Analog sensitive chemical inhibition of the DEAD‐box protein DDX3

Proper maintenance of RNA structure and dynamics is essential to maintain cellular health. Multiple families of RNA chaperones exist in cells to modulate RNA structure, RNA–protein complexes, and RNA granules. The largest of these families is the DEAD‐box proteins, named after their catalytic Asp‐Glu‐Ala‐Asp motif. The human DEAD‐box protein DDX3 is implicated in diverse biological processes including translation initiation and is mutated in numerous cancers. Like many DEAD‐box proteins, DDX3 is essential to cellular health and exhibits dosage sensitivity, such that both decreases and increases in protein levels can be lethal. Therefore, chemical inhibition would be an ideal tool to probe the function of DDX3. However, most DEAD‐box protein active sites are extremely similar, complicating the design of specific inhibitors. Here, we show that a chemical genetic approach best characterized in protein kinases, known as analog‐sensitive chemical inhibition, is viable for DDX3 and possibly other DEAD‐box proteins. We present an expanded active‐site mutant that is tolerated in vitro and in vivo, and is sensitive to chemical inhibition by a novel bulky inhibitor. Our results highlight a course towards analog sensitive chemical inhibition of DDX3 and potentially the entire DEAD‐box protein family.     

蕨类植物问荆DEAD box RNA解旋酶基因的克隆与分析

DEAD box RNA解旋酶参与RNA多方面的代谢,在植物生长发育和逆境反应中起重要作用。本研究从蕨类植物问荆(Equisetum arvense)中克隆到一条DEAD box RNA解旋酶cDNA全长序列,命名为EaRH1,并在GenBank注册登记（KJ734026）。序列分析显示：该cDNA全长3230bp,包含一个从487bp到2799bp编码770个氨基酸的开放读码框,其对应的蛋白序列包含9个保守模块结构。EaRH1与其它物种DEAD box RNA 解旋酶蛋白序列比对结果显示：模块Ⅰa、Ⅱ和Ⅲ序列几乎完全相同,模块Q、Ⅰ和 Ⅳ序列存在一些差异。EaRH1与江南卷柏(Selaginella moellendorffii)基因组一条假定序列相似度高达69%,其中相似度最高的区域集中在包含9个保守模块的结构域。系统进化树分析显示：EaRH1与拟南芥(Arabidopsis thaliana)DEAD box RNA解旋酶At3g22320在氨基酸序列上有相对较高的同源性。序列结构比较和进化分析可推测出EaRH1可能参与植物体生长发育、miRNA生物合成、与RNA结合蛋白的相互作用和非生物胁迫应答。本文的研究为探索问荆DEAD box RNA解旋酶的进一步功能提供参考。     

Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes

Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5’ to 3’ exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.     

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.     

DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint.     

Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog

Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp‐Glu‐Ala‐Asp; DExD/H) box‐type helicases in mammals, among which retinoic acid‐inducible gene 1 (RIG‐I) and melanoma differentiation‐associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG‐I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN‐inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double‐stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG‐I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG‐I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG‐I, the RNA‐sensing system of chicken lacks RIG‐I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG‐I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens.     

Hepatitis C virus core protein abrogates the DDX3 function that enhances IPS-1-mediated IFN-beta induction

The DEAD box helicase DDX3 assembles IPS-1 (also called Cardif, MAVS, or VISA) in non-infected human cells where minimal amounts of the RIG-I-like receptor (RLR) protein are expressed. DDX3 C-terminal regions directly bind the IPS-1 CARD-like domain as well as the N-terminal hepatitis C virus (HCV) core protein. DDX3 physically binds viral RNA to form IPS-1-containing spots, that are visible by confocal microscopy. HCV polyU/UC induced IPS-1-mediated interferon (IFN)-beta promoter activation, which was augmented by co-transfected DDX3. DDX3 spots localized near the lipid droplets (LDs) where HCV particles were generated. Here, we report that HCV core protein interferes with DDX3-enhanced IPS-1 signaling in HEK293 cells and in hepatocyte Oc cells. Unlike the DEAD box helicases RIG-I and MDA5, DDX3 was constitutively expressed and colocalized with IPS-1 around mitochondria. In hepatocytes (O cells) with the HCV replicon, however, DDX3/IPS-1-enhanced IFN-beta-induction was largely abrogated even when DDX3 was co-expressed. DDX3 spots barely merged with IPS-1, and partly assembled in the HCV core protein located near the LD in O cells, though in some O cells IPS-1 was diminished or disseminated apart from mitochondria. Expression of DDX3 in replicon-negative or core-less replicon-positive cells failed to cause complex formation or LD association. HCV core protein and DDX3 partially colocalized only in replicon-expressing cells. Since the HCV core protein has been reported to promote HCV replication through binding to DDX3, the core protein appears to switch DDX3 from an IFN-inducing mode to an HCV-replication mode. The results enable us to conclude that HCV infection is promoted by modulating the dual function of DDX3.     

The expression of RNA helicase DDX5 is transcriptionally upregulated by calcitriol through a vitamin D response element in the proximal promoter in SiHa cervical cells

Molecular and Cellular Biochemistry - The DEAD box RNA helicase DDX5 is a multifunctional protein involved in the regulatory events of gene expression. Herein, we presented evidence indicating that...     

O‐GlcNAcylation promotes colorectal cancer progression by regulating protein stability and potential catcinogenic function of DDX5

The RNA helicase p68 (DDX5), a key player in RNA metabolism, belongs to the DEAD box family and is involved in the development of colorectal cancer. Here, we found both DDX5 and O‐GlcNAcylation are up‐regulated in colorectal cancer. In addition, DDX5 protein level is significantly positively correlated with the expression of O‐GlcNAcylation. Although it was known DDX5 protein could be regulated by post‐translational modification (PTM), how O‐GlcNAcylation modification regulated of DDX5 remains unclear. Here we show that DDX5 interacts directly with OGT in the SW480 cell line, which is the only known enzyme that catalyses O‐GlcNAcylation in humans. Meanwhile, O‐GlcNAcylation could promote DDX5 protein stability. The OGT‐DDX5 axis affects colorectal cancer progression mainly by regulating activation of the AKT/mTOR signalling pathway. Taken together, these results indicated that OGT‐mediated O‐GlcNAcylation stabilizes DDX5, promoting activation of the AKT/mTOR signalling pathway, thus accelerating colorectal cancer progression. This study not only reveals the novel functional of O‐GlcNAcylation in regulating DDX5, but also reveals the carcinogenic effect of the OGT‐DDX5 axis in colorectal cancer.     

GABA<Subscript>A</Subscript> receptor-associated protein (GABARAP) induces apoptosis by interacting with DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 47 (DDX 47)

GABA_A receptor-associated protein (GABARAP) is a 14-kDa cytoplasmic protein initially identified as a molecular chaperone for GABA_A receptor in cortical neurons. However, evidence indicates that the function of GABARAP is much broader than a specific role in neuronal cells. As an initial step to define the biological role of GABARAP, we searched for binding partners of GABARAP using co-immunoprecipitation coupled with LC-MS/MS analysis. As a result, DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 47 (DDX 47), recently identified as RNA helicase, is identified as a binding partner of GABARAP. An interaction between GABARAP and DDX 47 was further confirmed by yeast two-hybrid systems. Further, co-transfection of GABARAP and DDX47 cDNA into a tumor cell line induces apoptosis. This result may be the first evidence showing that GABARAP and DDX 47 are involved in the apoptotic process.