Molecular cloning,functional characterization and antiviral activity of porcine DDX3X

Human DDX3X is a newly discovered DEAD-box RNA helicase. In addition to involvement of eukaryotic gene expression regulation, human DDX3X has recently been demonstrated to be a critical molecule in innate immune signaling pathways and to contribute to type I interferon (IFN) induction. In the present study, porcine DDX3X was cloned by RT-PCR from PK-15 cells and its function in regulating IFN-β was characterized. The putative porcine DDX3X ORF encodes 662 amino acids possessing several conserved motifs. Sequence alignments indicated that porcine DDX3X has high identity at the amino acid level to those of horse (96.7%), mouse (97.6%), cattle (98.5%), dog (98.6%) and human (98.9%). Ectopic expression of porcine DDX3X significantly activated IFN-β expression, whereas knockdown of porcine DDX3X inhibited dsRNA- or Sendai virus (SeV)-induced IFN-β. Furthermore, porcine DDX3X co-localized with IPS-1, TBK1 and IKKε, and enhanced IFN-β promoter activation induced by these molecules. We also investigated the role of porcine DDX3X during porcine reproductive and respiratory syndrome virus (PRRSV) infection and found that overexpression of DDX3X significantly inhibited PRRSV replication, indicating that DDX3X is a potential antiviral agent.     

人DDX36和小鼠Ddx36基因在成年睾丸组织中的表达研究

果蝇是结构基因组学和功能基因组学研究的最为理想的一种模式生物,采用同源克隆的策略,应用生物信息学分析和实验技术相结合的方法分别从人和小鼠中克隆了同源于果蝇MLE蛋白的新基因DDX36和Ddx36。为进一步研究DDX36和Ddx36基因与精子发生的关系,再应用Northrn blotting,RT-PCR和组织原位杂交技术探讨了DDX36和Ddx36基因的表达情况,结果发现人DDX36和小鼠Ddx36基因在成年睾丸组织中高表达。初步证明DDX36和Ddx36基因在精子发生中亦可能发挥重要作用。     

Gene structure of the human DDX3 and chromosome mapping of its related sequences.

The human DDX3 gene (GenBank accession No. U50553) is the human homologue of the mouse Ddx3 gene and is a member of the gene family that contains DEAD motifs. Previously, we mapped the gene to the Xp11.3-11.23. In this report, we describe the structural organization of the human DDX3 gene. It consisted of 17 exons that span approximately 16 kb. An Alu element was present in the intron 13. Its organization was the same as that of the human DBY gene, a closely related sequence present on the Y chromosome. We also identified two processed pseudogenes (DDX3) with a sequence that is highly homologous to those of DDX3 cDNAs, but contain a translation termination codon within its open-reading frame. Pseudogenes are mapped on human chromosomes 4 and X, respectively. In this paper, we discuss the relationships between DDX3 and its related sequences that have been isolated.     

The human DDX and DHX gene families of putative RNA helicases

Nucleic acid helicases are characterized by the presence of the helicase domain containing eight motifs. The sequence of the helicase domain is used to classify helicases into families. To identify members of the DEAD and DEAH families of human RNA helicases, we used the helicase domain sequences to search the nonredundant peptide sequence database. We report the identification of 36 and 14 members of the DEAD and DEAH families of putative RNA helicases, including several novel genes. The gene symbol DDX had been used previously for both DEAD- and DEAH-box families. We have now adopted DDX and DHX symbols to denote DEAD- and DEAH-box families, respectively. Members of human DDX and DHX families of putative RNA helicases play roles in differentiation and carcinogenesis.     

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.     

A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication

DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.     

DDX21 is a p38-MAPK-sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification

Successful navigation of the mouse preimplantation stages of development, during which three distinct blastocyst lineages are derived, represents a prerequisite for continued development. We previously identified a role for p38-mitogen-activated kinases (p38-MAPK) regulating blastocyst inner cell mass (ICM) cell fate, specifically primitive endoderm (PrE) differentiation, that is intimately linked to rRNA precursor processing, polysome formation and protein translation regulation. Here, we develop this work by assaying the role of DEAD-box RNA helicase 21 (DDX21), a known regulator of rRNA processing, in the context of p38-MAPK regulation of preimplantation mouse embryo development. We show nuclear DDX21 protein is robustly expressed from the 16-cell stage, becoming exclusively nucleolar during blastocyst maturation, a localization dependent on active p38-MAPK. siRNA-mediated clonal Ddx21 knockdown within developing embryos is associated with profound cell-autonomous and non-autonomous proliferation defects and reduced blastocyst volume, by the equivalent peri-implantation blastocyst stage. Moreover, ICM residing Ddx21 knockdown clones express the EPI marker NANOG but rarely express the PrE differentiation marker GATA4. These data contribute further significance to the emerging importance of lineage-specific translation regulation, as identified for p38-MAPK, during mouse preimplantation development.     

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.     

Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1

RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1′s enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an ‘open’ to a ‘closed’-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change.     

Cloning and expression analysis of the chicken DEAD box gene DDX1

DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. While some DEAD box genes expressed in higher eukaryotes are ubiquitous, others have distribution profiles that suggest a cell-, tissue-, or developmental-specific role. The DEAD box gene, DDX1, was identified by differential screening of a subtracted retinoblastoma cDNA library. A limited survey of human fetal tissues indicated that DDX1 mRNA has a widespread distribution but is not uniformly expressed in all tissues. To further document the spatial and temporal distribution of DDX1 during embryonic development, we cloned the chicken DDX1 cDNA. The predicted amino acid sequence of chicken DDX1 was 93% identical to that of human DDX1. All DEAD box motifs, as well as a SPRY domain, were present in chicken DDX1. Northern and Western blot analyses showed highest levels of DDX1 at early stages of development. Tissue maturation was generally accompanied by a decrease in expression, although DDX1 levels remained elevated in late embryonic retina and brain. In situ hybridization of retinal tissue sections revealed widespread distribution of DDX1 mRNA at early developmental stages with preferential expression in amacrine and ganglion cells of the differentiated tissue. Preferential expression of DDX1 was also observed in specific areas of the brain in older embryos, such as the external granule layer of the cerebellum. These results suggest a specific role for DDX1 in subsets of differentiated cells as well as a more general role in undifferentiated cells.     

The Ded1/DDX3 subfamily of DEAD-box RNA helicases

Abstract

In eukaryotic organisms, the orthologs of the DEAD-box RNA helicase Ded1p from yeast and DDX3 from human form a well-defined subfamily that is characterized by high sequence conservation in their helicase core and their N- and C- termini. Individual members of this Ded1/DDX3 subfamily perform multiple functions in RNA metabolism in both nucleus and cytoplasm. Ded1/DDX3 subfamily members have also been implicated in cellular signaling pathways and are targeted by diverse viruses. In this review, we discuss the considerable body of work on the biochemistry and biology of these proteins, including the recently discovered link of human DDX3 to tumorigenesis.     

Investigating nucleo-cytoplasmic shuttling of the human DEAD-box helicase DDX3

The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3’s nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3’s subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3’s subcellular distribution as determined by High Content Analysis. However, DDX3’s nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer.