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PubMed1992

A conserved double-stranded RNA-binding domain.

We have identified a double-stranded (ds)RNA-binding domain in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in staufen and one in Xlrbpa, we delimited the minimal region that binds dsRNA. On the basis of the binding studies and computer analysis, we have derived a consensus sequence that defines a 65- to 68-amino acid dsRNA-binding domain.

Amino Acid Sequence↗
PubMed1999

Subsets of the zinc finger motifs in dsRBP-ZFa can bind double-stranded RNA.

dsRBP-ZFa is a Xenopus zinc finger protein that binds dsRNA and RNA-DNA hybrids with high affinity and in a sequence-independent manner. The protein consists of a basic N-terminal region with seven C2H2 zinc finger motifs and an acidic C-terminal region that is not required for binding. The last four zinc finger motifs, and the linkers that join them, are nearly identical repeats, while the first three motifs and their linkers are each unique. To identify which regions of the protein are involved in nucleic acid binding, we examined the ability of five protein fragments to bind dsRNA and RNA-DNA hybrids. Our studies reveal that a fragment encompassing the three N-terminal, unique zinc finger motifs and another encompassing the last three of the nearly identical motifs have binding properties similar to the full-length protein. Since these two fragments do not share zinc finger motifs of the same sequence, dsRBP-ZFa must contain more than one type of zinc finger motif capable of binding dsRNA. As with the full-length protein, ssRNA and DNA do not significantly compete for dsRNA binding by the fragments.

Amino Acid Sequence↗
PubMed2003

RNA binding and intramolecular interactions modulate the regulation of gene expression by nuclear factor 110.

Nuclear factor 110 (NF110) belongs to the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA) binding proteins that regulate gene expression at the transcriptional level in vertebrates. The proteins are identical at their N terminus, which functions as a negative regulatory region, but have distinct C termini as a result of alternate splicing. Maximal transcriptional activity of NF110 requires its C-terminal domain and a central domain that contains a nuclear localization signal and two dsRNA-binding motifs (dsRBMs). We find that dsRNA binding is reduced by RGG and GQSY motifs present in the C-terminal region. To directly evaluate the role of RNA binding in transactivation, we conducted site-directed mutagenesis to substitute conserved residues in one or both of the dsRBMs. The mutations reduced the ability of NF110 to stimulate gene expression to an extent that paralleled the mutants' reduced ability to bind dsRNA. Full activity was restored when the dsRBM-containing region of NF110 was replaced with the RNA-binding region of the protein kinase PKR. Finally, NF110-mediated transactivation was inhibited by cotransfection of a plasmid encoding an artificial highly structured RNA. These data suggest that NF110 and its homologs are regulated by cis-acting domains present in some of the protein isoforms, and via interactions with RNAs that bind to their dsRBMs. We propose a model in which structured RNAs regulate gene expression by modulating transcription through interactions with members of the NF90 protein family.

Base Sequence↗
PubMed2006

Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling.

The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-alpha/beta) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) . poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) . poly(rC), poly(rA) . poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-beta promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKepsilon and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.

Animals↗
PubMed1995

Human PKR transfected into murine cells stimulates expression of genes under control of the HIV1 or HTLV-I LTR.

We have analyzed the effect of transfection into murine NIH/3T3 cells of the human dsRNA-activated kinase PKR on the expression of the beta-galactosidase reporter gene, placed under control of the HIV1 or the HTLV-I LTR. beta-Galactosidase expression is stimulated when the reporter plasmids are cotransfected with wild-type PKR but inhibited when cotransfected with a catalytically inactive mutant PKR. In the case of HIV1, beta-galactosidase expression was not stimulated when cotransfection was carried out with PKR harboring mutations in the dsRNA binding domains, indicating that stimulation depends on the classical mode of PKR activation through dsRNA binding. In contrast, the dsRNA binding mutants of PKR could still partially stimulate beta-galactosidase expression from the HTLV-I LTR, suggesting that PKR activation in this case may involve different/additional mechanisms. These results show that, in addition to the known down-regulation of protein synthesis through elF2 phosphorylation, PKR can also positively stimulate gene expression in vivo, most probably through phosphorylation of a substrate distinct from elF2.

3T3 Cells↗
PubMed2002

Ribonuclease activity and RNA binding of recombinant human Dicer.

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes.

Animals↗
PubMed2001

Investigating the structure of human RNase H1 by site-directed mutagenesis.

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.

Amino Acid Sequence↗
PubMed1995

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Amino Acid Sequence↗
PubMed2000

Regulation of the RNA-dependent protein kinase by triple helix formation.

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phos-phorylates the alpha-subunit of the translation initiation factor eIF-2, inhibiting its function. PKR is activated in vitro by binding to double-stranded RNA (dsRNA) molecules of approximately 30 bp or longer. Here we show that triple helix forming oligonucleotides (TFOs) inhibit dsRNA binding to the isolated RNA binding domain of PKR. The inhibition is specific to the targeted RNA and dependent on TFO length. Binding to a 30 bp duplex is inhibited by a 28 nt TFO and a 20 nt TFO with an IC(50) of 35 +/- 2 and 210 +/- 22 nM, respectively. An 18 nt TFO partially inhibits binding. The activation of the kinase domain of PKR by a 30 bp RNA duplex is also inhibited by a 28 nt TFO. Inhibition of binding is most effective when the triple helix is formed prior to addition of the protein. These results indicate that triplex formation can be used to prevent the binding of an RNA binding protein with dsRNA-binding motifs.

