Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding

Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of one dsRBM and the deaminase domain was capable of deaminating a short 15-bp substrate. In contrast, full-length hADAR2 was inactive on this short substrate. In addition, we observed that the N terminus, which was deleted from the truncated protein, inhibits editing activity when added in trans. We propose that the N-terminal domain of hADAR2 contains sequences that cause auto-inhibition of the enzyme. Our results suggest activation requires binding to an RNA substrate long enough to accommodate interactions with both dsRBMs.     

Selective binding by the RNA binding domain of PKR revealed by affinity cleavage

The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.     

Substrate-dependent contribution of double-stranded RNA-binding motifs to ADAR2 function

ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine (A-to-I). ADAR2 contains two tandem double-stranded RNA-binding motifs (dsRBMs) that are not only important for efficient editing of RNA substrates but also necessary for localizing ADAR2 to nucleoli. The sequence and structural similarity of these motifs have raised questions regarding the role(s) that each dsRBM plays in ADAR2 function. Here, we demonstrate that the dsRBMs of ADAR2 differ in both their ability to modulate subnuclear localization as well as to promote site-selective A-to-I conversion. Surprisingly, dsRBM1 contributes to editing activity in a substrate-dependent manner, indicating that dsRBMs recognize distinct structural determinants in each RNA substrate. Although dsRBM2 is essential for the editing of all substrates examined, a point mutation in this motif affects editing for only a subset of RNAs, suggesting that dsRBM2 uses unique sets of amino acid(s) for functional interactions with different RNA targets. The dsRBMs of ADAR2 are interchangeable for subnuclear targeting, yet such motif alterations do not support site-selective editing, indicating that the unique binding preferences of each dsRBM differentially contribute to their pleiotropic function.     

dsRBM1 and a proline-rich domain of RNA helicase A can form a composite binder to recognize a specific dsDNA

The double-stranded RNA-binding motif (dsRBM) is a widely distributed motif frequently found within proteins with sequence non-specific RNA duplex-binding activity. In addition to the binding of double-stranded RNA, some dsRBMs also participate in complex formation via protein–protein interactions. Interestingly, a lot of proteins containing multiple dsRBMs have only some of their dsRBMs with the expected RNA duplex-binding competency proven, while the functions of the other dsRBMs remain unknown. We show here that the dsRBM1 of RNA helicase A (RHA) can cooperate with a C-terminal domain of proline-rich content to gain novel nucleic acid-binding activities. This integrated nucleic acid-binding module is capable of associating with the consensus sequences of the constitutive transport element (CTE) RNA of type D retrovirus against RNA duplex competitors. Remarkably, binding activity for double-stranded DNA corresponding to the consensus sequences of the cyclic-AMP responsive element also resides within this composite nucleic acid binder. It thus suggests that the dsRBM fold can be used as a platform for the building of a ligand binding module capable of non-RNA macromolecule binding with an accessory sequence, and functional assessment for a newly identified protein containing dsRBM fold should be more cautious.     

Identification of binding sites for both dsRBMs of PKR on kinase-activating and kinase-inhibiting RNA ligands

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR has a double-stranded RNA-binding domain (dsRBD) composed of two copies of the dsRNA binding motif (dsRBM). PKR's dsRBD is involved in the regulation of the enzyme as dsRNAs of cellular and viral origins bind to the dsRBD, leading to either activation or inhibition of PKR's kinase activity. In this study, we site-specifically modified each of the dsRBMs of PKR's dsRBD with the hydroxyl radical generator EDTA small middle dotFe and performed cleavage studies on kinase-activating and kinase-inhibiting RNAs. These experiments led to the identification of binding sites for the individual dsRBMs on various RNA ligands including a viral activating RNA (TAR from HIV-1), a viral inhibiting RNA (VA(I) RNA from adenovirus), an aptamer RNA that activates PKR, and a small synthetic inhibiting RNA. These results indicate that some RNAs interact only with one dsRBM, while others can bind both dsRBMs of PKR. In addition, EDTA small middle dotFe modification coupled with site-directed mutagenesis was used to assess the extent of cooperativity in the binding of the two dsRBMs. These experiments support the hypothesis that simultaneous binding of both dsRBMs of PKR occurs on kinase activating RNA ligands.     

Functional characterization of and cooperation between the double-stranded RNA-binding motifs of the protein kinase PKR

The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR is regulated by dsRNAs that interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus. The dsRBM is a conserved protein motif found in many proteins from most organisms. In this study, we investigated the biochemical functions and cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and the cooperation between them. We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2. In addition, dsRBM1 has RNA-annealing activity that is not displayed by dsRBM2. Both dsRBMs have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1). Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and strongly inhibits the dimerization of the intact PKR N terminus (p20) containing both dsRBMs. dsRBM1, like p20, activates reporter gene expression in transfection assays, and it plays a determinative role in localizing PKR to the nucleolus and cytoplasm of the cell. Thus, dsRBM2 has weak or no activity in dsRNA binding, stimulation of gene expression, and PKR localization, but it strongly enhances these functions of dsRBM1 when contained in p20. However, dsRBM2 does not enhance the annealing activity of dsRBM1. This study shows that the dsRBMs of PKR possess distinct properties and that some, but not all, of the functions of the enzyme depend on cooperation between the two motifs.     

