Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA.

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

NMR characterization of RNA binding property of the DEAD-box RNA helicase DDX3X and its implications for helicase activity.

The DEAD-box RNA helicase (DDX) plays a central role in many aspects of RNA metabolism by remodeling the defined structure of RNA molecules. While a number of structural studies have revealed the atomistic details of the interaction between DDX and RNA ligands, the molecular mechanism of how this molecule unwinds a structured RNA into an unstructured single-stranded RNA (ssRNA) has largely remained elusive. This is due to challenges in structurally characterizing the unwinding intermediate state and the lack of thermodynamic details underlying this process. In this study, we use solution nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction of human DDX3X, a member of the DDX family, with various RNA ligands. Our results show that the inherent binding affinity of DDX3X for ssRNA is significantly higher than that for structured RNA elements. This preferential binding, accompanied by the formation of a domain-closed conformation in complex with ssRNA, effectively stabilizes the denatured ssRNA state and thus underlies the unwinding activity of DDX3X. Our results provide a thermodynamic and structural basis for the DDX function, whereby DDX can recognize and remodel a distinct set of structured RNAs to participate in a wide range of physiological processes.

DNA G-Quadruplex Is a Transcriptional Control Device That Regulates Memory.

The conformational state of DNA fine-tunes the transcriptional rate and abundance of RNA. Here, we report that G-quadruplex DNA (G4-DNA) accumulates in neurons, in an experience-dependent manner, and that this is required for the transient silencing and activation of genes that are critically involved in learning and memory in male C57/BL6 mice. In addition, site-specific resolution of G4-DNA by dCas9-mediated deposition of the helicase DHX36 impairs fear extinction memory. Dynamic DNA structure states therefore represent a key molecular mechanism underlying memory consolidation.One-Sentence Summary: G4-DNA is a molecular switch that enables the temporal regulation of the gene expression underlying the formation of fear extinction memory.

Terminal regions of UAP56 and URH49 are required for their distinct complex formation functioning to an essential role in mRNA processing and export.

UAP56 and URH49 are closely related RNA helicases that function in selective mRNA processing and export pathways to fine-tune gene expression through distinct complex formations. The complex formation of UAP56 and URH49 is believed to play a crucial role in regulating target mRNAs. However, the mechanisms underlying this complex formation have not been fully elucidated. Here we identified the regions essential for the complex formation of both helicases. The terminal regions of UAP56 and the C-terminal region of URH49 were indispensable for their respective complex formation. Further analysis revealed that a specific amino acid at the C-terminus of UAP56 is critical for its complex formation. Alanine substitution of this amino acid impairs its complex formation and subsequently affected its mRNA processing and export activity. Our study provides a deeper understanding of the basis for the complex formation between UAP56 and URH49.

The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways.

In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.

Conserved Residues at the Mtr4 C-Terminus Coordinate Helicase Activity and Exosome Interactions.

Mtr4 is an essential RNA helicase involved in nuclear RNA processing and degradation and is a member of the Ski2-like helicase family. Ski2-like helicases share a common core architecture that includes two RecA-like domains, a winged helix, and a helical bundle (HB) domain. In Mtr4, a short C-terminal tail immediately follows the HB domain and is positioned at the interface of the RecA-like domains. The tail ends with a SLYΦ sequence motif that is highly conserved in a subset of Ski2-like helicases. Here, we show that this sequence is critical for Mtr4 function. Mutations in the C-terminus result in decreased RNA unwinding activity. Mtr4 is a key activator of the RNA exosome complex, and mutations in the SLYΦ motif produce a slow growth phenotype when combined with a partial exosome defect in S. cerevisiae, suggesting an important role of the C-terminus of Mtr4 and the RNA exosome. We further demonstrate that C-terminal mutations impair RNA degradation activity by the major RNA exosome nuclease Rrp44 in vitro. These data demonstrate a role for the Mtr4 C-terminus in regulating helicase activity and coordinating Mtr4-exosome interactions.

Nucleolus activity-dependent recruitment and biomolecular condensation by pH sensing.

DEAD-box ATPases are major regulators of biomolecular condensates and orchestrate diverse biochemical processes that are critical for the functioning of cells. How DEAD-box proteins are selectively recruited to their respective biomolecular condensates is unknown. We explored this in the context of the nucleolus and DEAD-box protein DDX21. We find that the pH of the nucleolus is intricately linked to the transcriptional activity of the organelle and facilitates the recruitment and condensation of DDX21. We identify an evolutionarily conserved feature of the C terminus of DDX21 responsible for nucleolar localization. This domain is essential for zebrafish development, and its intrinsically disordered and isoelectric properties are necessary and sufficient for the ability of DDX21 to respond to changes in pH and form condensates. Molecularly, the enzymatic activities of poly(ADP-ribose) polymerases contribute to maintaining the nucleolar pH and, consequently, DDX21 recruitment and nucleolar partitioning. These observations reveal an activity-dependent physicochemical mechanism for the selective recruitment of biochemical activities to biomolecular condensates.

Crystal structures of the DExH-box RNA helicase DHX9.

DHX9 is a DExH-box RNA helicase with versatile functions in transcription, translation, RNA processing and regulation of DNA replication. DHX9 has recently emerged as a promising target for oncology, but to date no mammalian structures have been published. Here, crystal structures of human, dog and cat DHX9 bound to ADP are reported. The three mammalian DHX9 structures share identical structural folds. Additionally, the overall architecture and the individual domain structures of DHX9 are highly conserved with those of MLE, the Drosophila orthologue of DHX9 previously solved in complex with RNA and a transition-state analogue of ATP. Due to differences in the bound substrates and global domain orientations, the localized loop conformations and occupancy of dsRNA-binding domain 2 (dsRBD2) differ between the mammalian DHX9 and MLE structures. The combined effects of the structural changes considerably alter the RNA-binding channel, providing an opportunity to compare active and inactive states of the helicase. Finally, the mammalian DHX9 structures provide a potential tool for structure-based drug-design efforts.

Pervasive downstream RNA hairpins dynamically dictate start-codon selection.

Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites1-4. However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.

Development of assays to support identification and characterization of modulators of DExH-box helicase DHX9.

DHX9 is a DExH-box RNA helicase that utilizes hydrolysis of all four nucleotide triphosphates (NTPs) to power cycles of 3' to 5' directional movement to resolve and/or unwind double stranded RNA, DNA, and RNA/DNA hybrids, R-loops, triplex-DNA and G-quadraplexes. DHX9 activity is important for both viral amplification and maintaining genomic stability in cancer cells; therefore, it is a therapeutic target of interest for drug discovery efforts. Biochemical assays measuring ATP hydrolysis and oligonucleotide unwinding for DHX9 have been developed and characterized, and these assays can support high-throughput compound screening efforts under balanced conditions. Assay development efforts revealed DHX9 can use double stranded RNA with 18-mer poly(U) 3' overhangs and as well as significantly shorter overhangs at the 5' or 3' end as substrates. The enzymatic assays are augmented by a robust SPR assay for compound validation. A mechanism-derived inhibitor, GTPγS, was characterized as part of the validation of these assays and a crystal structure of GDP bound to cat DHX9 has been solved. In addition to enabling drug discovery efforts for DHX9, these assays may be extrapolated to other RNA helicases providing a valuable toolkit for this important target class.

Structural basis for RNA-duplex unwinding by the DEAD-box helicase DbpA.

DEAD-box RNA helicases are implicated in most aspects of RNA biology, where these enzymes unwind short RNA duplexes in an ATP-dependent manner. During the central step of the unwinding cycle, the two domains of the helicase core form a distinct closed conformation that destabilizes the RNA duplex, which ultimately leads to duplex melting. Despite the importance of this step for the unwinding process no high-resolution structures of this state are available. Here, I used nuclear magnetic resonance spectroscopy and X-ray crystallography to determine structures of the DEAD-box helicase DbpA in the closed conformation, complexed with substrate duplexes and single-stranded unwinding product. These structures reveal that DbpA initiates duplex unwinding by interacting with up to three base-paired nucleotides and a 5' single-stranded RNA duplex overhang. These high-resolution snapshots, together with biochemical assays, rationalize the destabilization of the RNA duplex and are integrated into a conclusive model of the unwinding process.

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

Is the Association of the Rare rs35667974 IFIH1 Gene Polymorphism With Autoimmune Diseases a Case of RNA Epigenetics?

