Double-stranded RNA-binding Domain Research Articles

Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism

RNA helicase A (RHA) promotes multiple steps in HIV-1 production including transcription and translation of viral RNA, annealing of primer tRNALys3 to viral RNA, and elevating the ratio of unspliced to spliced viral RNA. At its amino terminus are two double-stranded RNA binding domains (dsRBDs) that are essential for RHA-viral RNA interaction. Linking the dsRBDs to the core helicase domain is a linker region containing 6 predicted helices. Working in vitro with purified mutant RHAs containing deletions of individual helices reveals that this region may regulate the enzyme's helicase activity, since deletion of helix 2 or 3 reduces the rate of unwinding RNA by RHA. The biological significance of this finding was then examined during HIV-1 production. Deletions in the linker region do not significantly affect either RHA-HIV-1 RNA interaction in vivo or the incorporation of mutant RHAs into progeny virions. While the partial reduction in helicase activity of mutant RHA containing a deletion of helices 2 or 3 does not reduce the ability of RHA to stimulate viral RNA synthesis, the promotion of tRNALys3 annealing to viral RNA is blocked. In contrast, deletion of helices 4 or 5 does not affect the ability of RHA to promote tRNALys3 annealing, but reduces its ability to stimulate viral RNA synthesis. Additionally, RHA stimulation of viral RNA synthesis results in an increased ratio of unspliced to spliced viral RNA, and this increase is not inhibited by deletions in the linker region, nor is the pattern of splicing changed within the ∼ 4.0 kb or ∼ 1.8 kb HIV-1 RNA classes, suggesting that RHA's effect on suppressing splicing is confined mainly to the first 5′-splice donor site. Overall, the differential responses to the mutations in the linker region of RHA reveal that RHA participates in HIV-1 RNA metabolism by multiple distinct mechanisms.

A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis

Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges.

Efficient siRNA transfection to the inner ear through the intact round window by a novel proteidic delivery technology in the chinchilla

The use of small-interfering RNA (siRNA) has great potential for the development of drugs designed to knock down the expression of damage- or disease-causing genes. However, because of the high molecular weight and negative charge of siRNA, it is restricted from crossing the blood–cochlear barrier, which limits the concentration and size of molecules that are able to gain access to cells of the inner ear. Intratympanic approaches, which deliver siRNA to the middle ear, rely on permeation through the round window for access to the structures of the inner ear. We developed an innovative siRNA delivery recombination protein, TAT double-stranded RNA-binding domains (TAT-DRBDs), which can transfect Cy3-labeled siRNA into cells of the inner ear, including the inner and outer hair cells, crista ampullaris, macula utriculi and macula sacculi, through intact round-window permeation in the chinchilla in vivo, and there were no apparent morphological damages for the time of observation. We also found that Cy3-labeled siRNA could directly enter spiral ganglion neurons and the epithelium of the stria vascularis independently; however, the mechanism is unknown. Therefore, as a non-viral vector, TAT-DRBD is a good candidate for the delivery of double-stranded siRNAs for treating various inner ear ailments and preservation of hearing function.

The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

Staufen-1 Interacts with the Human Endogenous Retrovirus Family HERV-K(HML-2) Rec and Gag Proteins and Increases Virion Production

The human endogenous retrovirus family HERV-K(HML-2) Rec protein is an RNA transport factor that enhances nuclear export of intron-containing retroviral transcripts. Using the yeast two-hybrid approach, we have newly identified human Staufen-1 as a Rec-interacting protein. The interaction was confirmed by coimmunoprecipitation experiments, and the relevant site in Staufen-1 has been mapped to double-stranded RNA binding domain 4 (RBD4). Staufen-1 is in several aspects functionally related to retroviral RNA transport proteins. It binds mRNAs and targets its ribonuclear cargo to polysomes for efficient translation. We observed an accumulation of Staufen-1 in the nucleus of Rec-expressing cells and colocalization in the nucleoli as well as in the cytoplasm. Overexpression of Staufen-1 resulted in a 5-fold enhancement in nuclear export and/or translation of unspliced HERV-K(HML-2) viral RNAs in the presence of Rec and its Rec-responsive element (RcRE) binding site together with a clear increase in virus production. Staufen-1 was previously shown to interact with the Gag protein of HIV-1, promoting Gag oligomerization and RNA encapsidation. We demonstrate here that Staufen-1 also binds to the Gag protein of HERV-K(HML-2). Under stress conditions, Rec colocalizes with Staufen-1 in stress granules in cells that express viral RNA but not in mRNA-decay-related processing bodies. Our results suggest a new role for Staufen-1 as a cellular Rec and HERV-K(HML-2) Gag cofactor.

