Structural basis for Z-DNA binding and stabilization by the zebrafish Z-DNA dependent protein kinase PKZ

The RNA-dependent protein kinase PKR plays a central role in the antiviral defense of vertebrates by shutting down protein translation upon detection of viral dsRNA in the cytoplasm. In some teleost fish, PKZ, a homolog of PKR, performs the same function, but surprisingly, instead of dsRNA binding domains, it harbors two Z-DNA/Z-RNA-binding domains belonging to the Zalpha domain family. Zalpha domains have also been found in other proteins, which have key roles in the regulation of interferon responses such as ADAR1 and DNA-dependent activator of IFN-regulatory factors (DAI) and in viral proteins involved in immune response evasion such as the poxviral E3L and the Cyprinid Herpesvirus 3 ORF112. The underlying mechanism of nucleic acids binding and stabilization by Zalpha domains is still unclear. Here, we present two crystal structures of the zebrafish PKZ Zalpha domain (DrZalpha^PKZ) in alternatively organized complexes with a (CG)₆ DNA oligonucleotide at 2 and 1.8 Å resolution. These structures reveal novel aspects of the Zalpha interaction with DNA, and they give insights on the arrangement of multiple Zalpha domains on DNA helices longer than the minimal binding site.     

The crystal structure of the Zbeta domain of the RNA-editing enzyme ADAR1 reveals distinct conserved surfaces among Z-domains

The Zalpha domains represent a growing subfamily of the winged helix-turn-helix (HTH) domain family whose members share a remarkable ability to bind specifically to Z-DNA and/or Z-RNA. They have been found exclusively in proteins involved in interferon response and, while their importance in determining pox viral pathogenicity has been demonstrated, their actual target and biological role remain obscure. Cellular proteins containing Zalpha domains bear a second homologous domain termed Zbeta, which appears to lack the ability to bind left-handed nucleic acids. Here, we present the crystal structure of the Zbeta domain from the human double-stranded RNA adenosine deaminase ADAR1 at 0.97 A, determined by single isomorphous replacement including anomalous scattering. Zbeta maintains a winged-HTH fold with the addition of a C-terminal helix. Mapping of the Zbeta conservation profile on the Zbeta surface reveals a new conserved surface formed partly by the terminal helix 4, involved in metal binding and dimerization and absent from Zalpha domains. Our results show how two domains similar in fold may have evolved into different functional entities even in the context of the same protein.     

Modulation of ADAR1 editing activity by Z-RNA in vitro

RNA editing by A-to-I modification has been recognized as an important molecular mechanism for generating RNA and protein diversity. In mammals, it is mediated by a family of adenosine deaminases that act on RNAs (ADARs). The large version of the editing enzyme ADAR1 (ADAR1-L), expressed from an interferon-responsible promoter, has a Z-DNA/Z-RNA binding domain at its N-terminus. We have tested the in vitro ability of the enzyme to act on a 50 bp segment of dsRNA with or without a Z-RNA forming nucleotide sequence. A-to-I editing efficiency is markedly enhanced in presence of the sequence favoring Z-RNA. In addition, an alteration in the pattern of modification along the RNA duplex becomes evident as reaction times decrease. These results suggest that the local conformation of dsRNA molecules might be an important feature for target selectivity by ADAR1 and other proteins with Z-RNA binding domains.     

The Zalpha domain from human ADAR1 binds to the Z-DNA conformer of many different sequences.

Z-DNA, the left-handed conformer of DNA, is stabilized by the negative supercoiling generated during the movement of an RNA polymerase through a gene. Recently, we have shown that the editing enzyme ADAR1 (double-stranded RNA adenosine deaminase, type 1) has two Z-DNA binding motifs, Zalpha and Zbeta, the function of which is currently unknown. Here we show that a peptide containing the Zalpha motif binds with high affinity to Z-DNA as a dimer, that the binding site is no larger than 6 bp and that the Zalpha domain can flip a range of sequences, including d(TA)3, into the Z-DNAconformation. Evidence is also presented to show that Zalpha and Zbeta interact to form a functional DNA binding site. Studies with atomic force microscopy reveal that binding of Zalpha to supercoiled plasmids is associated with relaxation of the plasmid. Pronounced kinking of DNA is observed, and appears to be induced by binding of Zalpha. The results reported here support a model where the Z-DNA binding motifs target ADAR1 to regions of negative supercoiling in actively transcribing genes. In this situation, binding by Zalpha would be dependent upon the local level of negative superhelicity rather than the presence of any particular sequence.     

Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2

Adenosine deaminases acting on RNA (ADARs) hydrolytically deaminate adenosines (A) in a wide variety of duplex RNAs and misregulation of editing is correlated with human disease. However, our understanding of reaction selectivity is limited. ADARs are modular enzymes with multiple double-stranded RNA binding domains (dsRBDs) and a catalytic domain. While dsRBD binding is understood, little is known about ADAR catalytic domain/RNA interactions. Here we use a recently discovered RNA substrate that is rapidly deaminated by the isolated human ADAR2 deaminase domain (hADAR2-D) to probe these interactions. We introduced the nucleoside analog 8-azanebularine (8-azaN) into this RNA (and derived constructs) to mechanistically trap the protein–RNA complex without catalytic turnover for EMSA and ribonuclease footprinting analyses. EMSA showed that hADAR2-D requires duplex RNA and is sensitive to 2′-deoxy substitution at nucleotides opposite the editing site, the local sequence and 8-azaN nucleotide positioning on the duplex. Ribonuclease V1 footprinting shows that hADAR2-D protects ∼23 nt on the edited strand around the editing site in an asymmetric fashion (∼18 nt on the 5′ side and ∼5 nt on the 3′ side). These studies provide a deeper understanding of the ADAR catalytic domain–RNA interaction and new tools for biophysical analysis of ADAR–RNA complexes.     

Conformational changes that occur during an RNA-editing adenosine deamination reaction

ADARs are adenosine deaminases responsible for RNA-editing reactions that occur within duplex RNA. Currently little is known regarding the nature of the protein-RNA interactions that lead to site-selective adenosine deamination. We previously reported that ADAR2 induced changes in 2-aminopurine fluorescence of a modified substrate, consistent with a base-flipping mechanism. Additional data have been obtained using full-length ADAR2 and a protein comprising only the RNA binding domain (RBD) of ADAR2. The increase in 2-aminopurine fluorescence is specific to the editing site and dependent on the presence of the catalytic domain. Hydroxyl radical footprinting demonstrates that the RBD protects a region of the RNA duplex around the editing site, suggesting a significant role for the RBD in identifying potential ADAR2 editing sites. Nucleotides near the editing site on the non-edited strand become hypersensitive to hydrolytic cleavage upon binding of ADAR2 RBD. Therefore, the RBD may assist base flipping by increasing the conformational flexibility of nucleotides in the duplex adjacent to its binding site. In addition, an increase in tryptophan fluorescence is observed when ADAR2 binds duplex RNA, suggesting a conformational change in the catalytic domain of the enzyme. Furthermore, acrylamide quenching experiments indicate that RNA binding creates heterogeneity in the solvent accessibility of ADAR2 tryptophan residues, with one out of five tryptophans more solvent-accessible in the ADAR2.RNA complex.     

Editing independent effects of ADARs on the miRNA/siRNA pathways

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR‐B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir‐376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase‐inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA‐binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.     

ADAR1 RNA deaminase limits short interfering RNA efficacy in mammalian cells

Double-stranded RNA induces the homology-dependent degradation of cognate mRNA in the cytoplasm via RNA interference (RNAi) but also is a target for adenosine-to-inosine (A-to-I) RNA editing by adenosine deaminases acting on RNA (ADARs). An interaction between the RNAi and the RNA editing pathways in Caenorhabditis elegans has been suggested recently, but the precise mode of interaction remains to be established. In addition, it is unclear whether this interaction is possible in mammalian cells with their somewhat different RNAi pathways. Here we show that ADAR1 and ADAR2, but not ADAR3, avidly bind short interfering RNA (siRNA) without RNA editing. In particular, the cytoplasmic full-length isoform of ADAR1 has the highest affinity among known ADARs, with a subnanomolar dissociation constant. Gene silencing by siRNA is significantly more effective in mouse fibroblasts homozygous for an ADAR1 null mutation than in wild-type cells. In addition, suppression of RNAi effects are detected in fibroblast cells overexpressing functional ADAR1 but not when overexpressing mutant ADAR1 lacking double-stranded RNA-binding domains. These results identify ADAR1 as a cellular factor that limits the efficacy of siRNA in mammalian cells.     

Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR+/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR-/- cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.     

RNA binding-independent dimerization of adenosine deaminases acting on RNA and dominant negative effects of nonfunctional subunits on dimer functions

RNA editing that converts adenosine to inosine in double-stranded RNA (dsRNA) is mediated by adenosine deaminases acting on RNA (ADAR). ADAR1 and ADAR2 form respective homodimers, and this association is essential for their enzymatic activities. In this investigation, we set out experiments aiming to determine whether formation of the homodimer complex is mediated by an amino acid interface made through protein-protein interactions of two monomers or via binding of the two subunits to a dsRNA substrate. Point mutations were created in the dsRNA binding domains (dsRBDs) that abolished all RNA binding, as tested for two classes of ADAR ligands, long and short dsRNA. The mutant ADAR dimer complexes were intact, as demonstrated by their ability to co-purify in a sequential affinity-tagged purification and also by their elution at the dimeric fraction position on a size fractionation column. Our results demonstrated ADAR dimerization independent of their binding to dsRNA, establishing the importance of protein-protein interactions for dimer formation. As expected, these mutant ADARs could no longer perform their catalytic function due to the loss in substrate binding. Surprisingly, a chimeric dimer consisting of one RNA binding mutant monomer and a wild type partner still abolished its ability to bind and edit its substrate, indicating that ADAR dimers require two subunits with functional dsRBDs for binding to a dsRNA substrate and then for editing activity to occur.     

Increased RNA editing and inhibition of hepatitis delta virus replication by high-level expression of ADAR1 and ADAR2

Hepatitis delta virus (HDV) is a subviral human pathogen that uses specific RNA editing activity of the host to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the longer form (HDAg-L), which is required for RNA packaging but which is a potent trans-dominant inhibitor of HDV RNA replication. Editing in infected cells is thought to be catalyzed by one or more of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). We examined the effects of increased ADAR1 and ADAR2 expression on HDV RNA editing and replication in transfected Huh7 cells. We found that both ADARs dramatically increased RNA editing, which was correlated with strong inhibition of HDV RNA replication. While increased HDAg-L production was the primary mechanism of inhibition, we observed at least two additional means by which ADARs can suppress HDV replication. High-level expression of both ADAR1 and ADAR2 led to extensive hyperediting at non-amber/W sites and subsequent production of HDAg variants that acted as trans-dominant inhibitors of HDV RNA replication. Moreover, we also observed weak inhibition of HDV RNA replication by mutated forms of ADARs defective for deaminase activity. Our results indicate that HDV requires highly regulated and selective editing and that the level of ADAR expression can play an important role: overexpression of ADARs inhibits HDV RNA replication and compromises virus viability.     

Enhancement of replication of RNA viruses by ADAR1 via RNA editing and inhibition of RNA-activated protein kinase

Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA binding protein and RNA-editing enzyme that modifies cellular and viral RNAs, including coding and noncoding RNAs. This interferon (IFN)-induced protein was expected to have an antiviral role, but recent studies have demonstrated that it promotes the replication of many RNA viruses. The data from these experiments show that ADAR1 directly enhances replication of hepatitis delta virus, human immunodeficiency virus type 1, vesicular stomatitis virus, and measles virus. The proviral activity of ADAR1 occurs through two mechanisms: RNA editing and inhibition of RNA-activated protein kinase (PKR). While these pathways have been found independently, the two mechanisms can act in concert to increase viral replication and contribute to viral pathogenesis. This novel type of proviral regulation by an IFN-induced protein, combined with some antiviral effects of hyperediting, sheds new light on the importance of ADAR1 during viral infection and transforms our overall understanding of the innate immune response.