Biological Functions of DEAD/DEAH-box RNA helicases in Health and Disease

Abstract

On 15–16 November 2021 the National Institute of Allergy and Infectious Diseases (NIAID), the National Cancer Institute (NCI), and the National Heart, Lung, and Blood Institute (NHLBI) hosted a virtual workshop on DEAD/DEAH-box RNA helicases in health and disease. The goal of the workshop was to review current advances, and to identify knowledge gaps and future research directions for improving our understanding of the function of RNA helicases, and leveraging these molecules as molecular targets with translational potential.

DEAD/H-box RNA helicases (RHs) are highly conserved RNA-binding proteins with ATPase activity. They are crucial for RNA metabolism¹, and belong to the RH superfamily 2 (SF2) which is characterized by nine conserved motifs. These motifs mediate ATP-binding and hydrolysis, RNA-binding, and ATP-dependent intramolecular RNA remodeling. These RHs are defined by their the Asp-Glu-Ala-Asp (DEAD) or Asp-Glu-Ala-His (DEAH) amino acid sequence². Although some of these RHs unwind duplex RNA, others can unwind dsDNA and RNA-DNA duplexes, while others exhibit little to no unwinding activity. The human genome encodes 37 DEAD-box and 16 DEAH-box RHs that exhibit diverse biological functions, including regulation of embryonic development, cell proliferation, hematopoiesis, metabolism, innate immunity and immune programming, cancer pathogenesis, inflammation, and autoimmune diseases (Fig. 1 and Table 1). Several RHs sense cytosolic non-self viral RNAs, initiating anti-viral innate immune signaling³. Moreover, RHs play critical roles in genome integrity⁴; mutations in RHs link to cancers, autoimmunity, and other diseases⁵. Thus, DEAD/H-box RHs are attractive therapeutic targets^{3, 6}. Despite the progress of this field, key knowledge gaps remain, including, cofactors of RH regulation; the nature of their RNA substrate; post-translational modifications regulating RH function; and the role of RHs in signaling cascades of biological processes. This NIH workshop focused on recent advances in understanding the functions of DEAD/H-box RHs.

Fig. 1.

Phylogeny of human DEAD/H-box RNA helicases. Phylogenetic analysis performed using the Poisson model of amino acid (AA) substitution with the neighbor-joining method and scaled with the branch lengths representing the evolutionary distances. The evolutionary distances to infer the phylogenetic tree were in the unit of the number of substituting amino acids per site.

Table 1.

RHs and their functions featured in the “Biological Functions of DEAD/DEAH-box RNA helicases in Health and Disease” workshop. Note that DDX3 and DDX3X represent the same molecule.

DEAD/DEAH RNA helicases			Function
SF1	Mov10		Supports gastrulation and central nervous system development
SF2	DDX3 DDX3X		Suppresses AR translation in ARlow/neg castration-resistant prostate cancer Inhibition induces cell death and suppresses latent HIV infection Involved in liquid -liquid phase separation (LLPS) and stress granule formation Is a tumor suppressor in generating medulloblastoma subtypes Knockdown triggers anti-tumor immunity via IFN/STAT production
	DDX5		Resolves DNA/RNA hybrids (R-loops) Promotes MYC transcription by unfolding a G-quadruplex of the MYC promoter Supports cellular respiration in small cell lung cancer cell lines Forms an RNA-driven silencing epigenetic complex with innate nuclear receptor IFI16 in hepatocytes
	Ded1		Binds and unfolds RNA and DNA quadruplexes Coordinates with eIF4 to stimulate preinitiation complex formation
	Me31B		Forms viscous P body condensates with bicoid mRNA in Drosophila
	DDX41		Senses retroviruses, supports innate immunity and HSPC differentiation; limits R-loops. Mutations cause human myeloid neoplasms and myelodysplastic syndromes
	Antiviral Dicer (AviD)		Enriched in stem cells within adult tissues; protects adult stem cells from RNA viruses via canonical antiviral RNAi response
	DDX56		Maintains stemness in normal and cancer stem cells
	RLRs	DDX58 (RIG-I) DHX58 (LGP2) IFIH1 (MDA5)	Pathogen Recognition Initiator of innate immunity Immune system activation and programming; RLR agonists exhibit anti-tumor effects
	DHX15		Immune system activation; senses rotavirus infection in intestinal epithelia; shown to sense RNA viruses in specific cell types

