to binding and activation but the mechanism is not understood. Here, we demonstrate that single-stranded RNAs bind to PKR with micromolar dissociation constants and can induce activation. Addition of a 5'-triphosphate slightly enhances binding affinity. Single-stranded RNAs also activate PKR constructs lacking the double-stranded RNA binding domain and bind to a basic region adjacent to the N-terminus of the kinase. However, the isolated kinase is not activated by and does not bind single-stranded RNA. Photocrosslinking measurements demonstrate that that the basic region interacts with RNA in the context of full length PKR. We propose that bivalent interactions with the double stranded RNA binding domain and the basic region underlie the ability of RNAs containing limited structure to activate PKR by enhancing binding affinity and thereby increasing the population of productive complexes containing two PKRs bound to a single RNA.
| S-EPMC5469796 | biostudies-literature
Project description:The double-stranded (ds) RNA-activated protein kinase, PKR, has a key role in the innate immunity response to viral infection in higher eukaryotes. PKR contains an N-terminal dsRNA-binding domain and a C-terminal kinase domain. In the prevalent autoinhibition model for PKR activation, dsRNA binding induces a conformational change that leads to the release of the dsRNA-binding domain from the kinase, thus relieving the inhibition of the latent enzyme. Structural and biophysical data now favor a model whereby dsRNA principally functions to induce dimerization of PKR via the kinase domain. This dimerization model has implications for other PKR regulatory mechanisms and represents a new structural paradigm for control of protein kinase activity.
| S-EPMC2703476 | biostudies-literature
Project description:PKR is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Some highly structured viral RNAs do not activate and function as PKR inhibitors. In order to define the mechanisms of activation and inhibition of PKR by RNA, it is necessary to characterize the stoichiometries, affinities, and free energy couplings governing the assembly of the relevant complexes. We have found sedimentation velocity analytical ultracentrifugation to be particularly useful in the study of PKR-RNA interactions. Here, we describe protocols for designing and analyzing sedimentation velocity experiments that are generally applicable to studies of protein-nucleic acid interactions. Initially, velocity data obtained at multiple protein:RNA ratios are analyzed using the dc/dt method's to define the association model and to test whether the system is kinetically limited. The sedimentation velocity data obtained at multiple loading concentrations are then globally fitted to this model to determine the relevant association constants. The frictional ratios of the complexes are calculated using the fitted sedimentation coefficients to determine whether the hydrodynamic properties are physically reasonable. We demonstrate the utility of this approach using examples from our studies of PKR interactions with simple dsRNAs, the HIV TAR RNA, and the VAI RNA from adenovirus.
| S-EPMC3058617 | biostudies-literature