Interferon induced with helicase C domain-containing protein 1 (IFIH1) gene encodes a cytoplasmic RNA helicase otherwise known as melanoma differentiation-associated 5 (MDA5), a RIG-1-like RNA helicase that recognizes viral RNA and is involved in innate immunity through recognition of viral RNA. Upon binding to double-stranded (ds) RNA, MDA5 forms a filamentous assembly along the length of dsRNA and utilizes molecular signatures to discriminate self, versus non-self on the basis of dsRNA length and methylation. Its missense variant rs35667974 is protective for type 1 diabetes, psoriasis, and psoriatic arthritis, but is also found to be associated with an increased risk for ankylosing spondylitis, Crohn's disease, and ulcerative colitis. To gain insight into the complex role of this variant we performed a structural analysis of MDA5 in complex with dsRNA using molecular dynamics simulations. Our data suggest that while the Ile923Val mutation of the rs35667974 variant does not affect binding to native dsRNA significantly, it displays a destabilizing effect in the presence of 2'-O uridine methylation. Thus, the presence of 2'-O-methylation at the dsRNA introduces a sensing signature that leads to selective reduction of the overall MDA catalytic activity. This study represents an evaluation of the role of the shared rs35667974 variant of autoimmune locus IFIH1, reported to lead to selectively reduced catalytic activity of the modified MDA5 phenotype and, as a consequence, reduced negative feedback on cytokine and chemokine signaling and selectively protection against autoimmunity.

RHAU Peptides Specific for Parallel G-Quadruplexes: Potential Applications in Chemical Biology.

G-quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich sequences, which are ubiquitously found in the human genome and transcriptome. Targeting G4s by specific ligands provides a powerful tool to monitor and regulate G4s-associated biological processes. RHAU peptides, derived from the G4-binding motif of "RNA Helicase associated with AU-rich element" (RHAU), have emerged as extraordinary ligands for specific recognition of parallel G4s. This review highlights the significances of recent studies investigating potential applications of the engineered RHAU peptides incorporated to different functional moieties.

Protein purification, crystallization, and structure determination of human DEAD-box RNA helicase DDX21 in different unwinding states.

RNA helicase DDX21 plays vital roles in ribosomal RNA processing and the regulation of host innate immunity during virus infection. Here, we describe the optimized protocols for nucleic acid-free protein purification and crystallization of DDX21 in its different unwinding states. Rational design of the flexible region within the helicase core, and biophysical approach to characterize interactions between DDX21 and RNA, leads to successful crystallization of DDX21. This protocol can be applied to the crystallography of other DExD/H-box RNA helicases. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

Unraveling activity of crucial domain HABD protein in dengue virus.

Dengue virus (DENV) causes dengue, which is a very common mosquito-borne viral disease. The global incidence of dengue has increased dramatically in recent decades. About half of the world's population is now at risk. This virus is widespread throughout the tropics, which are influenced by rainfall, temperature, and humidity; however, severe dengue has a higher risk of death when not managed timely. To describe Dengue virus helicase ATP binding domain (HABD) protein in biochemically characterized. Sequences analysis, structure modeling, secondary structure prediction, ATPase assay, unwinding assay, RNA binding assay. HABD has RNA-dependent ATPase and helicase activity which are crucial proteins that participate in the unwinding of double-stranded DNA or RNA by utilizing ATP. RNA binding proteins and DEAD-box RNA helicases have been revealed to contribute to viral replication. Moreover, DEAD-box RNA helicases have been demonstrated to be involved in several features of cellular metabolism of RNA, for example, transcription, splicing, biogenesis, ribosomal processing of RNA, etc. In the present study, we have mainly focused on the Dengue virus's helicase ATP binding domain (HABD) and observed that HABD contains RNA-dependent ATPase and unwinding activity at different concentrations and time points.

Measurement of ATP utilization in RNA unwinding and RNA chaperone activities by DEAD-box helicase proteins.

RNA helicase proteins perform coupled reactions in which cycles of ATP binding and hydrolysis are used to drive local unwinding of double-stranded RNA (dsRNA). For some helicases in the ubiquitous DEAD-box family, these local unwinding events are integral to folding transitions in structured RNAs, and thus these helicases function as RNA chaperones. An important measure of the efficiency of the helicase-catalyzed reaction is the ATP utilization value, which represents the average number of ATP molecules hydrolyzed during RNA unwinding or a chaperone-assisted RNA structural rearrangement. Here we outline procedures that can be used to measure the ATP utilization value in RNA unwinding or folding transitions. As an example of an RNA folding transition, we focus on the refolding of the Tetrahymena thermophila group I intron ribozyme from a long-lived misfolded structure to its native structure, and we outline strategies for adapting this assay to other RNA folding transitions. For a simple dsRNA unwinding event, the ATP utilization value provides a measure of the coupling between the ATPase and RNA unwinding activities, and for a complex RNA structural transition it can give insight into the scope of the rearrangement and the efficiency with which the helicase uses the energy from ATPase cycles to promote the rearrangement.

To unwind the biological knots: The DNA/RNA G-quadruplex resolvase RHAU (DHX36) in development and disease.

The G-quadruplex (G4) sequences are short fragments of 4-interval triple guanine (G) with frequent and ubiquitous distribution in the genome and RNA transcripts. The G4 sequences are usually folded into secondary "knot" structure via Hoogsteen hydrogen bond to exert negative regulation on a variety of biological processes, including DNA replication and transcription, mRNA translation, and telomere maintenance. Recent structural biological and mouse genetics studies have demonstrated that RHAU (DHX36) can bind and unwind the G4 "knots" to modulate embryonic development and postnatal organ function. Deficiency of RHAU gives rise to embryonic lethality, impaired organogenesis, and organ dysfunction. These studies uncovered the pivotal G4 resolvase function of RHAU to release the G4 barrier, which plays fundamental roles in development and physiological homeostasis. This review discusses the latest advancements and findings in deciphering RHAU functions using animal models.

Vasa Is a Potential Germ Cell Marker in Leopard Coral Grouper (Plectropomus leopardus).

Vasa (Ddx4, DEAD box polypeptide 4), an extremely specific marker of germ cells in vivo, is an ATP-dependent RNA helicase that plays an essential role in germ cell development and gametogenesis. However, the expression and function information about this gene in groupers remains lacking. Here, vasa homolog termed Plvasa was isolated and identified Plvasa as a putative germ cell marker in the leopard coral grouper, (Plectropomus leopardus). Results indicated that Plvasa contained 17 exons in the genomic sequence and 9 conserved motifs of the DEAD-box protein by sequence analysis. The sequence comparison, phylogenetic analyses and synteny analyses showed that Plvasa was homologous with other teleosts. Additionally, the expression of Plvasa was significantly higher in gonads than in other tissues in adult individuals (p < 0.05). Further, the distribution of Plvasa revealed that it was only expressed in the germ cells, such as spermatids, germline stem cells and oocytes at different stages, and could not be detected in the somatic cells of gonads. The current study verified that the Plvasa gene is a valuable molecular marker of germ cells in leopard coral grouper, which potentially plays an important role in investigating the genesis and development of teleost germ cells.

A loosened gating mechanism of RIG-I leads to autoimmune disorders.

DDX58 encodes RIG-I, a cytosolic RNA sensor that ensures immune surveillance of nonself RNAs. Individuals with RIG-IE510V and RIG-IQ517H mutations have increased susceptibility to Singleton-Merten syndrome (SMS) defects, resulting in tissue-specific (mild) and classic (severe) phenotypes. The coupling between RNA recognition and conformational changes is central to RIG-I RNA proofreading, but the molecular determinants leading to dissociated disease phenotypes remain unknown. Herein, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) and single molecule magnetic tweezers (MT) to precisely examine how subtle conformational changes in the helicase insertion domain (HEL2i) promote impaired ATPase and erroneous RNA proofreading activities. We showed that the mutations cause a loosened latch-gate engagement in apo RIG-I, which in turn gradually dampens its self RNA (Cap2 moiety:m7G cap and N1-2-2'-O-methylation RNA) proofreading ability, leading to increased immunopathy. These results reveal HEL2i as a unique checkpoint directing two specialized functions, i.e. stabilizing the CARD2-HEL2i interface and gating the helicase from incoming self RNAs; thus, these findings add new insights into the role of HEL2i in the control of antiviral innate immunity and autoimmunity diseases.