PolyQ-expanded ataxin-2 aggregation impairs cellular processing-body homeostasis via sequestering the RNA helicase DDX6.

Ataxin-2 (Atx2) is a polyglutamine (polyQ) tract-containing RNA-binding protein, while its polyQ expansion may cause protein aggregation that is implicated in the pathogenesis of neurodegenerative diseases such as spinocerebellar ataxia type 2 (SCA2). However, the molecular mechanism underlying how Atx2 aggregation contributes to the proteinopathies remains elusive. Here, we investigated the influence of Atx2 aggregation on the assembly and functionality of cellular processing bodies (P-bodies) by using biochemical and fluorescence imaging approaches. We have revealed that polyQ-expanded (PQE) Atx2 sequesters the DEAD-box RNA helicase (DDX6), an essential component of P-bodies, into aggregates or puncta via some RNA sequences. The N-terminal like-Sm (LSm) domain of Atx2 (residues 82-184) and the C-terminal helicase domain of DDX6 are responsible for the interaction and specific sequestration. Moreover, sequestration of DDX6 may aggravate pre-mRNA mis-splicing, and interfere with the assembly of cellular P-bodies, releasing the endoribonuclease MARF1 that promotes mRNA decay and translational repression. Rescuing the DDX6 protein level can recover the assembly and functionality of P-bodies, preventing targeted mRNA from degradation. This study provides a line of evidence for sequestration of the P-body components and impairment of the P-body homeostasis in dysregulating RNA metabolism, which is implicated in the disease pathologies and a potential therapeutic target.

Rab3B enhances the stabilization of DDX6 to promote lung adenocarcinoma aggressiveness.

BACKGROUND: Liver kinase B1 (LKB1) is frequently mutated in lung adenocarcinoma, and its loss contributes to tumor progression. METHODS: To identify LKB1 downstream genes that promote lung adenocarcinoma aggressiveness, we performed bioinformatical analysis using publicly available datasets. RESULTS: Rab3B was upregulated in LKB1-depleted lung adenocarcinoma cells and suppressed by LKB1 overexpression. CREB protein was enriched at the promoter of Rab3B in lung cancer cells. Silencing of CREB abrogated the upregulation of Rab3B upon LKB1 loss. Immunohistochemistry revealed the elevated expression of Rab3B in lung adenocarcinomas relative to adjacent normal tissues. Upregulation of Rab3B was significantly associated with lymph node metastasis, advanced tumor stage, and reduced overall survival in lung adenocarcinoma patients. Knockdown of Rab3B suppressed and overexpression of Rab3B promoted the proliferation, colony formation, and migration of lung adenocarcinoma cells in vitro. In a mouse xenograft model, Rab3B depletion restrained and Rab3B overexpression augmented the growth of lung adenocarcinoma tumors. Mechanistically, Rab3B interacted with DDX6 and enhanced its protein stability. Ectopic expression of DDX6 significantly promoted the proliferation, colony formation, and migration of lung adenocarcinoma cells. DDX6 knockdown phenocopied the effects of Rab3B depletion on lung adenocarcinoma cells. Additionally, DDX6 overexpression partially rescued the aggressive phenotype of Rab3B-depleted lung adenocarcinoma cells. CONCLUSION: LKB1 deficiency promotes Rab3B upregulation via a CREB-dependent manner. Rab3B interacts with and stabilizes DDX6 protein to accelerate lung adenocarcinoma progression. The Rab3B-DDX6 axis may be potential therapeutic target for lung adenocarcinoma.

DDX6 is involved in the pathogenesis of inflammatory diseases via NF-κB activation.

