Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR

PKR (double-stranded RNA-dependent protein kinase) is an important component of host defense to virus infection. Binding of dsRNA to two dsRBDs (double-stranded RNA binding domains) of PKR modulates its own kinase activation. How structural features of natural target RNAs, such as bulges and loops, have an effect on the binding to two dsRBDs of PKR still remains unclear. By using ITC and NMR, we show here that both the bulge and loop of TAR RNA are necessary for the high affinity binding to dsRBD1-dsRBD2 of PKR with 1:1 stoichiometry. The binding site for the dsRBD1-dsRBD2 spans from upper bulge to lower stem of the TAR RNA, based on chemical shift mapping. The backbone resonances in the 40 kDa TAR.dsRBD1-dsRBD2 were assigned. NMR chemical shift perturbation data suggest that the beta1-beta2 loop of the dsRBD1 interacts with the TAR RNA, whereas that of the dsRBD2 is less involved in the TAR RNA recognition. In addition, the residues of the interdomain linker between the dsRBD1 and the dsRBD2 also show large chemical perturbations indicating that the linker is involved in the recognition of TAR RNA. The results presented here provide the biophysical and spectroscopic basis for high-resolution structural studies, and show how local RNA structural features modulate recognition by dsRBDs.     

The double-stranded RNA-binding protein X1rbpa promotes RNA strand annealing.

RNA-annealing activity is a common feature of several RNA-binding proteins. The Xenopus RNA-binding protein X1rbpa is composed of three tandemly arranged double-stranded RNA-binding domains (dsRBDs) but lacks any other catalytic or functional domains, therefore making the assessment of biological functions of this protein rather difficult. Here we show that full-length X1rbpa but also isolated dsRBDs from this protein can facilitate RNA strand annealing. RNA annealing can be efficiently inhibited by heparin. However, dsRBDs with a neutral pI still promote strand annealing, suggesting that charged residues within the dsRBD are important for strand annealing. Additionally, mutant versions of the dsRBD, unable to bind dsRNA in northwestern assays, were tested. Of these, some show RNA-annealing activity while others fail to do so, indicating that RNA annealing and dsRNA binding are separable functions. Our data, together with the previously reported association of the protein with most cellular RNAs, suggests an RNA chaperone-like function of X1rbpa.     

Determination of the structure of the RNA complex of a double-stranded RNA-binding domain from Drosophila Staufen protein

We have determined using NMR the structure of the complex between the third double-stranded RNA-binding domain (dsRBD3) of Drosophila Staufen protein and a RNA stem-loop with optimal binding properties in vitro. This work was designed to understand how dsRBD proteins bind RNA and to investigate the role of Staufen dsRBDs in the localization of maternal RNAs during early embryonic development. The structure determination was challenging, because of weak, nonsequence specific binding and residual conformational flexibility at the RNA-protein interface. In order to overcome the problems originated by the weak interaction, we used both new and more traditional approaches to obtain distance and orientation information for the protein and RNA components of the complex. The resulting structure allowed the verification of aspects of RNA recognition by dsRBDs matching the information obtained by a related crystallographic study. We were also able to generate new observations that are likely to be relevant to dsRBD-RNA binding and to the physiological role of Staufen protein.Copyright 2001 John Wiley & Sons, Inc.     

Structures of the first and second double-stranded RNA-binding domains of human TAR RNA-binding protein

The TAR RNA-binding Protein (TRBP) is a double-stranded RNA (dsRNA)-binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA-binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α-β-β-β-α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)-binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 nM, respectively. In contrast, the full-length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 nM, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue.     

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.     

Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha).

During viral infection, phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) by the interferon-induced RNA-dependent protein kinase, PKR, leads to inhibition of translation initiation and viral proliferation. Activation of PKR is mediated by association of virally encoded double-stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) located in the N-terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild-type and mutant versions of human PKR expressed and purified from yeast. The catalytic rate of eIF2alpha phosphorylation by our purified PKR was increased in response to dsRNA, but not single-stranded RNA or DNA, consistent with the properties previously described for PKR purified from mammalian sources. While both dsRBD1 and dsRBD2 were required for activation of PKR by dsRNA, only deletion of dsRBD1 severely reduced the basal eIF2alpha kinase activity. Removal of as few as 25 residues at the C-terminal junction of dsRBD2 dramatically increased eIF2alpha kinase activity and characterization of larger deletions that included dsRBD1 demonstrated that removal of these negative-acting sequences could bypass the dsRBD1 requirement for in vitro phosphorylation of eIF2alpha. Heparin, a known in vitro activator of PKR, enhanced eIF2alpha phosphorylation by PKR mutants lacking their entire N-terminal sequences, including the dsRBDs. The results indicate that induction of PKR activity is mediated by multiple mechanisms, one of which involves release of inhibition by negative-acting sequences in PKR.     

Inherent conformational plasticity in dsRBDs enables interaction with topologically distinct RNAs

Many double-stranded RNA-binding domains (dsRBDs) interact with topologically distinct dsRNAs in biological pathways pivotal to viral replication, cancer causation, neurodegeneration, and so on. We hypothesized that the adaptability of dsRBDs is essential to target different dsRNA substrates. A model dsRBD and a few dsRNAs, slightly different in shape from each other, were used to test the systematic shape dependence of RNA on the dsRBD-binding using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. NMR-based titrations showed a distinct binding pattern for the dsRBD with the topologically distinct dsRNAs. The line broadening upon RNA binding was observed to cluster in the residues lying in close proximity, thereby suggesting an RNA-induced conformational exchange in the dsRBD. Further, while the intrinsic microsecond dynamics observed in the apo-dsRBD were found to quench upon binding with the dsRNA, the microsecond dynamics got induced at residues spatially proximal to quench sites upon binding with the dsRNA. This apparent relay of conformational exchange suggests the significance of intrinsic dynamics to help adapt the dsRBD to target various dsRNA-shapes. The conformational pool visualized in MD simulations for the apo-dsRBD reported here has also been observed to sample the conformations seen previously for various dsRBDs in apo- and in dsRNA-bound state structures, further suggesting the conformational adaptability of the dsRBDs. These investigations provide a dynamic basis for the substrate promiscuity for dsRBD proteins.     