Base Sequence↗
PubMed1998

The vaccinia virus E3L gene product interacts with both the regulatory and the substrate binding regions of PKR: implications for PKR autoregulation.

The vaccinia virus E3L gene product, pE3, is a dsRNA binding protein that prevents activation of the interferon-induced, dsRNA-activated protein kinase, PKR. Activation of PKR, which results in phosphorylation of the translation initiation factor, eIF2alpha, leads to the inhibition of protein synthesis, a process involved in defense against virus infection. The E3L gene product has a conserved dsRNA binding domain (DRBD) in its carboxyl-terminal region and has been shown to function in vitro by sequestration of dsRNA. We have utilized in vitro binding assays and the yeast two-hybrid system to demonstrate direct interactions of pE3 with PKR. By these methods, we demonstrate that pE3 interacts with two distinct regions in PKR, the amino-terminal (amino acids 1-99) located in the regulatory domain and the carboxyl-terminal (amino acids 367-523) located in the catalytic domain. The amino-terminal region of PKR that interacts with pE3 contains a conserved DRBD, suggesting that PKR can form nonfunctional heterodimers with pE3, analogous to those seen with other dsRNA binding proteins. Interaction of pE3 with the amino-terminal region of PKR is enhanced by dsRNA. In contrast, dsRNA reduces the interaction of pE3 with the carboxyl-terminal region of PKR. Competition experiments demonstrate that the carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with which eIF2alpha and the pseudosubstrate pK3 interact, suggesting that pE3 may also prevent PKR activation by masking the substrate binding domain. Like pE3, the amino-terminal region of PKR also interacts with the carboxyl-terminal domain of PKR. These interactions increase our understanding of the mechanisms by which pE3 downregulates PKR. In addition, the PKR-PKR interactions observed leads us to suggest a novel autoregulatory mechanism for activation of PKR in which dsRNA binding to the DRBD(s) induces a conformational change that results in release of the amino terminal region from the substrate binding domain, allowing access to eIF2alpha and its subsequent phosphorylation.

Binding Sites↗
PubMed2006

Double-stranded RNA binding of influenza B virus nonstructural NS1 protein inhibits protein kinase R but is not essential to antagonize production of alpha/beta interferon.

Expression of alpha/beta interferon (IFN-alpha/beta) in virus-infected vertebrate cells is a key event in the establishment of a sustained antiviral response, which is triggered by double-stranded RNA (dsRNA) produced during viral replication. These antiviral cytokines initiate the expression of cellular proteins with activities that limit the replication and spread of the invading viruses. Within this response, the dsRNA-dependent protein kinase R (PKR) that is expressed at constitutive levels and upregulated by IFN-alpha/beta acts as an important antiviral effector that can block the cellular translational machinery. We previously demonstrated that efficient replication of influenza B virus depends on the viral dsRNA-binding NS1 protein that inhibits the transcriptional activation of IFN-alpha/beta genes. Here we tested the postulate that the viral NS1 protein counteracts antiviral responses through sequestering intracellular dsRNA by analyzing a collection of recombinant influenza B viruses. As expected, viruses expressing dsRNA-binding-defective NS1 proteins were strongly attenuated for replication in IFN-competent hosts. Interestingly, these virus mutants failed to prevent activation of PKR but could effectively limit IFN induction. Conversely, a mutant virus expressing the N-terminal dsRNA-binding domain of NS1 prevented PKR activation, but not IFN induction, suggesting an important role for the NS1 C-terminal part in silencing the activation route of IFN-alpha/beta genes. Thus, our findings indicate an unexpected mechanistic dichotomy of the influenza B virus NS1 protein in the suppression of antiviral responses, which involves at least one activity that is largely separable from dsRNA binding.

Cell Line↗
PubMed2000

Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR.

The human interferon-induced protein kinase, PKR, is an antiviral agent that is activated by long stretches of double-stranded (ds)RNA. PKR has an N-terminal dsRNA-binding domain that contains two tandem copies of the dsRNA-binding motif and interacts with dsRNA in a nonsequence-specific fashion. Surprisingly, PKR can be regulated by certain viral and cellular RNAs containing non-Watson-Crick features. We found that RNAs containing bulges in the middle of a helix can bind to p20, a C-terminal truncated PKR containing the dsRNA-binding domain. Bulges are known to change the global geometry of RNA by bending the helical axis; therefore, we investigated the conformational changes of bulged RNA caused by PKR binding. A 66-mer DNA-RNA(+/- A(3) bulge)-DNA chimera was constructed and annealed to a complementary RNA strand. This duplex forces the protein to bind in the middle. A 66-mer duplex with a top strand composed of DNA-DNA(+/-A(3) bulge)-RNA was used as a control. Gel mobility-shift changes among the RNA-protein complexes are consistent with straightening of bulged RNA on protein binding. In addition, a van't Hoff analysis of p20 binding to bulged RNA reveals a favorable DeltaDeltaH degrees and an unfavorable DeltaDeltaS degrees relative to binding to straight dsRNA. These thermodynamic parameters are in good agreement with predictions from a nearest-neighbor analysis for RNA straightening and support a model in which the helical junction flanking the bulge stacks on protein binding. The ability of dsRNA-binding motif proteins to recognize and straighten bent RNA has implications for modulating the topology of RNAs in vivo.

Base Sequence↗
PubMed2006

Activation of PKR: an open and shut case?

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.

Animals↗
PubMed2000

Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR.

Myotonic dystrophy is caused by an expanded CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The expanded repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK probably does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats may interfere with nuclear function. Here we show by thermal melting and nuclease digestion studies that CUG repeats form highly stable hairpins. Furthermore, CUG repeats bind to the dsRNA-binding domain of PKR, the dsRNA-activated protein kinase. The threshold for binding to PKR is approximately 15 CUG repeats, and the affinity increases with longer repeat lengths. Finally, CUG repeats that are pathologically expanded can activate PKR in vitro. These results raise the possibility that the disease mechanism could be, in part, a gain of function by mutant DMPK transcripts that involves sequestration or activation of dsRNA binding proteins.

Base Pairing↗
PubMed2003

Recognition of double-stranded RNA by proteins and small molecules.

Molecular recognition of double-stranded RNA (dsRNA) is a key event for numerous biological pathways including the trafficking, editing, and maturation of cellular RNA, the interferon antiviral response, and RNA interference. Over the past several years, our laboratory has studied proteins and small molecules that bind dsRNA with the goal of understanding and controlling the binding selectivity. In this review, we discuss members of the dsRBM class of proteins that bind dsRNA. The dsRBM is an approximately 70 amino acid sequence motif found in a variety of dsRNA-binding proteins. Recent results have led to a new appreciation of the ability of these proteins to bind selectivity to certain sites on dsRNA. This property is discussed in light of the RNA selectivity observed in the function of two proteins that contain dsRBMs, the RNA-dependent protein kinase (PKR) and an adenosine deaminase that acts on dsRNA (ADAR2). In addition, we introduce peptide-acridine conjugates (PACs), small molecules designed to control dsRBM-RNA interactions. These intercalating molecules bear variable peptide appendages at opposite edges of an acridine heterocycle. This design imparts the potential to exploit differences in groove characteristics and/or base-pair dynamics at binding sites to achieve selective binding.

Acridines↗
PubMed2003

RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum.

RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick-host interface.

Animals↗
PubMed2003

C114 is a novel IL-11-inducible nuclear double-stranded RNA-binding protein that inhibits protein kinase R.

We have identified a cDNA (named C114) that encodes novel transcripts induced by IL-11 in mouse 3T3 L1 cells. Northern analysis of RNAs from multiple mouse tissues detects two C114 transcripts of approximately 1.0 and approximately 2.0 kb with the highest expression in liver, testis, brain, and kidney. The C114 cDNA contains an open reading frame of 187 amino acids with a predicted mass of 21 kDa. Three putative nuclear localization signals are predicted at amino acids 83-88, 126-131, and 167-178. Using green fluorescent protein (GFP)-C114 fusion plasmids, amino acids 126-131 are shown to be essential for the nuclear localization of C114. An arginine-rich region (amino acids 98-143) spanning the nuclear localization signals (amino acids 126-131) exhibits a double-stranded RNA (dsRNA) binding activity. Competition experiments with different RNA homopolymers demonstrate that C114 preferentially binds to poly(I.C). Similar to other dsRNA-binding proteins, C114 binds to the dsRNA-activated protein kinase, protein kinase R (PKR), via dsRNA-binding domains of PKR and the N-terminal region of the C114 protein. In vitro kinase assays indicate that C114 inhibits PKR activation via a dsRNA-independent mechanism. Overexpression of C114 protein inhibits the induction of eIF-2alpha phosphorylation following poly(I.C) treatment. This is the first demonstration of a novel PKR modulator induced by a gp130 superfamily cytokine that may play a role in cytokine-mediated biological functions.

3T3 Cells↗
PubMed2000

A dynamically tuned double-stranded RNA binding mechanism for the activation of antiviral kinase PKR.

A key step in the activation of interferon-inducible antiviral kinase PKR involves differential binding of viral double-stranded RNA (dsRNA) to its two structurally similar N-terminal dsRNA binding motifs, dsRBM1 and dsRBM2. We show here, using NMR spectroscopy, that dsRBM1 with higher RNA binding activity exhibits significant motional flexibility on a millisecond timescale as compared with dsRBM2 with lower RNA binding activity. We further show that dsRBM2, but not dsRBM1, specifically interacts with the C-terminal kinase domain. These results suggest a dynamically tuned dsRNA binding mechanism for PKR activation, where motionally more flexible dsRBM1 anchors to dsRNA, thereby inducing a cooperative RNA binding for dsRBM2 to expose the kinase domain.

Amino Acid Sequence↗