The IL-6 amplifier was originally discovered as a mechanism for the enhanced activation of NF-κB in non-immune cells. In the IL-6 amplifier, IL-6-STAT3 and NF-κB stimulation is followed by an excessive production of IL-6, chemokines, and growth factors to develop chronic inflammation preceding the development of inflammatory diseases. Previously, using a shRNA-mediated genome-wide screening, we found that DEAD-Box Helicase 6 (DDX6) is a candidate positive regulator of the amplifier. Here, we investigate whether DDX6 is involved in the pathogenesis of inflammatory diseases via the IL-6 amplifier. We found that DDX6-silencing in non-immune cells suppressed the NF-κB pathway and inhibited activation of the IL-6 amplifier, while the forced expression of DDX6 enhanced NF-κB promoter activity independent of the RNA helicase activity of DDX6. The imiquimod-mediated dermatitis model was suppressed by the siRNA-mediated gene downregulation of DDX6. Furthermore, silencing DDX6 significantly reduced the TNF-α-induced phosphorylation of p65/RelA and IκBα, nuclear localization of p65, and the protein levels of IκBα. Mechanistically, DDX6 is strongly associated with p65 and IκBα, but not TRADD, RIP, or TRAF2, suggesting a novel function of DDX6 as an adaptor protein in the NF-κB pathway. Thus, our findings demonstrate a possible role of DDX6 beyond RNA metabolism and suggest DDX6 is a therapeutic target for inflammatory diseases.

IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap.

Inositol hexakisphosphate kinase 1 (IP6K1) is a small molecule kinase that catalyzes the conversion of the inositol phosphate IP6 to 5-IP7. We show that IP6K1 acts independently of its catalytic activity to upregulate the formation of processing bodies (P-bodies), which are cytoplasmic ribonucleoprotein granules that store translationally repressed mRNA. IP6K1 does not localise to P-bodies, but instead binds to ribosomes, where it interacts with the mRNA decapping complex - the scaffold protein EDC4, activator proteins DCP1A/B, decapping enzyme DCP2 and RNA helicase DDX6. Along with its partner 4E-T, DDX6 is known to nucleate protein-protein interactions on the 5' mRNA cap to facilitate P-body formation. IP6K1 binds the translation initiation complex eIF4F on the mRNA cap, augmenting the interaction of DDX6 with 4E-T (also known as EIF4ENIF1) and the cap-binding protein eIF4E. Cells with reduced IP6K1 show downregulated microRNA-mediated translational suppression and increased stability of DCP2-regulated transcripts. Our findings unveil IP6K1 as a novel facilitator of proteome remodelling on the mRNA cap, tipping the balance in favour of translational repression over initiation, thus leading to P-body assembly. This article has an associated First Person interview with the first author of the paper.

Antagonism between DDX6 and PI3K-AKT signaling is an oocyte-intrinsic mechanism controlling primordial follicle growth†.

Oocyte maturation and subsequent ovulation during the reproductive lifespan ensure long-term reproduction in mammalian females. This is achieved by tight regulation for the maintenance and growth of primordial follicles. However, the underlying mechanisms remain unsolved. We herein report that posttranscriptional gene regulation mediated by an RNA helicase, DEAD-box helicase 6 (DDX6), and phosphoinositide-3-kinase (PI3K)-AKT signaling exhibits an antagonistic interaction in mouse primordial follicles. DDX6 forms P-body-like cytoplasmic foci in oocytes, which colocalize to a P-body component, DCP1A. Interestingly, the P-body-like granules predominantly assemble in primordial follicles, but disperse once follicle growth is initiated, suggesting that they play a role in the maintenance of primordial follicles. Oocyte-specific knockout of Ddx6 using Gdf9-iCre revealed that Ddx6-deficient oocytes are defective in foci assembly and are abnormally enlarged, resulting in premature depletion of primordial follicles. These results indicate that DDX6 is required to maintain primordial follicles. The abnormal oocyte enlargement is because of enhanced PI3K-AKT signaling, a pivotal signaling pathway in the growth of primordial follicles. Conversely, the forced activation of PI3K-AKT signaling by knocking out Pten disassembles P-body-like granules in primordial follicles. These data suggest that DDX6 and PI3K-AKT signaling mutually antagonize the assembly of P-body-like granules and the growth of primordial follicles. We propose this mutual antagonism as an oocyte-intrinsic mechanism controlling the maintenance and growth of primordial follicles, ensuring the longevity of female reproduction.

A New Long Noncoding RNA, MAHAT, Inhibits Replication of Porcine Reproductive and Respiratory Syndrome Virus by Recruiting DDX6 To Bind to ZNF34 and Promote an Innate Immune Response.

Long noncoding RNAs (lncRNAs) have increasingly been recognized as being integral to cellular processes, including the antiviral immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is costly to the global swine industry. To identify PRRSV-related lncRNAs, we performed RNA deep sequencing and compared the profiles of lncRNAs in PRRSV-infected and uninfected Marc-145 cells. We identified a novel lncRNA called MAHAT (maintaining cell morphology-associated and highly conserved antiviral transcript; LTCON_00080558) that inhibits PRRSV replication. MAHAT binds and negatively regulates ZNF34 expression by recruiting and binding DDX6, an RNA helicase forming a complex with ZNF34. Inhibition of ZNF34 expression results in increased type I interferon expression and decreased PRRSV replication. This finding reveals a novel mechanism by which PRRSV evades the host antiviral innate immune response by downregulating the MAHAT-DDX6-ZNF34 pathway. MAHAT could be a host factor target for antiviral therapies against PRRSV infection. IMPORTANCE Long noncoding RNAs (lncRNAs) play important roles in viral infection by regulating the transcription and expression of host genes, and interferon signaling pathways. Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses in the swine industry worldwide, but the mechanisms of its pathogenesis and immunology are not fully understood. Here, a new lncRNA, designated MAHAT, was identified as a regulator of host innate immune responses. MAHAT negatively regulates the expression of its target gene, ZNF34, by recruiting and binding DDX6, an RNA helicase, forming a complex with ZNF34. Inhibition of ZNF34 expression increases type I interferon expression and decreases PRRSV replication. This finding suggests that MAHAT has potential as a new target for developing antiviral drugs against PRRSV infection.

Host Cellular RNA Helicases Regulate SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the largest RNA genome, approximately 30 kb, among RNA viruses. The DDX DEAD box RNA helicase is a multifunctional protein involved in all aspects of RNA metabolism. Therefore, host RNA helicases may regulate and maintain such a large viral RNA genome. In this study, I investigated the potential role of several host cellular RNA helicases in SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, indicating that DDX21 strongly restricts the SARS-CoV-2 infection. In addition, MOV10 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA helicases as well as XRN1 exonuclease, while the viral infection did not induce stress granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted with DDX1, DDX3, DDX5, DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that SARS-CoV-2 N hijacks DDX6 to carry out viral replication. Conversely, DDX21 and MOV10 restricted SARS-CoV-2 infection through an interaction of SARS-CoV-2 N with host cellular RNA helicases. Altogether, host cellular RNA helicases seem to regulate the SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has a large RNA genome, of approximately 30 kb. To regulate and maintain such a large viral RNA genome, host RNA helicases may be involved in SARS-CoV-2 replication. In this study, I have demonstrated that DDX21 and MOV10 RNA helicases limit viral infection and replication. In contrast, DDX1, DDX5, and DDX6 are required for SARS-CoV-2 infection. Interestingly, SARS-CoV-2 infection disrupted P-body formation and attenuated or suppressed stress granule formation. Thus, SARS-CoV-2 seems to hijack host cellular RNA helicases to play a proviral role by facilitating viral infection and replication and by suppressing the host innate immune system.

RNA Helicase DDX6 Regulates A-to-I Editing and Neuronal Differentiation in Human Cells.

The DEAD-box proteins, one family of RNA-binding proteins (RBPs), participate in post-transcriptional regulation of gene expression with multiple aspects. Among them, DDX6 is an essential component of the cytoplasmic RNA processing body (P-body) and is involved in translational repression, miRNA-meditated gene silencing, and RNA decay. In addition to the cytoplasmic function, DDX6 is also present in the nucleus, but the nuclear function remains unknown. To decipher the potential role of DDX6 in the nucleus, we performed mass spectrometry analysis of immunoprecipitated DDX6 from a HeLa nuclear extract. We found that adenosine deaminases that act on RNA 1 (ADAR1) interact with DDX6 in the nucleus. Utilizing our newly developed dual-fluorescence reporter assay, we elucidated the DDX6 function as negative regulators in cellular ADAR1p110 and ADAR2. In addition, depletion of DDX6 and ADARs results in the opposite effect on facilitation of RA-induced differentiation of neuronal lineage cells. Our data suggest the impact of DDX6 in regulation of the cellular RNA editing level, thus contributing to differentiation in the neuronal cell model.