Development of a specific siRNA delivery system into HeLa cells using an IgG-binding fusion protein

Stable carriers are required for delivering siRNA to cells. The use of polyethyleneimine (PEI) as gene carrier has been researched extensively; however, it does not provide sufficient protection from RNase degradation and is not suitable for targeted siRNA delivery to specific cells. In this study, two repeats of Fc binding domain of protein G (C2) were used to introduce a specific antibody to PEI-based carrier of siRNA. In addition, we used the double-stranded RNA binding domain (DRBD) that can bind to siRNA. The complex, consisting of PEI, siRNA and constructed fusion protein, TrxC2DRBD including C2 and DRBD domains, could protect siRNA from RNase degradation. Furthermore, cell specific siRNA delivery into HeLa cells could be performed by the complex fusion with specific antibodies via C2 domain.

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and double-stranded RNA into ∼21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic.

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

Staufen1 dimerizes through a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay

Staufen (STAU)1-mediated mRNA decay (SMD) degrades mammalian-cell mRNAs that bind the double-stranded (ds)RNA-binding protein STAU1 in their 3′-untranslated region. We report a new motif, which typifies STAU homologs from all vertebrate classes, that is responsible for human (h)STAU1 homodimerization. Our crystal structure and mutagenesis analyses reveal that this motif, now named the Staufen-swapping motif (SSM), and dsRNA-binding domain 5 (‘RBD’5) mediate protein dimerization: the two SSM α-helices of one molecule interact primarily through a hydrophobic patch with the two ‘RBD’5 α-helices of a second molecule. ‘RBD’5 adopts the canonical α-β-β-β-α fold of a functional RBD, but it lacks residues and features needed to bind duplex RNA. In cells, SSM-mediated hSTAU1 dimerization increases the efficiency of SMD by augmenting hSTAU1 binding to the ATP-dependent RNA helicase hUPF1. Dimerization regulates keratinocyte-mediated wound-healing and, undoubtedly, many other cellular processes.

The RNA-binding region of human TRBP interacts with microRNA precursors through two independent domains

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through RNA interference. Human miRNAs are generated through a series of enzymatic processing steps. The precursor miRNA (pre-miRNA) is recognized and cleaved by a complex containing Dicer and several non-catalytic accessory proteins. HIV TAR element binding protein (TRBP) is a constituent of the Dicer complex, which augments complex stability and potentially functions in substrate recognition and product transfer to the RNA-induced silencing complex. Here we have analysed the interaction between the RNA-binding region of TRBP and an oncogenic human miRNA, miR-155, at different stages in the biogenesis pathway. We show that the region of TRBP that binds immature miRNAs comprises two independent double-stranded RNA-binding domains connected by a 60-residue flexible linker. No evidence of contact between the two double-stranded RNA-binding domains was observed either in the apo- or RNA-bound state. We establish that the RNA-binding region of TRBP interacts with both pre-miR-155 and the miR-155/miR-155* duplex through the same binding surfaces and with similar affinities, and that two protein molecules can simultaneously interact with each immature miRNA. These data suggest that TRBP could play a role before and after processing of pre-miRNAs by Dicer.

Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stem-loops; HIV-1 TAR and adenovirus VA(I)-AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA-protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.

RNA Targets and Specificity of Staufen, a Double-stranded RNA-binding Protein in Caenorhabditis elegans

The Staufen family consists of proteins that possess double-stranded RNA-binding domains (dsRBDs). Staufen proteins of Drosophila and mammals regulate mRNA localization, translation, and decay. We report analysis of Staufen in Caenorhabditis elegans, which we have designated STAU-1. We focus on its biochemical properties, mRNA targets, and possible role in RNAi. We show that STAU-1 is expressed as mRNA and protein at all stages of C. elegans development. The wild-type, full-length protein, purified from bacteria, binds duplex RNA with high affinity in vitro. Purified, mutant proteins lacking single dsRBDs still bind RNA efficiently, demonstrating that no single domain is required for binding to duplex RNA (although dsRBD2 could not be tested). STAU-1 mRNA targets were identified via immunoprecipitation with specific anti-STAU-1 antibodies, followed by microarray analysis (RIP-Chip). These studies define a set of 418 likely STAU-1 mRNA targets. Finally, we demonstrate that stau-1 mutants enhance exogenous RNAi and that stau-1;eri-1 double mutants exhibit sterility and synthetic germ line defects.