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.     

The Role of Human Dicer-dsRBD in Processing Small Regulatory RNAs

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded – double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.     

Induced folding in RNA recognition by Arabidopsis thaliana DCL1

DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus. Here we show that the first of these domains binds precursor RNA fragments when isolated and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, this domain is intrinsically disordered. We found that it acquires a folded conformation when bound to its substrate, being the first report of a complete dsRBD folding upon binding. The free unfolded form shows tendency to adopt folded conformations, and goes through an unfolded bound state prior to the folding event. The significance of these results is discussed by comparison with the behavior of other dsRBDs.     

Solution structures of the double-stranded RNA-binding domains from RNA helicase A

RNA helicase A (RHA) is a highly conserved protein with multifaceted functions in the gene expression of cellular and viral mRNAs. RHA recognizes highly structured nucleotides and catalytically rearranges the various interactions between RNA, DNA, and protein molecules to provide a platform for the ribonucleoprotein complex. We present the first solution structures of the double-stranded RNA-binding domains (dsRBDs), dsRBD1 and dsRBD2, from mouse RHA. We discuss the binding mode of the dsRBDs of RHA, in comparison with the known dsRBD structures in their complexes. Our structural data provide important information for the elucidation of the molecular reassembly mediated by RHA.     

Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.     

Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9)

Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.     

New and old roles of the double-stranded RNA-binding domain

RNA-binding proteins can strongly regulate and influence the cellular function and fate of an RNA molecule. Of the many described nucleic acid-binding domains, the double-stranded RNA-binding domain (dsRBD) is a highly specialized example found in a wide variety of proteins with diverse cellular functions. Mostly present in multiple copies and highly homologous to one another, the individual functional specificity of dsRBDs is now becoming apparent. Here we review recent evidence showing that single dsRBDs within individual proteins are capable of distinct in vivo functions. Not only does this enable dsRBD-containing proteins to increase their functional diversity but it also reveals novel and unexpected roles that dsRBDs can perform.     

Helicase associated 2 domain is essential for helicase activity of RNA helicase A

RNA helicase A (RHA), a DExD/H box protein, plays critical roles in a wide variety of cellular or viral functions. RHA contains a conserved core helicase domain that is flanked by five other domains. Two double-stranded RNA binding domains (dsRBD1 and dsRBD2) are at the N-terminus, whereas HA2 (helicase associated 2), OB-fold (oligonucleotide- or oligosaccharide-binding fold), and RGG (repeats of arginine and glycine–glycine residues) domains are at the C-terminus. The role of these domains in the helicase activity of RHA is still elusive due to the difficulty of obtaining enzymatically active mutant RHA. Here, we purified a series of mutant RHAs containing deletions in either N-terminus or C-terminus. Analysis of these mutant RHAs reveals that the dsRBDs are not required for RNA unwinding, but can enhance the helicase activity by promoting the binding of RHA to substrate RNA. In contrast, deletion of C-terminal domains including RGG, OB-fold, and HA2 does not significantly affect the binding of RHA to substrate RNA. However, HA2 is essential for the RNA unwinding by RHA whereas the RGG and OB-fold are dispensable. The results indicate that the core helicase domain alone is not enough for RHA to execute the unwinding activity.     

Deformability in the cleavage site of primary microRNA is not sensed by the double‐stranded RNA binding domains in the microprocessor component DGCR8

The prevalence of double‐stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA‐binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD‐containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8‐Core, consists of its two dsRBDs and a C‐terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N‐terminal dsRBD of DGCR8 in isolation nor the DGCR8‐Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single‐stranded segments flanking the stem. Extending DGCR8‐Core to include an N‐terminal heme‐binding region does not change our conclusions. Thus, our data suggest that although the DGCR8‐Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency. Proteins 2015; 83:1165–1179. © 2015 Wiley Periodicals, Inc.     

Structure of a yeast RNase III dsRBD complex with a noncanonical RNA substrate provides new insights into binding specificity of dsRBDs

dsRBDs often bind dsRNAs with some specificity, yet the basis for this is poorly understood. Rnt1p, the major RNase III in Saccharomyces cerevisiae, cleaves RNA substrates containing hairpins capped by A/uGNN tetraloops, using its dsRBD to recognize a conserved tetraloop fold. However, the identification of a Rnt1p substrate with an AAGU tetraloop raised the question of whether Rnt1p binds to this noncanonical substrate differently than to A/uGNN tetraloops. The solution structure of Rnt1p dsRBD bound to an AAGU-capped hairpin reveals that the tetraloop undergoes a structural rearrangement upon binding to Rnt1p dsRBD to adopt a backbone conformation that is essentially the same as the AGAA tetraloop, and indicates that a conserved recognition mode is used for all Rnt1p substrates. Comparison of free and RNA-bound Rnt1p dsRBD reveals that tetraloop-specific binding requires a conformational change in helix α1. Our findings provide a unified model of binding site selection by this dsRBD.