DEAD box 1 (DDX1) protein binds to and protects cytoplasmic stress response mRNAs in cells exposed to oxidative stress

The integrated stress response is a network of highly orchestrated pathways activated when cells are exposed to environmental stressors. While global repression of translation is a well-recognized hallmark of the integrated stress response, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD box 1 (DDX1) protein accumulates at DNA double-strand breaks during genotoxic stress and promotes DNA double-strand break repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors, including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing, we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs are increased during stress in a DDX1-dependent manner. Even though DDX1’s RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.     

Knockdown of Cellular RNA Helicase DDX3 by Short Hairpin RNAs Suppresses HIV-1 Viral Replication Without Inducing Apoptosis

The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.     

Determination of the Role of DDX3 a Factor Involved in Mammalian RNAi Pathway Using an shRNA-Expression Library

RNA interference (RNAi) is an endogenous RNA-destruction phenomenon induced by certain double-stranded RNAs (dsRNAs). In RNAi, dsRNAs are processed into small interfering RNAs (siRNAs) which in turn trigger the cleavage of the target mRNA. Here, using a short hairpin RNA-expression library, we identified a DEAD-box helicase 3, DDX3, as an essential factor involved in RNAi pathway and revealed that DDX3 is colocalized with Ago2, an essential factor in RNAi pathway that cleaves target mRNA. Results of experiments with a dominant negative mutant of DDX3 further confirmed that this factor affects the RNAi activity. Together, DDX3 functions to assure mammalian RNAi pathway. Together, our results indicate that DDX3 is a new key molecule to understand the molecular mechanism underlying RNAi pathway in mammals.     

Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.     

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.     

Human DDX3 functions in translation and interacts with the translation initiation factor eIF3

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3.     

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

Upon environmental insults, SGs (stress granules) aid cell survival by serving as sites of translational silencing. RNA helicase DDX3 was reported to associate with SGs. However, its role in SG physiology remains undefined. We have demonstrated previously that DDX3 acts as an eIF4E (eukaryotic initiation factor 4E)-inhibitory protein to suppress translation. In the present study, we indentified the SG marker PABP1 [poly(A)-binding protein 1] as another direct interaction partner of DDX3. We established various stimuli as novel stressors that direct DDX3 with eIF4E and PABP1 into SGs, but not to processing bodies. Interestingly, down-regulation of DDX3 interfered with SG assembly, led to nuclear accumulation of PABP1 and reduced cell viability following stress. Conversely, supplementation with a shRNA (short hairpin RNA)-resistant DDX3 restored SG formation, the translocation of PABP1 into SGs and cell survival. Notably, the SG-inducing capacity of DDX3 is independent of its ATPase and helicase activities, but mapped to the eIF4E-binding region. Moreover, the eIF4E-binding-defective mutant DDX3 was impaired in its SG-inducing ability and protective effect on cell survival under adverse conditions. All together, the present study has characterized DDX3 as a pivotal SG-nucleating factor and illustrates co-ordinative roles for DDX3, eIF4E and PABP1 in integrating environmental stress with translational regulation.     

The Viral Protein K7 Inhibits Biochemical Activities and Condensate Formation by the DEAD-box Helicase DDX3X

The DEAD-box RNA helicase DDX3X promotes translation initiation and associates with stress granules. A range of diverse viruses produce proteins that target DDX3X, including hepatitis C, dengue, vaccinia, and influenza A. The interaction of some of these viral proteins with DDX3X has been shown to affect antiviral intracellular signaling, but it is unknown whether and how viral proteins impact the biochemical activities of DDX3X and its physical roles in cells. Here we show that the protein K7 from vaccinia virus, which binds to an intrinsically disordered region in the N-terminus of DDX3X, inhibits RNA helicase and RNA-stimulated ATPase activities, as well as liquid–liquid phase separation of DDX3X in vitro. We demonstrate in HCT 116 cells that K7 inhibits association of DDX3X with stress granules, as well as the formation of aberrant granules induced by expression of DDX3X with a point mutation linked to medulloblastoma and DDX3X syndrome. The results show that targeting of the intrinsically disordered N-terminus is an effective viral strategy to modulate the biochemical functions and subcellular localization of DDX3X. Our findings also have potential therapeutic implications for diseases linked to aberrant DDX3X granule formation.     

Comparative Structural Analysis of Human DEAD-Box RNA Helicases

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.     

NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47

Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP binding protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabolism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA precursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus.     

Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3

DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.     

The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA

Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5′ m⁷GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m⁷GTP 5′ cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled.