Nuclear protein p68 is an RNA-dependent ATPase.

The human nuclear antigen p68 cross reacts with a monoclonal antibody to SV40 large-T antigen. Its deduced amino acid sequence contains short motifs which place it in a large superfamily of proteins of known or putative helicase activity. Recently, a p68 subfamily (DEAD box proteins) which share more extensive regions of homology has been identified in mouse, Drosophila, Saccharomyces cerevisiae and Escherichia coli. These proteins are involved in translation, ribosome assembly, mitochondrial splicing, spermatogenesis and embryogenesis. We show here that immunopurified human p68 has RNA dependent ATPase activity. In addition, we show that the protein undergoes dramatic changes in cellular location during the cell cycle.     

Identification of a novel human member of the DEAD box protein family

The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM). From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family. DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division. RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family. Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets. In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated.     

p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts.

The human p68 protein is an RNA-dependent ATPase and RNA helicase which was first identified because of its immunological cross-reaction with a viral RNA helicase, simian virus 40 large T antigen. It belongs to a recently discovered family of proteins (DEAD box proteins) that share extensive regions of amino acid sequence homology, are ubiquitous in living organisms, and are involved in many aspects of RNA metabolism, including splicing, translation, and ribosome assembly. We have shown by immunofluorescent microscopy that mammalian p68, which is excluded from the nucleoli during interphase, translocates to prenucleolar bodies during telophase. We have cloned 55% identical genes from both Schizosaccharomyces pombe and Saccharomyces cerevisiae and shown that they are essential in both yeasts. The human and yeast genes contain a large intron whose position has been precisely conserved. In S. cerevisiae, the intron is unusual both because of its size and because of its location near the 3' end of the gene. We discuss possible functional roles for such an unusual intron in an RNA helicase gene.     

p72: a human nuclear DEAD box protein highly related to p68.

P72, a novel human member of the DEAD box family of putative RNA-dependent ATPases and ATP-dependent RNA helicases was isolated from a HeLa cDNA library. The predicted amino acid sequence of p72 is highly homologous to that of the prototypic DEAD box protein p68. In addition to the conserved core domains characteristic of DEAD box proteins, p72 contains several N-terminal RGG RNA-binding domains and a serine/glycine rich C-terminus likely involved in mediating protein-protein interactions. A p72-specific probe detects two mRNAs of approximately 5300 and 9300 bases which, although ubiquitously expressed, show variability in their expression levels in different tissues. Purified recombinant p72 exhibits ATPase activity in the presence of a range of RNA moieties. Immunocytochemical studies of p68 and p72 show that these proteins localise to similar locations in the nucleus of HeLa cells, suggesting their involvement in a nuclear process.     

Ddx42p--a human DEAD box protein with RNA chaperone activities

The human gene ddx42 encodes a human DEAD box protein highly homologous to the p68 subfamily of RNA helicases. In HeLa cells, two ddx42 poly(A)⁺ RNA species were detected both encoding the nuclear localized 938 amino acid Ddx42p polypeptide. Ddx42p has been heterologously expressed and its biochemical properties characterized. It is an RNA binding protein, and ATP and ADP modulate its RNA binding affinity. Ddx42p is an NTPase with a preference for ATP, the hydrolysis of which is enhanced by various RNA substrates. It acts as a non-processive RNA helicase. Interestingly, RNA unwinding by Ddx42p is promoted in the presence of a single-strand (ss) binding protein (T4gp32). Ddx42p, particularly in the ADP-bound form (the state after ATP hydrolysis), also mediates efficient annealing of complementary RNA strands thereby displacing the ss binding protein. Ddx42p therefore represents the first example of a human DEAD box protein possessing RNA helicase, protein displacement and RNA annealing activities. The adenosine nucleotide cofactor bound to Ddx42p apparently acts as a switch that controls the two opposing activities: ATP triggers RNA strand separation, whereas ADP triggers annealing of complementary RNA strands.     