Contribution of Double-Stranded RNA and CPSF30 Binding Domains of Influenza Virus NS1 to the Inhibition of Type I Interferon Production and Activation of Human Dendritic Cells

The influenza virus nonstructural protein 1 (NS1) inhibits innate immunity by multiple mechanisms. We previously reported that NS1 is able to inhibit the production of type I interferon (IFN) and proinflammatory cytokines in human primary dendritic cells (DCs). Here, we used recombinant viruses expressing mutant NS1 from the A/Texas/36/91 and A/Puerto Rico/08/34 strains in order to analyze the contribution of different NS1 domains to its antagonist functions. We show that the polyadenylation stimulating factor 30 (CPSF30) binding function of the NS1 protein from A/Texas/36/91 influenza virus, which is absent in the A/Puerto Rico/08/34 strain, is essential for counteracting these innate immune events in DCs. However, the double-stranded RNA (dsRNA) binding domain, present in both strains, specifically inhibits the induction of type I IFN genes in infected DCs, while it is essential only for inhibition of type I IFN proteins and proinflammatory cytokine production in cells infected with influenza viruses lacking a functional CPSF30 binding domain, such as A/Puerto Rico/08/34.

ATP-independent diffusion of double-stranded RNA binding proteins

The proteins harboring double-stranded RNA binding domains (dsRBDs) play diverse functional roles such as RNA localization, splicing, editing, export, and translation, yet mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response RNA binding protein (TRBP) consisting of three dsRBDs, which functions in HIV replication, protein kinase R(PKR)-mediated immune response, and RNA silencing. Here we report an ATP-independent diffusion activity of TRBP exclusively on dsRNA in a length-dependent manner. The first two dsRBDs of TRBP are essential for diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR activator and R3D1-L, displayed the same diffusion, implying a universality of the diffusion activity among this protein family. Furthermore, a Dicer-TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with Dicer's catalytic activity. These results implicate the dsRNA-specific diffusion activity of TRBP that contributes to enhancing siRNA and miRNA processing by Dicer.

Second Double-Stranded RNA Binding Domain of Dicer-like Ribonuclease 1: Structural and Biochemical Characterization

Dicer-like ribonuclease III enzymes are involved in different paths related to RNA silencing in plants. Little is known about the structural aspects of these processes. Here we present a structural characterization of the second double-stranded RNA binding domain (dsRBD) of DCL1, which is presumed to participate in pri-micro-RNA recognition and subcellular localization of this protein. We determined the solution structure and found that it has a canonical fold but bears some variation with respect to other homologous domains. We also found that this domain binds both double-stranded RNA and double-stranded DNA, in contrast to most dsRBDs. Our characterization shows that this domain likely has functions other than substrate recognition and binding.

Intrinsic Dynamics of an Extended Hydrophobic Core in the S. cerevisiae RNase III dsRBD Contributes to Recognition of Specific RNA Binding Sites

The Saccharomyces cerevisiae RNase III enzyme Rnt1p preferentially binds to double-stranded RNA hairpin substrates with a conserved (A/u)GNN tetraloop fold, via shape-specific interactions by its double-stranded RNA-binding domain (dsRBD) helix α1 to the tetraloop minor groove. To investigate whether conformational flexibility in the dsRBD regulates the binding specificity, we determined the backbone dynamics of the Rnt1p dsRBD in the free and AGAA hairpin-bound states using NMR spin-relaxation experiments. The intrinsic microsecond-to-millisecond timescale dynamics of the dsRBD suggests that helix α1 undergoes conformational sampling in the free state, with large dynamics at some residues in the α1–β1 loop (α1–β1 hinge). To correlate free dsRBD dynamics with structural changes upon binding, we determined the solution structure of the free dsRBD used in the previously determined RNA-bound structures. The Rnt1p dsRBD has an extended hydrophobic core comprising helix α1, the α1–β1 loop, and helix α3. Analysis of the backbone dynamics and structures of the free and bound dsRBD reveals that slow-timescale dynamics in the α1–β1 hinge are associated with concerted structural changes in the extended hydrophobic core that govern binding of helix α1 to AGAA tetraloops. The dynamic behavior of the dsRBD bound to a longer AGAA hairpin reveals that dynamics within the hydrophobic core differentiate between specific and nonspecific sites. Mutations of residues in the α1–β1 hinge result in changes to the dsRBD stability and RNA-binding affinity and cause defects in small nucleolar RNA processing invivo. These results reveal that dynamics in the extended hydrophobic core are important for binding site selection by the Rnt1p dsRBD.

SON Regulates GATA-2 Through Transcriptional Control of the Mir-23a∼27a∼24-2 Cluster

Abstract 110While characterizing AML1-ETO domains important for leukemia development and identifying proteins interacting with these domains, we discovered SON as a novel AML1-ETO binding protein (PNAS, 2008, 105:17103). SON is a large, poorly characterized serine/arginine rich SR protein localized to nuclear speckles. SON has DNA, single stranded RNA, and double stranded RNA binding domains and long repeats of amino acids. Overexpression of a partial fragment of SON in a transformed cell line decreased the tumorigenic potential of the cell in nude mice and protected yeast from apoptosis. However, partially due to its large size, the SON protein has not been well characterized. Recently, we reported that SON plays an important role in RNA splicing of a specific set of cell cycle related genes: ones that possess weak splice sites (Molecular Cell, 2011, 42:185).While SON is expressed ubiquitously, its expression level is noticeably higher in hematopoietic organs/tissues and blood cells compared to other tissues, suggesting important roles of SON in the hematopoietic system. To examine whether SON expression is regulated during hematopoietic differentiation, we measured relative mRNA level of mouse Son in different stages of hematopoiesis. Son mRNA level is higher in lineage marker negative (Lin-) bone marrow cells when compared to total bone marrow cells. Macrophages showed less expression of Son, suggesting that Son is down-regulated along the hematopoietic differentiation. We further sorted Lin- cells and measured the Son level in LSK (Lin-, Sca1+, cKit+), CMP (commom myeloid progenitors), GMP (granulocyte/monocyte progenitors) and MEP (megakaryocyte/erythroid progenitors) populations. LSK cells, which precede other progenitors, showed the highest level of Son. In addition, we confirmed that SON is down-regulated during TPA-induced monocytic differentiation of U937 myeloid cells. Taken together, SON is more abundantly expressed in immature hematopoietic cells and down-regulated during differentiation.Since SON is differentially expressed during hematopoietic differentiation, we examined whether SON is involved in regulation of hematopoietic transcription factors that are key dictators of hematopoietic differentiation. Among the several transcription factors analyzed, we found that Gata-2 mRNA was consistently reduced by two different Son shRNAs in Lin- bone marrow cells. Down regulation of the GATA-2 mRNA level was further confirmed in human K562 leukemic cell line. More interestingly, while having 20∼40% reduction of mRNA level, the GATA-2 protein level is more remarkably down-regulated upon SON knockdown, resulting in 75∼90% reduction. These results indicate that upon SON knockdown, GATA-2 protein level is mainly regulated at the post-transcriptional steps.Sequence analysis of the 3’ UTR of the human GATA-2 gene predicted several candidates of targeting microRNAs. Among them, we confirmed that the mature form of miR-27a and miR-24 are up-regulated upon SON knockdown. Next, we tested the effect of over-expression of miR-27a and miR-24 on GATA-2 expression using a GATA-2 3’ UTR-containing luciferase reporter construct and demonstrated that miR-27a indeed inhibits GATA-2 mRNA level. miR-27a is a member of the miR-23a∼27a∼24-2 cluster. RTqPCR showed that primary miRNA of the miR-23a∼27a∼24 cluster is upregulated upon SON knockdown. To test whether the increase of pri-miR of this cluster is due to promoter activation, we used a reporter construct containing the promoter sequence of the miR-23a∼27a∼24-2 cluster fused to the luciferase reporter. The expression of luciferase driven by this promoter is significantly elevated upon SON knockdown, suggesting that that SON functions to repress transcription of the miR-23a-27a-24-2 cluster, thereby relieving GATA-2 mRNA from targeting by miR-27a, and contributes to maintaining the GATA-2 protein level. Taken together, our results reveal a previously unidentified function of SON in microRNA transcription and controlling the GATA-2 protein level in hematopoietic cells. Disclosures:No relevant conflicts of interest to declare.

Specificity of the Double-Stranded RNA-Binding Domain from the RNA-Activated Protein Kinase PKR for Double-Stranded RNA: Insights from Thermodynamics and Small-Angle X-ray Scattering

The interferon-inducible, double-stranded (ds) RNA-activated protein kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key roles in viral pathogenesis and innate immunity. Activation of PKR is typically mediated by long dsRNA, and regulation of PKR is disfavored by most RNA imperfections, including bulges and internal loops. Herein, we combine isothermal titration calorimetry (ITC), electrophoretic mobility shift assays, and small-angle X-ray scattering (SAXS) to dissect the thermodynamic basis for the specificity of the dsRBD termed "p20" for various RNAs and to detect any RNA conformational changes induced upon protein binding. We monitor binding of p20 to chimeric duplexes containing terminal RNA-DNA hybrid segments and a central dsRNA segment, which was either unbulged ("perfect") or bulged. The ITC data reveal strong binding of p20 to the perfect duplex (K(d) ~ 30 nM) and weaker binding to the bulged duplex (K(d) ~ 2-5 μM). SAXS reconstructions and p(r) distance distribution functions further uncover that p20 induces no significant conformational change in perfect dsRNA but largely straightens bulged dsRNA. Together, these observations support the dsRBD's ability to tightly bind to only A-form RNA and suggest that in a noninfected cell, PKR may be buffered via weak interactions with various bulged and looped RNAs, which it may straighten. This work suggests that PKR-regulating RNAs with complex secondary and tertiary structures likely mimic dsRNA and/or engage portions of PKR outside of the dsRBD.

Roles of individual domains in the function of DHX29, an essential factor required for translation of structured mammalian mRNAs

On most eukaryotic mRNAs, initiation codon selection involves base-by-base inspection of 5' UTRs by scanning ribosomal complexes. Although the eukaryotic initiation factors 4A/4B/4G can mediate scanning through medium-stability hairpins, scanning through more stable structures additionally requires DHX29, a member of the superfamily 2 DEAH/RNA helicase A (RHA) helicase family that binds to 40S subunits and possesses 40S-stimulated nucleoside triphosphatase (NTPase) activity. Here, sequence alignment and structural modeling indicated that DHX29 comprises a unique 534-aa-long N-terminal region (NTR), central catalytic RecA1/RecA2 domains containing a large insert in the RecA2 domain, and the C-terminal part, which includes winged-helix, ratchet, and oligonucleotide/oligosaccharide-binding (OB) domains that are characteristic of DEAH/RHA helicases. Functional characterization revealed that specific ribosomal targeting is required for DHX29's activity in initiation and is determined by elements that map to the NTR and to the N-terminal half of the winged-helix domain. The ribosome-binding determinant located in the NTR was identified as a putative double-stranded RNA-binding domain. Mutational analyses of RecA1/RecA2 domains confirmed the essential role of NTP hydrolysis for DHX29's function in initiation and validated the significance of a β-hairpin protruding from RecA2. The large RecA2 insert played an autoinhibitory role in suppressing DHX29's intrinsic NTPase activity but was not essential for its 40S-stimulated NTPase activity and function in initiation. Deletion of the OB domain also increased DHX29's basal NTPase activity, but more importantly, abrogated the responsiveness of the NTPase activity to stimulation, which abolished DHX29's function in initiation. This finding suggests that the OB domain, which is specific for DEAH/RHA helicases, plays an important role in their NTPase cycle.

In Vitro and In Vivo Analysis of the Interaction between RNA Helicase A and HIV-1 RNA

RNA helicase A (RHA) promotes multiple steps of HIV-1 RNA metabolism during viral replication, including transcription, translation, and the annealing of primer tRNA(3)(Lys) to the viral RNA. RHA is a member of the DExH subclass of RNA helicases that uniquely contains two double-stranded RNA binding domains (dsRBDs) at its N terminus. Here, we performed a genome-wide analysis of the interaction of RHA with HIV-1 RNA both in vitro, using fluorescence polarization, and during viral replication, using an RNA-protein coprecipitation assay. In vitro, RHA binds to all the isolated regions of the HIV-1 RNA genome tested, with K(d) (equilibrium dissociation constant) values ranging from 44 to 178 nM. In contrast, during viral replication, RNA-protein coprecipitation assays detected only a major interaction of RHA with the 5'-untranslated region (5'-UTR) and a minor interaction with the Rev response element (RRE) of HIV-1 RNA. Since RHA does not associate well with all the highly structured regions of HIV-1 RNA tested in vivo, the results suggest that other viral or cellular factors not present in vitro may modulate the direct interaction of RHA with HIV-1 RNA during virus replication. Nevertheless, a role for duplex RNA as a target for RHA binding in vivo is suggested by the fact that the deletion of either one or both dsRBDs eliminates the in vivo interaction of RHA with HIV-1 RNA. Furthermore, these mutant RHAs do not promote the in vivo annealing of tRNA(3)(Lys) to viral RNA, nor are they packaged into virions, demonstrating that the dsRBDs are essential for the role of RHA in HIV-1 replication.