Biology and basic functions of DEAD/DEAH-box RHs

Rick Russel kicked off the meeting with n overview of structure, activities and functional roles of RHs in mediating RNA structural transitions and maintaining RNA in unfolded/or underfolded states.). Examples of the role of RHs in regulating gene expression include how deacetylation of helicase DDX3X by HDAC6 regulates stress granule (SG) formation through liquid-liquid protein phase separation (LLPS), presented by Patrick Matthias. SGs, membraneless organelles, are sites of stalled mRNA translation, involved in the stress-response. Stephane Richard discussed the role of DDX5 in resolving R-loops, DNA: RNA hybrids and associated non-template single-stranded DNA. DDX5 methylation by protein arginine methyltransferase 5 within its RGG/RG motif enables interaction with XRN2 ribonuclease, leading to R-loop resolution and ensuring DNA repair. Kevin Raney described Ded1 (hDDX3) binding to G-quadruplex (G4) DNA, leaking from nucleus or mitochondria, initiating LLPS and SG formation. Timothy Weil discussed ribonucleoprotein processing (P)-bodies in mRNA storage in Drosophila oocytes, showing that Me31B (hDDX6) in P-body condensates stores bicoid mRNA, and releasing it for translation upon egg activation. Danzhou Yang discussed how DDX5 unfolds G4-structure at the MYC promoter, regulating MYC transcription. This session described specific function of distinct DEAD/H-box helicases in resolving complex RNA/DNA structures to facilitate genome integrity and regulate gene expression.

Organ-specific Infection and Cancer

Deregulated expression of various RHs has a role in cancer pathogenesis, as well as in infection and cellular differentiation. Specifically, Elizabeth Tran reported linkage of DDX5 with small cell lung cancer (SCLC), and that DDX5 depletion reduced growth of a chemoresistant SCLC cell line by altering mitochondrial function. On the other hand, DDX5 forms in hepatocytes an epigenetic silencing complex with IFI16, an interferon-stimulated innate immunity gene, via an RNA-dependent mechanism, suggesting loss of this complex enables Hepatitis B virus transcription and hepatocellular carcinoma progression⁷, as presented by Ourania Andrisani. William Ricke described studies of how DDX3 promotes androgen receptor (AR)^neg/low castration-resistant prostate cancer (CRPC) by binding to AR mRNA and suppressing AR translation, thereby identifying DDX3 as a promising therapy target for CRPC. DDX3 control of translation for mRNAs enriched in RNA structures, alters transcriptome output in both cancer and developmental disorders, discussed by Stephen Floor. Further, Richard Gilbertson described DDX3X mutations in medulloblastoma, indicating that DDX3X is a tumor suppressor restricting cell lineages in generating medulloblastoma subtypes⁵. Susan Ross discussed the role of Ddx41, in recognizing retroviral RNA/DNA products of reverse transcription, and acting as an upstream regulator of innate immune signaling. Ddx41 is also critical for hematopoietic stem and progenitor cell (HSPC) differentiation. ddx41 loss-of-function mutations lead to loss of myeloid cells and susceptibility to Myelodysplastic Syndromes (MDS), as discussed by Teresa Bowman. Zebrafish ddx41 mutants have aberrant HSPC expansion, R-loop imbalance, and elevated inflammatory gene expression, triggering innate immune activation and HSPCs expansion⁸. Daniel Starczynowski discussed mutations linked to MDS in mice, showing that monoallelic mutations of Ddx41 result in features of MDS in an age-dependent manner. This session revealed that RHs mediate cell context-dependent, specialized functions, linked to cofactor and RNA substrate interactions.

Development and Cell Regulation.