The ATPase, RNA unwinding, and RNA binding activities of recombinant p68 RNA helicase

p68 RNA helicase, a nuclear RNA helicase, was identified 2 decades ago. The protein plays very important roles in cell development and organ maturation. However, the biological functions and enzymology of p68 RNA helicase are not well characterized. We report the expression and purification of recombinant p68 RNA helicase in a bacterial system. The recombinant p68 is an ATP-dependent RNA helicase. ATPase assays demonstrated that double-stranded RNA (dsRNA) is much more effective than single-stranded RNA in stimulating ATP hydrolysis by the recombinant protein. Consistently, RNA-binding assays showed that p68 RNA helicase binds single-stranded RNA weakly in an ATP-dependent manner. On the other hand, the recombinant protein has very high affinity for dsRNA. Binding of the protein to dsRNA is ATP-independent. The data indicate that p68 may directly target dsRNA as its natural substrate. Interestingly, the recombinant p68 RNA helicase unwinds dsRNA in both 3' --> 5' and 5' --> 3' directions. This is the second example of a Asp-Glu-Ala-Asp (DEAD) box RNA helicase that unwinds RNA duplexes in a bi-directional manner.     

Allosteric activation of the ATPase activity of the Escherichia coli RhlB RNA helicase

Helicase B (RhlB) is one of the five DEAD box RNA-dependent ATPases found in Escherichia coli. Unique among these enzymes, RhlB requires an interaction with the partner protein RNase E for appreciable ATPase and RNA unwinding activities. To explore the basis for this activating effect, we have generated a di-cistronic vector that overexpresses a complex comprising RhlB and its recognition site within RNase E, corresponding to residues 696-762. Complex formation has been characterized by isothermal titration calorimetry, revealing an avid, enthalpy-favored interaction between the helicase and RNase E-(696-762) with an equilibrium binding constant (Ka) of at least 1 x 10(8) m(-1). We studied ATPase activity of mutants with substitutions within the ATP binding pocket of RhlB and on the putative interaction surface that mediates recognition of RNase E. For comparisons, corresponding mutations were prepared in two other E. coli DEAD box ATPases, RhlE and SrmB. Strikingly, substitutions at a phenylalanine near the Q-motif found in DEAD box proteins boosts the ATPase activity of RhlB in the absence of RNA, but completely inhibits it in its presence. The data support the proposal that the protein-protein and RNA-binding surfaces both communicate allosterically with the ATPase catalytic center. We conjecture that this communication may govern the mechanical power and efficiency of the helicases, and is tuned in individual helicases in accordance with cellular function.     

恶性疟原虫abstrakt同源基因的克隆及DEAD盒家族蛋白的序列分析

DEAD盒蛋白家族的ATP依赖性的RNA解旋酶类参与细胞内几乎所有的RNA代谢过程 ,在几乎所有生物的细胞生长发育过程中扮演着众多不可或缺的角色。在本实验中 ,通过PCR和探针杂交相结合的筛选方法 ,筛选恶性疟原虫 (Plasmodiumfalciparum)的基因组文库 ,克隆了FH1F———abstrakt同源基因的完整序列。通过搜索已经完成测序的恶性疟原虫基因组数据库 ,推测FH1F序列定位在第 5条染色体上。FH1F全长2 80 4bp,包含一个 1 1 6 1bp的完整阅读框 ,编码一个由 386个氨基酸组成的蛋白。对FH1F蛋白序列用BlastP进行搜索和分析以及用DNAStar与许多典型的DEAD盒蛋白序列进行比对分析 ,结果均提示FH1F蛋白应该是DEAD盒家族的一个Abstrakt蛋白。另一方面 ,用DNAStar对已知所有完整的DEAD盒蛋白进行详细的序列分析以及用Mega对这些序列进行系统发育研究的结果都显示 :DEAD盒家族的蛋白聚类成为若干不同的亚群 ;与DEAD盒蛋白的一般保守序列相比 ,Abstrakt,eIF 4A ,Vasa ,P6 8等不同亚群的DEAD盒蛋白在保守区具有各自不同的结构特征。本文对不同的DEAD盒蛋白的结构特征进行了总结并试图给出不同亚群分类上的结构标准 ,对Abstrakt蛋白在本应高度保守的位点上异常于其它DEAD盒蛋白的氨基酸残基的取代也进行了相关的初步分析     

Interaction of the Escherichia coli DEAD box protein DbpA with 23 S ribosomal RNA.

The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA. We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity. Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted. These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA. We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested.     

Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.     

P68 RNA helicase mediates PDGF-induced epithelial mesenchymal transition by displacing Axin from beta-catenin

The nuclear p68 RNA helicase (referred to as p68) is a prototypical member of the DEAD box family of RNA helicases. The protein plays a very important role in early organ development. In the present study, we characterized the tyrosine phosphorylation of p68 under platelet-derived growth factor (PDGF) stimulation. We demonstrated that tyrosine phosphorylation of p68 at Y593 mediated PDGF-stimulated epithelial-mesenchymal transition (EMT). We showed that PDGF treatment led to phosphorylation of p68 at Y593 in the cell nucleus. The Y593-phosphorylated p68 (referred to as phosphor-p68) promotes beta-catenin nuclear translocation via a Wnt-independent pathway. The phosphor-p68 facilitates beta-catenin nuclear translocation by blocking phosphorylation of beta-catenin by GSK-3beta and displacing Axin from beta-catenin. The beta-catenin nuclear translocation and subsequent interaction with the LEF/TCF was required for the EMT process. These data demonstrated a novel mechanism of phosphor-p68 in mediating the growth factor-induced EMT and uncovered a new pathway to promote beta-catenin nuclear translocation.     

p68 DEAD box RNA helicase expression in keratinocytes. Regulation, nucleolar localization, and functional connection to proliferation and vascular endothelial growth factor gene expression

Nitric oxide (NO) represents a short lived mediator that pivotally drives keratinocyte movements during cutaneous wound healing. In this study, we have identified p68 DEAD box RNA helicase (p68) from an NO-induced differential keratinocyte cDNA library. Subsequently, we have analyzed regulation of p68 by wound-associated mediators in human and murine keratinocytes. NO, serum, growth factors, and pro-inflammatory cytokines were potent inducers of p68 expression in the cells. p68 was constitutively expressed in the epithelial compartment of murine skin. Upon injury, we found a transient down-regulation of overall p68 protein in wound tissue. However, p68 did not completely disappear during early wound repair, as we found an expression of p68 protein in isolated wound margin tissue 24 h after wounding. Moreover, immunohistochemistry and cell fractionation analysis revealed a restricted localization of p68 in keratinocyte nuclei of the developing epithelium. Accordingly, cultured keratinocytes also showed a nuclear localization of the helicase. Moreover, confocal microscopy revealed a strong localization of p68 protein within the nucleoli of the cells. Functional analyses demonstrated that p68 strongly participated in keratinocyte proliferation and gene expression. Keratinocytes that constitutively overexpressed p68 protein were characterized by a marked increase in serum-induced proliferation and vascular endothelial growth factor expression, whereas down-regulation of endogenous p68 using small interfering RNA markedly attenuated serum-induced proliferation and vascular endothelial growth factor expression. Altogether, our results suggest a tightly controlled expression and nucleolar localization of p68 in keratinocytes in vitro and during skin repair in vivo that functionally contributes to keratinocyte proliferation and gene expression.     

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。     

rhlB, a new Escherichia coli K-12 gene with an RNA helicase-like protein sequence motif, one of at least five such possible genes in a prokaryote

A newly recognized gene we name rhlB, after RNA helicase-like genes, has been found in the 85-minute region of the Escherichia coli chromosome. This gene encodes protein sequence motifs similar to those known for "D-E-A-D box" gene products. Proteins in this gene family occur in eukaryotes as well as prokaryotes, and, as far as tested, have been found to participate in ATP-dependent RNA helicase or RNA-dependent ATPase activities. The functions of these enzymes are poorly understood. In yeast, mutant phenotypes of various D-E-A-D genes suggest that they function in RNA splicing, processing, or translation. We find that rhlB is necessary for viability only in some genetic backgrounds. Conditional rhlB lethality is not complemented by another E. coli RNA helicase-like gene (srmB). Using primers with homology to consensus sequences in D-E-A-D box proteins, we have recovered DNA fragments amplified from E. coli genomic DNA by polymerase chain reactions. Sequence analysis of these fragments suggests that E. coli possesses at least five RNA helicase-like (rhl) D-E-A-D box genes at widely separated chromosomal locations. The multiplicity of such genes in a prokaryote raises the possibility of important roles for the corresponding class of biologically widespread proteins.     

Hepatitis C virus core protein binds to a DEAD box RNA helicase.

Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation.