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The DEAD-box helicase DDX3X is a critical component of the TANK-binding kinase 1-dependent innate immune response.

ABSTRACT: TANK-binding kinase 1 (TBK1) is of central importance for the induction of type-I interferon (IFN) in response to pathogens. We identified the DEAD-box helicase DDX3X as an interaction partner of TBK1. TBK1 and DDX3X acted synergistically in their ability to stimulate the IFN promoter, whereas RNAi-mediated reduction of DDX3X expression led to an impairment of IFN production. Chromatin immunoprecipitation indicated that DDX3X is recruited to the IFN promoter upon infection with Listeria monocytogenes, suggesting a transcriptional mechanism of action. DDX3X was found to be a TBK1 substrate in vitro and in vivo. Phosphorylation-deficient mutants of DDX3X failed to synergize with TBK1 in their ability to stimulate the IFN promoter. Overall, our data imply that DDX3X is a critical effector of TBK1 that is necessary for type I IFN induction.

SUBMITTER: Soulat D

PROVIDER: S-EPMC2453059 | biostudies-literature |

REPOSITORIES: biostudies-literature

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DEAD-box RNA helicase 21 negatively regulates cytosolic RNA-mediated innate immune signaling.

Project description:DEAD-box RNA helicase 21 (DDX21), also known as RHII/Gu, is an ATP-dependent RNA helicase. In addition to playing a vital role in regulating cellular RNA splicing, transcription, and translation, accumulated evidence has suggested that DDX21 is also involved in the regulation of innate immunity. However, whether DDX21 induces or antagonizes type I interferon (IFN-I) production has not been clear and most studies have been performed through ectopic overexpression or RNA interference-mediated knockdown. In this study, we generated DDX21 knockout cell lines and found that knockout of DDX21 enhanced Sendai virus (SeV)-induced IFN-β production and IFN-stimulated gene (ISG) expression, suggesting that DDX21 is a negative regulator of IFN-β. Mechanistically, DDX21 competes with retinoic acid-inducible gene I (RIG-I) for binding to double-stranded RNA (dsRNA), thereby attenuating RIG-I-mediated IFN-β production. We also identified that the 217-784 amino acid region of DDX21 is essential for binding dsRNA and associated with its ability to antagonize IFN production. Taken together, our results clearly demonstrated that DDX21 negatively regulates IFN-β production and functions to maintain immune homeostasis.

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Biochemical Differences and Similarities between the DEAD-Box Helicase Orthologs DDX3X and Ded1p.

Project description:DDX3X is a conserved DEAD-box RNA helicase involved in translation initiation and other processes of RNA metabolism. Mutations in human DDX3X and deregulation of its expression are linked to tumorigenesis and intellectual disability. The protein is also targeted by diverse viruses. Previous studies demonstrated helicase and NTPase activities for DDX3X, but important biochemical features of the enzyme remain unclear. Here, we systematically characterize enzymatic activities of human DDX3X and compare these to its closely related Saccharomyces cerevisiae ortholog Ded1p. We show that DDX3X, like Ded1p, utilizes exclusively adenosine triphosphates to unwind helices, oligomerizes to function as efficient RNA helicase, and does not unwind DNA duplexes. The ATPase activity of DDX3X is markedly stimulated by RNA and weaker by DNA, although DNA binds to the enzyme. For RNA unwinding, DDX3X shows a greater preference than Ded1p for substrates with unpaired regions 3' to the duplex over those with 5' unpaired regions. DDX3X separates longer RNA duplexes faster than Ded1p and is less potent than Ded1p in facilitating strand annealing. Our results reveal that the biochemical activities of human DDX3X are typical for DEAD-box RNA helicases, but diverge quantitatively from its highly similar S. cerevisiae ortholog Ded1p.

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The mechanism of RNA duplex recognition and unwinding by DEAD-box helicase DDX3X.

Project description:DEAD-box helicases (DDXs) regulate RNA processing and metabolism by unwinding short double-stranded (ds) RNAs. Sharing a helicase core composed of two RecA-like domains (D1D2), DDXs function in an ATP-dependent, non-processive manner. As an attractive target for cancer and AIDS treatment, DDX3X and its orthologs are extensively studied, yielding a wealth of biochemical and biophysical data, including structures of apo-D1D2 and post-unwound D1D2:single-stranded RNA complex, and the structure of a D2:dsRNA complex that is thought to represent a pre-unwound state. However, the structure of a pre-unwound D1D2:dsRNA complex remains elusive, and thus, the mechanism of DDX action is not fully understood. Here, we describe the structure of a D1D2 core in complex with a 23-base pair dsRNA at pre-unwound state, revealing that two DDXs recognize a 2-turn dsRNA, each DDX mainly recognizes a single RNA strand, and conformational changes induced by ATP binding unwinds the RNA duplex in a cooperative manner.

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2021-09-08 | PXD028122 | Pride

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| S-EPMC8396550 | biostudies-literature

RK-33 Is a Broad-Spectrum Antiviral Agent That Targets DEAD-Box RNA Helicase DDX3X.

Project description:Viral disease is one of the greatest burdens for human health worldwide, with an urgent need for efficacious antiviral strategies. While antiviral drugs are available, in many cases, they are prone to the development of drug resistance. A way to overcome drug resistance associated with common antiviral therapies is to develop antivirals targeting host cellular co-factors critical to viral replication, such as DEAD-box helicase 3 X-linked (DDX3X), which plays key roles in RNA metabolism and the antiviral response. Here, we use biochemical/biophysical approaches and infectious assays to show for the first time that the small molecule RK-33 has broad-spectrum antiviral action by inhibiting the enzymatic activities of DDX3X. Importantly, we show that RK-33 is efficacious at low micromolar concentrations in limiting infection by human parainfluenza virus type 3 (hPIV-3), respiratory syncytial virus (RSV), dengue virus (DENV), Zika virus (ZIKV) or West Nile virus (WNV)-for all of which, no Food and Drug Administration (FDA)-approved therapeutic is widely available. These findings establish for the first time that RK-33 is a broad-spectrum antiviral agent that blocks DDX3X's catalytic activities in vitro and limits viral replication in cells.

| S-EPMC7016805 | biostudies-literature

Medulloblastoma-associated mutations in the DEAD-box RNA helicase DDX3X/DED1 cause specific defects in translation.

Project description:Medulloblastoma is the most common pediatric brain cancer, and sequencing studies identified frequent mutations in DDX3X, a DEAD-box RNA helicase primarily implicated in translation. Forty-two different sites were identified, suggesting that the functional effects of the mutations are complex. To investigate how these mutations are affecting DDX3X cellular function, we constructed a full set of equivalent mutant alleles in DED1, the Saccharomyces cerevisiae ortholog of DDX3X, and characterized their effects in vivo and in vitro. Most of the medulloblastoma-associated mutants in DDX3X/DED1 (ded1-mam) showed substantial growth defects, indicating that functional effects are conserved in yeast. Further, while translation was affected in some mutants, translation defects affecting bulk mRNA were neither consistent nor correlated with the growth phenotypes. Likewise, increased formation of stress granules in ded1-mam mutants was common but did not correspond to the severity of the mutants' growth defects. In contrast, defects in translating mRNAs containing secondary structure in their 5' untranslated regions (UTRs) were found in almost all ded1-mam mutants and correlated well with growth phenotypes. We thus conclude that these specific translation defects, rather than generalized effects on translation, are responsible for the observed cellular phenotypes and likely contribute to DDX3X-mutant medulloblastoma. Examination of ATPase activity and RNA binding of recombinant mutant proteins also did not reveal a consistent defect, indicating that the translation defects are derived from multiple enzymatic deficiencies. This work suggests that future studies into medulloblastoma pathology should focus on this specific translation defect, while taking into account the wide spectrum of DDX3X mutations.

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Exportin-1-Dependent Nuclear Export of DEAD-box Helicase DDX3X is Central to its Role in Antiviral Immunity.

Project description:DEAD-box helicase 3, X-linked (DDX3X) regulates the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated antiviral response, but can also be a host factor contributing to the replication of viruses of significance to human health, such as human immunodeficiency virus type 1 (HIV-1). These roles are mediated in part through its ability to actively shuttle between the nucleus and the cytoplasm to modulate gene expression, although the trafficking mechanisms, and impact thereof on immune signaling and viral infection, are incompletely defined. We confirm that DDX3X nuclear export is mediated by the nuclear transporter exportin-1/CRM1, dependent on an N-terminal, leucine-rich nuclear export signal (NES) and the monomeric guanine nucleotide binding protein Ran in activated GTP-bound form. Transcriptome profiling and ELISA show that exportin-1-dependent export of DDX3X to the cytoplasm strongly impacts IFN-β production and the upregulation of immune genes in response to infection. That this is key to DDX3X's antiviral role was indicated by enhanced infection by human parainfluenza virus-3 (hPIV-3)/elevated virus production when the DDX3X NES was inactivated. Our results highlight a link between nucleocytoplasmic distribution of DDX3X and its role in antiviral immunity, with strong relevance to hPIV-3, as well as other viruses such as HIV-1.

| S-EPMC6848931 | biostudies-literature

Transcriptome analysis of Listeria monocytogenes infected bone marrow derived macrophages with or without DEAD-box helicase DDX3X

Project description:Bone marrow derived macrophages were infected with Listeria monocytogenes for 4 hours. We investigated differently expressed genes in the absence of DDX3X upon infection and also in steady state conditions.

2018-10-17 | GSE86591 | GEO

DEAD-box helicase eIF4A2 inhibits CNOT7 deadenylation activity.

Project description:The CCR4-NOT complex plays an important role in the translational repression and deadenylation of mRNAs. However, little is known about the specific roles of interacting factors. We demonstrate that the DEAD-box helicases eIF4A2 and DDX6 interact directly with the MA3 and MIF domains of CNOT1 and compete for binding. Furthermore, we now show that incorporation of eIF4A2 into the CCR4-NOT complex inhibits CNOT7 deadenylation activity in contrast to DDX6 which enhances CNOT7 activity. Polyadenylation tests (PAT) on endogenous mRNAs determined that eIF4A2 bound mRNAs have longer poly(A) tails than DDX6 bound mRNAs. Immunoprecipitation experiments show that eIF4A2 does not inhibit CNOT7 association with the CCR4-NOT complex but instead inhibits CNOT7 activity. We identified a CCR4-NOT interacting factor, TAB182, that modulates helicase recruitment into the CCR4-NOT complex, potentially affecting the outcome for the targeted mRNA. Together, these data show that the fate of an mRNA is dependent on the specific recruitment of either eIF4A2 or DDX6 to the CCR4-NOT complex which results in different pathways for translational repression and mRNA deadenylation.

| S-EPMC6736043 | biostudies-literature

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