Multiple RHs are required for eukaryotic translation and are important for maintaining the integrity of protein synthesis. Alan Hinnebusch discussed the mechanism of translation initiation in yeast by eukaryotic initiation factor eIF4A and Ded1. Ded1 in concert with eIF4A stimulates translation by resolving RNA structures that impede preinitiation complex attachment or 5-nontranslated scanning. Enzo Poirier discussed the identification of a novel mammalian antiviral Dicer (AviD), an alternatively spliced Dicer mRNA without a Hel2i domain. AviD is highly efficient at processing double-stranded (ds)RNA and protects adult stem cells from RNA viruses by degrading viral dsRNA, allowing cells to mount a canonical antiviral RNAi response. The SF1 helicase MOV10, presented by Stephanie Ceman, controls gastrulation and central nervous system development through binding G-rich structures and unwinding RNA in an ATP-dependent manner. Bret Pearson introduced DDX56 in regulating stemness by localizing to the nucleolus in normal and cancer stem cells, regulating rRNA expression, and nucleolar integrity⁹. In summary, RHs impart translational control and mediate the efficiency of protein synthesis, confer RNA chaperone and RNA localization functions, thus impacting cell growth and development. The discovery of AviD, a new member of antiviral effector helicases underscores the broad functions of RHs including their role in infection and immunity.

Triggering innate immunity and the immune response against RNA viruses.

The RIG-I-like receptors (RLRs) are a subfamily of RHs that play major roles as pathogen recognition receptors and initiators of innate immune activation. RLRs are increasingly implicated in immune programming wherein they facilitate innate-to-adaptive immunity crosstalk. Curt Horvath reviewed RLR biology, including retinoic acid-inducible gene I (RIG-I), laboratory of genetics and physiology-2 (LGP2), and melanoma differentiation-associated gene-5 (MDA5). RLRs serve as pathogen recognition receptors by recognizing and binding to nonself pathogen associated molecular patterns (PAMPs) within viral RNA, inducing and regulating antiviral innate immune defense and linking innate and adaptive immunity¹⁰. The immune response to virus infection typically initiates with innate immunity in which during RNA virus infection the RLRs signal innate immune activation. RLR recognition of viral RNA via binding to pathogen associate molecular pattern (PAMP) motifs within viral RNA serves to initiate the immune response to infection. Joseph Marcotrigiano discussed the structural features of RIG-I with an emphasis on viral PAMP RNA binding at the C terminal domain also known as the repressor domain (RD), thus triggering ATP hydrolysis and RIG-I conformation change to signaling-on state. While much is known on the structure-function of RIG-I, less is known of the structural properties of MDA5 regulation. Michael Gale showed that SARS-CoV-2 infection in lung epithelial cells is sensed by MDA5, upon its recognition and binding to specific viral RNA PAMP products. Greg Towers further described that RLR sensing of SARS-CoV-2 promotes macrophage activation and inflammatory actions. In particular, the Alpha (B.1.1.7) SARS-CoV-2 variant more effectively suppresses innate immune responses in airway epithelial cells by expressing high Orf9b and Orf6, which are innate immune antagonists operating to suppress downstream RLR signaling actions. These properties of SARS-CoV-2 reflect strategies in general across viral genera to target and suppress RLR function to avoid innate immunity, thus facilitating viral replication and spread. Indeed, RLR suppression by a broad range of RNA viruses is linked with viral pathogenesis and disease. Sun Hur then highlighted the diverse bivalent recognition of tripartite motif (TRIM) ubiquitin ligases (RIPLET, TRIM65) by RLRs. She showed how RIPLET recognizes active, filamentous forms of RIG-I, while TRIM65 recognizes filamentous MDA5¹¹. Remarkably, RLR interactions with RIPLET or TRIM proteins are known to be targeted and dysregulated by pathogenic viruses. Moreover, DHX15, is another RH involved in immune activation, and was shown by Zhiqiang Zhang to be essential for sensing rotavirus in intestinal epithelial cells, leading to interferon (IFN) and IL-18 production. Studies presented in this session demonstrate critical roles of RHs and their cofactors in initiating the immune response to virus infection, showing that targeting and disruption of these processes is a hallmark feature of pathogenic RNA viruses.

DEAD-H-box RHs in inflammation and immune programming.

