The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Foot-and-mouth disease virus(FMDV) can infect domestic and wild cloven-hoofed animals. The non-structural protein 3 D plays an important role in FMDV replication and pathogenesis. However, the interaction partners of 3 D, and the effects of those interactions on FMDV replication, remain incompletely elucidated. In the present study, using the yeast two-hybrid system, we identified a porcine cell protein, DEAD-box RNA helicase 1(DDX1), which interacted with FMDV 3 D. The DDX1-3 D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay(IFA) in porcine kidney 15(PK-15) cells. DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses. However, the roles of DDX1 during FMDV infection remain unclear. Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner. In addition, DDX1 stimulated IFN-b activation in FMDV-infected cells. Together, our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.     

MDM2 Mediates Nonproteolytic Polyubiquitylation of the DEAD-Box RNA Helicase DDX24

MDM2 mediates the ubiquitylation and thereby triggers the proteasomal degradation of the tumor suppressor protein p53. However, genetic evidence suggests that MDM2 contributes to multiple regulatory networks independently of p53 degradation. We have now identified the DEAD-box RNA helicase DDX24 as a nucleolar protein that interacts with MDM2. DDX24 was found to bind to the central region of MDM2, resulting in the polyubiquitylation of DDX24 both in vitro and in vivo. Unexpectedly, however, the polyubiquitylation of DDX24 did not elicit its proteasomal degradation but rather promoted its association with preribosomal ribonucleoprotein (pre-rRNP) processing complexes that are required for the early steps of pre-rRNA processing. Consistently with these findings, depletion of DDX24 in cells impaired pre-rRNA processing and resulted both in abrogation of MDM2 function and in consequent p53 stabilization. Our results thus suggest an unexpected role of MDM2 in the nonproteolytic ubiquitylation of DDX24, which may contribute to the regulation of pre-rRNA processing.     

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.     

SACY-1 DEAD-Box Helicase Links the Somatic Control of Oocyte Meiotic Maturation to the Sperm-to-Oocyte Switch and Gamete Maintenance in Caenorhabditis elegans

In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. In Caenorhabditis elegans, major sperm protein triggers meiotic resumption through a mechanism involving somatic Gα_s–adenylate cyclase signaling and soma-to-germline gap-junctional communication. Using genetic mosaic analysis, we show that the major effector of Gα_s–adenylate cyclase signaling, protein kinase A (PKA), is required in gonadal sheath cells for oocyte meiotic maturation and dispensable in the germ line. This result rules out a model in which cyclic nucleotides must transit through sheath-oocyte gap junctions to activate PKA in the germ line, as proposed in vertebrate systems. We conducted a genetic screen to identify regulators of oocyte meiotic maturation functioning downstream of Gα_s–adenylate cyclase–PKA signaling. We molecularly identified 10 regulatory loci, which include essential and nonessential factors. sacy-1, which encodes a highly conserved DEAD-box helicase, is an essential germline factor that negatively regulates meiotic maturation. SACY-1 is a multifunctional protein that establishes a mechanistic link connecting the somatic control of meiotic maturation to germline sex determination and gamete maintenance. Modulatory factors include multiple subunits of a CoREST-like complex and the TWK-1 two-pore potassium channel. These factors are not absolutely required for meiotic maturation or its negative regulation in the absence of sperm, but function cumulatively to enable somatic control of meiotic maturation. This work provides insights into the genetic control of meiotic maturation signaling in C. elegans, and the conserved factors identified here might inform analysis in other systems through either homology or analogy.     

A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature

DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold.     

Crystal Structure of Human RNA Helicase A (DHX9): Structural Basis for Unselective Nucleotide Base Binding in a DEAD-Box Variant Protein

RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long α-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5′-triphosphate, cytidine 5′-triphosphate, and uridine 5′-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.     

Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif

DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding.     

Identification and analysis of LNO1-Like and AtGLE1-Like Nucleoporins in plants

Nucleoporins (Nups) are building blocks of the nuclear pore complex (NPC) that mediate cargo trafficking between the nucleus and the cytoplasm. Although the physical structure of the NPC is well studied in yeast and vertebrates, little is known about the structure of NPCs or the function of most Nups in plants. Recently we demonstrated two Nups in Arabidopsis: LONO1 (LNO1), homolog of human NUP214 and yeast Nup159, and AtGLE1, homolog of yeast Gle1, are required for early embryogenesis and seed development. To identify LNO1 and AtGLE1 homologs in other plant species, we searched the protein databases and identified 30 LNO1-like and 35 AtGLE1-like proteins from lower plant species to higher plants. Furthermore, phylogenetic analyses indicate that the evolutionary trees of these proteins follow expected plant phylogenies. High sequence homology and conserved domain structure of these nucleoporins suggest important functions of these proteins in nucleocytoplasmic transport, growth and development in plants.     

Intra- and Intermolecular Regulatory Interactions in Upf1, the RNA Helicase Central to Nonsense-Mediated mRNA Decay in Yeast

RNA helicases are involved in almost every aspect of RNA metabolism, yet very little is known about the regulation of this class of enzymes. In Saccharomyces cerevisiae, the stability and translational fidelity of nonsense-containing mRNAs are controlled by the group I RNA helicase Upf1 and the proteins it interacts with, Upf2 and Upf3. Combining the yeast two-hybrid system with genetic analysis, we show here that the cysteine- and histidine-rich (CH) domain and the RNA helicase domain of yeast Upf1 can engage in two new types of molecular interactions: an intramolecular interaction between these two domains and self-association of each of these domains. Multiple observations indicate that these molecular interactions are crucial for Upf1 regulation. First, coexpression of the CH domain and the RNA helicase domain in trans can reconstitute Upf1 function in both promoting nonsense-mediated mRNA decay (NMD) and preventing nonsense suppression. Second, mutations that disrupt Upf1 intramolecular interaction cause loss of Upf1 function. These mutations weaken Upf2 interaction and, surprisingly, promote Upf1 self-association. Third, the genetic defects resulting from deficiency in Upf1 intramolecular interaction or RNA binding are suppressed by expression of Upf2. Collectively, these data reveal a set of sequential molecular interactions and their roles in regulating Upf1 function during activation of NMD and suggest that cis intramolecular interaction and trans self-association may be general mechanisms for regulation of RNA helicase functions.     

GTPase Activity of MxB Contributes to Its Nuclear Location,Interaction with Nucleoporins and Anti-HIV-1 Activity

The human myxovirus resistance 2(Mx2/Mx B) protein, a member of interferon(IFN)-inducible dynamin-like large GTPases, restricts a number of virus infections. Inhibition of these viruses occurs at poorly-defined steps after viral entry and has a common requirement for Mx B oligomerization. However, the GTPase activity is essential for the anti-viral effects of Mx B against herpesviruses and HBV but not HIV-1. To understand the role of Mx B GTPase activity, including GTP binding and GTP hydrolysis, in restriction of HIV-1 infection, we genetically separated these two functions and evaluated their contributions to restriction. We found that both the GTP binding and hydrolysis function of Mx B involved in the restriction of HIV-1 replication. The GTPase activity of Mx B contributed to its nuclear location, interaction with nucleoporins(NUPs) and HIV-1 capsids. Furthermore, Mx B disrupted the association between NUPs and HIV-1 cores dependently upon its GTPase activity. The function of GTPase activity was therefore multi-faceted, led to fundamentally distinct mechanisms employed by wild-type Mx B and GTPase activity defective Mx B mutations to restrict HIV-1 replication.