Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response

Shih, Jing Wen; Wang, Wei‐Ting; Tsai, Tsung‐Yuan; Kuo, Chu Yun; Li, Hao Kang; Lee, Yan Hwa Wu

doi:10.1042/bj20110739

Cited by 166 publications

(203 citation statements)

References 33 publications

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“…Assembly occurs through aggregation of proteins such as TIA1 or G3BP, which can interact with mRNA leading to translationally stalled aggregates. The DDX3 RNA helicase can associate with stress granules and suppress translation through the inhibition of eIF4E (89). Additionally, molecules that inhibit eIF4A can promote stress granule formation independently of eIF2␣ (reviewed in Ref.…”

Section: Discussionmentioning

confidence: 99%

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

Byrd¹,

Zybailov

Maddukuri³

et al. 2016

Journal of Biological Chemistry

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Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.

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Section: Discussionmentioning

confidence: 99%

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

Byrd¹,

Zybailov

Maddukuri³

et al. 2016

Journal of Biological Chemistry

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“…DDX3 has not only been implicated in contributing the innate immune response (48,51,62,79) but may also function in host cell translation initiation via an interaction with eukaryotic initiation factor 3 (eIF3) (38). Recent studies suggested that DDX3 contributes to HIV translation (39) and stress granule formation (64). eIF3 has been implicated to have a role in the unique mechanism of norovirus VPg-mediated translation initiation (11,12).…”

Section: Figmentioning

confidence: 99%

Identification of RNA-Protein Interaction Networks Involved in the Norovirus Life Cycle

Vashist

Ureña

Chaudhry

et al. 2012

J Virol

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Human noroviruses are one of the major causes of acute gastroenteritis in the developed world, yet our understanding of their molecular mechanisms of genome translation and replication lags behind that for many RNA viruses. Due to the nonculturable nature of human noroviruses, many related members of the Caliciviridae family of small RNA viruses are often used as model systems to dissect the finer details of the norovirus life cycle. Murine norovirus (MNV) has provided one such system with which to study the basic mechanisms of norovirus translation and replication in cell culture. In this report we describe the use of riboproteomics to identify host factors that interact with the extremities of the MNV genome. This network of RNA-protein interactions contains many well-characterized host factors, including PTB, La, and DDX3, which have been shown to play a role in the life cycle of other RNA viruses. By using RNA coimmunoprecipitation, we confirmed that a number of the factors identified using riboproteomics are associated with the viral RNA during virus replication in cell culture. We further demonstrated that RNA inhibition-mediated knockdown of the intracellular levels of a number of these factors inhibits or slows norovirus replication in cell culture, allowing identification of new intracellular targets for this important group of pathogens. The genomes of small positive-strand RNA viruses must not only encode the viral proteins required for viral genome replication and infectious virus production, but they must also contain cis-acting RNA sequences and structures that play critical roles in the virus life cycle (40). Host cell factors that interact with these RNA elements play important roles in all aspects of the virus life cycle (49); consequently, the manipulation of these interactions via the introduction of mutations into the viral genome is a viable approach to rational attenuation (24,26,57,78). In addition, the modification of these RNA-protein interactions by using small-molecule or peptide inhibitors has been shown to provide antiviral activity, at least in cell culture (8,19).Noroviruses, first identified in 1972 by Albert Kapikian (33), are now accepted as the major cause of viral gastroenteritis in the developed world (21). Reports indicate that in excess of 23 million cases occur each year in the United States, resulting in a significant economic impact. Now over 20 years since the first full genome sequence of a norovirus became available (77), investigators are still unable to efficiently propagate human noroviruses in cell culture (15, 37). Recent data have indicated that the viral RNA purified from the feces of individuals infected with Norwalk virus, the prototype human norovirus, can replicate when transfected into immortalized cells in culture (23). These data clearly demonstrate that one of the major blocks in the ability of human noroviruses to replicate in cell culture is the ability of the virus to enter and spread from cell to cell. It is also clear that the intracellular environ...

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“…Like other RNP bodies, P granules are enriched in RNA-binding proteins, including PGL-1,-3 and the RNA helicases CGH-1, GLH-1-4, LAF-1, and VBH-1 (11). Members of the highly conserved DDX3 subfamily of DEAD-box RNA helicases, including human DDX3X, yeast Ded1p, and Drosophila Belle, have demonstrated roles in the assembly and remodeling of RNPs (12)(13)(14). Interestingly, many of these RNA helicases are predicted to be partially disordered, consistent with bioinformatic analysis, suggesting disordered motifs are common in RNP bodies (15).…”

mentioning

confidence: 99%

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Elbaum‐Garfinkle

Kim

Szczepaniak

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

1,062

1,292

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P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA-protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.liquid droplets | intracellular phase transition | intrinsically disordered proteins | RNA granules I ntracellular RNA/protein (RNP) assemblies, including germ granules, processing bodies, stress granules, and nucleoli, are key players in the regulation of gene expression (1). RNP bodies, also referred to as RNA granules, function in diverse modes of RNA processing, including splicing, degradation, and translational repression of mRNA. These ubiquitous structures lack a membrane boundary but nonetheless represent a coherent organelle composed of thousands of molecules, manifesting as microscopically visible puncta in both the cytoplasm and the nucleus.Recent studies have demonstrated the apparent liquid-like behavior of various RNP bodies (2-5) including wetting, dripping, and relaxation to spherical structures upon fusion or shearing. The assembly and disassembly of liquid-like organelles appears to be governed by a phase separation process, demonstrated by a concentration-dependent condensation/dissolution of P granules (2, 6) and the assembly and size scaling of the nucleolus (7) in the Caenorhabditis elegans embryo. Liquid phase separation has also been suggested to play a role in stress granule assembly (4) and in multivalent signaling proteins (8). These studies lend increasing support to the hypothesis that liquid phases play a central role in intracellular organization. However, the specific molecular interactions that drive phase separation and the mechanisms by which liquid properties impart cellular function remain largely unclear. P granule...

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Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response

Cited by 166 publications

References 33 publications

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

Identification of RNA-Protein Interaction Networks Involved in the Norovirus Life Cycle

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

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