Beyond innate immune activation, RLRs and other RHs play important role in immune programming to direct or “polarize” the immune response toward specific effector actions. Mehul Suthar described how LGP2 and MAVS (the RLR signaling-adaptor), regulate T cell activation through different mechanisms- LGP2 is an RH that supports the antigen-induced T cell expansion whereas MAVS exerts a metabolic role by integrating signals from T cells receptor and RLRs to program the T cell toward an inflammatory/effector antiviral phenotype. Reflecting this expanding role in immune activation and immune programming, monogenic disorders of RLRs present dire clinical outcomes. As discussed by Tracy Briggs, heterozygous pathogenic mutation in IFIH1 (encoding MDA5) links to skin pathology, neurological disorders, and premature tooth loss. Increased ISG expression in these disorders indicates their linkage with constitutive type I interferon (IFN) signaling. MDA5/IFN-linked disorders or “interferonopathies” also include Aicardi-Goutières and Singleton-Merten syndromes¹². Marisa Gariglio described how human papillomavirus E6 and E7 oncoproteins evade host innate immunity through depletion of multiple innate immune effector RHs. Thus, RLRs and other RHs impart immune signaling and immune programming actions that when dysregulated impose severe autoimmune disorders.

DEAD/DEAH-box RHs in therapeutics and vaccination.

As enzymes, and owing to their broad functions in biological processes, DEAD/DEAH-box RHs present attractive therapeutic targets to consider in strategies aimed at mitigating disease. For example, Cecil Han discussed how DDX3X knockdown in cancer cells increased type I IFN production, STAT activation, and ISG expression via cytosolic accumulation of endogenous dsRNAs. She suggested that targeting DDX3X triggers antitumor immunity via the same mechanism and might serve as a therapeutic liability in cancer cells. Shringar Rao described how DDX3 inhibitors can induce cell death and serve as a therapeutic against latent HIV infected cells, thus, approaches to inhibit DDX3 can be considered in HIV cure strategies. Moreover, RIG-I signaling, as presented by Henry Poeck, is critical in supporting and enhancing immune checkpoint blockade (ICB)-targeted tumor therapy, underscoring an emerging therapeutic application for RLR agonists as anti-tumor immune adjuvants. Simon Rothenfusser then focused on synthetic strategies for RIG-I activation to improve immunotherapies for cancer. In an acute myeloid leukemia mouse model, 5’ triphosphate RNA-induced RIG-I signaling and reduced tumor burden by enhancing tumor-specific T cell responses in synergy with immune checkpoint blockade treatment (ICB). Lastly, Dahai Luo presented synthesized immune-modulatory RNAs (immRNA) as regulators of RIG-I mediated antiviral immune response for therapy and vaccination. Studies of immRNA showed impressive anti-tumor activity via enhancement of tumor-specific T cell responses in mouse melanoma models. This session showed that targeting RIG-I alone and in combination with immune checkpoint blockade such as anti-PD-1 can offer powerful anti-cancer therapeutic potential and immune-enhancement against virus infection and cancer. Moreover, mitigating RH expression, such as DDX3X, could serve as effective strategy to suppress certain types of solid tumors. Synthetic biology to produce and evaluate RH agonists and inhibitors, including RLR-specific compounds, represents an exciting new area of RH research for controlling biological processes of disease (Fig. 2).

Fig. 2.

Immuno-therapeutic considerations of RHs. Upper: RH inhibitors (left) or activators (right) to induce antiviral actions of specific helicases. Middle: RHs are linked to suppression of specific type tumors where RH inhibition (left) or activation (right) could impart tumor cell death through immunologic mechanisms. Lower: Aberrant activation and signaling by RIG-I and MDA5 leading to constitutive type 1 interferon production and actions are linked to a class of autoimmune conditions called interferonopathies where specific RH inhibition could ameliorate disease.

Implications and directions

The workshop concluded with a discussion of RH functions. Considering that specific RNAs are compartmentalized in the cells¹³ it remains to be determined how RH functions link with specific RNA location, recognition, RNA secondary structure, and binding. While the DEAD/H-box RH family is large (see Fig. 1), only a few RHs were discussed in this workshop (Table 1). Future directions should include expanding research across all members of the DEAD/H-box RH family. Defining the spectrum of substrate RNAs targeted and bound by specific RHs across tissues and under different conditions of stress, infection, cancer, autoimmunity, is a perceived priority. Likewise, defining RH-interacting proteins will continue to expand our understanding of RH regulation and function.