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double-stranded RNA binding domain of protein kinase R

Double-stranded RNA binding domains of human protein kinase R (dsRBD-PKR) regulate distinct cellular functions and the fate of an RNA molecule in the cell. This highly homologous domains present in multiple copies in a number of species, exhibit individual and specific functional specificity. Number of NMR and X-ray crystallographic structural studies reveals that such domains take a common alpha-beta-beta-beta-alpha tertiary fold. However, the functional specificities of these domains could be due to the dynamics of the individual amino acid residues, as has been shown earlier in the case of backbone dynamics of 15N-1H of dsRNA binding motifs (dsRBMs) of human protein kinase R (PKR) (Nanduri S, Rahman F, Williams BRG, Qin J. EMBO J 2000;19:5567-5574). To further investigate if the differences in dynamics of the two dsRBMs are restricted to only backbone, or if the side-chain motions are also different to the extent of influencing their packing of the two hydrophobic cores, we have investigated the methyl group dynamics using 13C-methyl relaxation measurements. The results show that the hydrophobic core of dsRBM1 is more tightly packed than dsRBM2, and it seems to undergo less fast scale motions in the subnanosecond regime.     

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.     

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.     

Mutational analysis of the vaccinia virus E3 protein defines amino acid residues involved in E3 binding to double-stranded RNA.

Alanine-substitution mutations were targeted to 14 amino acid residues within the double-stranded (ds) RNA binding motif (dsRBM) of the vaccinia virus E3 protein. Substitutions at six positions--Glu-124, Phe-135, Phe-148, Lys-167, Arg-168, and Lys-171--caused significant reductions in dsRNA binding. These six residues are conserved in the two dsRBMs for which structural information is available (Escherichia coli RNase III and Drosophila melanogaster staufen) and in many other members of the dsRBM protein family. Residues we show to be important for dsRNA binding by vaccinia virus E3 map to the same face of the dsRBM structure and are thus likely to compose part of the RNA binding site.     

Double-stranded RNA adenosine deaminases ADAR1 and ADAR2 have overlapping specificities

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to produce inosines within RNAs that are largely double-stranded (ds). Like most dsRNA binding proteins, the enzymes will bind to any dsRNA without apparent sequence specificity. However, once bound, ADARs deaminate certain adenosines more efficiently than others. Most of what is known about the intrinsic deamination specificity of ADARs derives from analyses of Xenopus ADAR1. In addition to ADAR1, mammalian cells have a second ADAR, named ADAR2; the deamination specificity of this enzyme has not been rigorously studied. Here we directly compare the specificity of human ADAR1 and ADAR2. We find that, like ADAR1, ADAR2 has a 5' neighbor preference (A approximately U > C = G), but, unlike ADAR1, also has a 3' neighbor preference (U = G > C = A). Simultaneous analysis of both neighbor preferences reveals that ADAR2 prefers certain trinucleotide sequences (UAU, AAG, UAG, AAU). In addition to characterizing ADAR2 preferences, we analyzed the fraction of adenosines deaminated in a given RNA at complete reaction, or the enzyme's selectivity. We find that ADAR1 and ADAR2 deaminate a given RNA with the same selectivity, and this appears to be dictated by features of the RNA substrate. Finally, we observed that Xenopus and human ADAR1 deaminate the same adenosines on all RNAs tested, emphasizing the similarity of ADAR1 in these two species. Our data add substantially to the understanding of ADAR2 specificity, and aid in efforts to predict which ADAR deaminates a given editing site adenosine in vivo.     

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.     

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.     

The double-stranded RNA-binding motif, a versatile macromolecular docking platform

The double-stranded RNA-binding motif (dsRBM) is an alphabetabetabetaalpha fold with a well-characterized function to bind structured RNA molecules. This motif is widely distributed in eukaryotic proteins, as well as in proteins from bacteria and viruses. dsRBM-containing proteins are involved in processes ranging from RNA editing to protein phosphorylation in translational control and contain a variable number of dsRBM domains. The structural work of the past five years has identified a common mode of RNA target recognition by dsRBMs and dissected this recognition into two functionally separated interaction modes. The first involves the recognition of specific moieties of the RNA A-form helix by two protein loops, while the second is based on the interaction between structural elements flanking the RNA duplex with the first helix of the dsRBM. The latter interaction can be tuned by other protein elements. Recent work has made clear that dsRBMs can also recognize non-RNA targets (proteins and DNA), and act in combination with other dsRBMs and non-dsRBM motifs to play a regulatory role in catalytic processes. The elucidation of functional networks coordinated by dsRBM folds will require information on the precise functional relationship between different dsRBMs and a clarification of the principles underlying dsRBM-protein recognition.     

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.     

In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site

The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that is completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. We have attempted to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question we studied binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and compared a wild-type molecule with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences was almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appeared nonspecific.     

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.