Rare De Novo Missense Variants in RNA Helicase DDX6 Cause Intellectual Disability and Dysmorphic Features and Lead to P-Body Defects and RNA Dysregulation.

The human RNA helicase DDX6 is an essential component of membrane-less organelles called processing bodies (PBs). PBs are involved in mRNA metabolic processes including translational repression via coordinated storage of mRNAs. Previous studies in human cell lines have implicated altered DDX6 in molecular and cellular dysfunction, but clinical consequences and pathogenesis in humans have yet to be described. Here, we report the identification of five rare de novo missense variants in DDX6 in probands presenting with intellectual disability, developmental delay, and similar dysmorphic features including telecanthus, epicanthus, arched eyebrows, and low-set ears. All five missense variants (p.His372Arg, p.Arg373Gln, p.Cys390Arg, p.Thr391Ile, and p.Thr391Pro) are located in two conserved motifs of the RecA-2 domain of DDX6 involved in RNA binding, helicase activity, and protein-partner binding. We use functional studies to demonstrate that the first variants identified (p.Arg373Gln and p.Cys390Arg) cause significant defects in PB assembly in primary fibroblast and model human cell lines. These variants' interactions with several protein partners were also disrupted in immunoprecipitation assays. Further investigation via complementation assays included the additional variants p.Thr391Ile and p.Thr391Pro, both of which, similarly to p.Arg373Gln and p.Cys390Arg, demonstrated significant defects in P-body assembly. Complementing these molecular findings, modeling of the variants on solved protein structures showed distinct spatial clustering near known protein binding regions. Collectively, our clinical and molecular data describe a neurodevelopmental syndrome associated with pathogenic missense variants in DDX6. Additionally, we suggest DDX6 join the DExD/H-box genes DDX3X and DHX30 in an emerging class of neurodevelopmental disorders involving RNA helicases.

Identification of an Antiretroviral Small Molecule That Appears To Be a Host-Targeting Inhibitor of HIV-1 Assembly.

Given the projected increase in multidrug-resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life cycle stages not targeted by drugs currently in use. Host-targeting compounds are of particular interest because they can offer a high barrier to resistance. Here, we report identification of two related small molecules that inhibit HIV-1 late events, a part of the HIV-1 life cycle for which potent and specific inhibitors are lacking. This chemotype was discovered using cell-free protein synthesis and assembly systems that recapitulate intracellular host-catalyzed viral capsid assembly pathways. These compounds inhibit replication of HIV-1 in human T cell lines and peripheral blood mononuclear cells, and are effective against a primary isolate. They reduce virus production, likely by inhibiting a posttranslational step in HIV-1 Gag assembly. Notably, the compound colocalizes with HIV-1 Gag in situ; however, unexpectedly, selection experiments failed to identify compound-specific resistance mutations in gag or pol, even though known resistance mutations developed upon parallel nelfinavir selection. Thus, we hypothesized that instead of binding to Gag directly, these compounds localize to assembly intermediates, the intracellular multiprotein complexes containing Gag and host factors that form during immature HIV-1 capsid assembly. Indeed, imaging of infected cells shows compound colocalized with two host enzymes found in assembly intermediates, ABCE1 and DDX6, but not two host proteins found in other complexes. While the exact target and mechanism of action of this chemotype remain to be determined, our findings suggest that these compounds represent first-in-class, host-targeting inhibitors of intracellular events in HIV-1 assembly.IMPORTANCE The success of antiretroviral treatment for HIV-1 is at risk of being undermined by the growing problem of drug resistance. Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulates the intracellular events of HIV-1 assembly, including virus-host interactions that promote assembly. This effort led to the identification of a new chemotype that inhibits HIV-1 replication at nanomolar concentrations, likely by acting on assembly. This compound colocalized with Gag and two host enzymes that facilitate capsid assembly. However, resistance selection did not result in compound-specific mutations in gag, suggesting that the chemotype does not directly target Gag. We hypothesize that this chemotype represents a first-in-class inhibitor of virus production that acts by targeting a virus-host complex important for HIV-1 Gag assembly.