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RNA Helicase (rna + helicase)
Terms modified by RNA Helicase Selected AbstractsmRNA Encoding a Putative RNA Helicase of the DEAD-Box Gene Family is Up-Regulated in Trypomastigotes of Trypanosoma cruziTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2000ALBERTO M. DÍAZ AÑEL ABSTRACT. Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands. [source] Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domainsFEBS JOURNAL, Issue 2 2007Anne R. Karow RNA helicases mediate structural rearrangements of RNA or RNA,protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis,Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication. [source] Leishmania infantum LeIF protein is an ATP-dependent RNA helicase and an eIF4A-like factor that inhibits translation in yeastFEBS JOURNAL, Issue 22 2006Mourad Barhoumi LeIF, a Leishmania protein similar to the eukaryotic initiation factor eIF4A, which is a prototype of the DEAD box protein family, was originally described as a Th1-type natural adjuvant and as an antigen that induces an IL12-mediated Th1 response in the peripheral blood mononuclear cells of leishmaniasis patients. This study aims to characterize this protein by comparative biochemical and genetic analysis with eIF4A in order to assess its potential as a target for drug development. We show that a His-tagged, recombinant, LeIF protein of Leishmania infantum, which was purified from Escherichia coli, is both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described previously for other members of the DEAD box helicase protein family. In vivo experiments show that the LeIF gene cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. In contrast, expression of LeIF inhibits yeast growth when endogenous eIF4A is expressed off only one of its two encoding genes. Furthermore, in vitro binding assays show that LeIF interacts with yeast eIF4G. These results show an unproductive interaction of LeIF with translation initiation factors in yeast. Furthermore, the 25 amino terminal residues were shown to enhance the ability of LeIF to interfere with the translation machinery in yeast. [source] Changes in Drosophila melanogaster midgut proteins in response to dietary Bowman,Birk inhibitorINSECT MOLECULAR BIOLOGY, Issue 5 2007H.-M. Li Abstract The midgut proteome of Drosophila melanogaster was compared in larvae fed dietary Bowman,Birk inhibitor (BBI) vs. larvae fed a control diet. By using two-dimensional gel electrophoresis, nine differentially expressed proteins were observed, which were associated with enzymes or transport functions such as sterol carrier protein X (SCPX), ubiquitin-conjugating enzyme, endopeptidase, receptor signalling protein kinase, ATP-dependent RNA helicase and ,-tocopherol transport. Quantitative real-time PCR verified differential expression of transcripts coding for six of the proteins observed from the proteomic analysis. BBI evidently affects expression of proteins associated with protein degradation, transport and fatty acid catabolism. We then tested the hypothesis that SCPX was critical for the Drosophila third instars' response to BBI treatment. Inhibition of SCPX caused the third instars to become more susceptible to dietary BBI. [source] The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complexMOLECULAR MICROBIOLOGY, Issue 4 2010Martin Lehnik-Habrink Summary In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram-positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein,protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA-like domains that are found in all DEAD box RNA helicases and a C-terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C-terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome. [source] Interaction of the plant glycine-rich RNA-binding protein MA16 with a novel nucleolar DEAD box RNA helicase protein from Zea maysTHE PLANT JOURNAL, Issue 6 2004Elisenda Gendra Summary The maize RNA-binding MA16 protein is a developmentally and environmentally regulated nucleolar protein that interacts with RNAs through complex association with several proteins. By using yeast two-hybrid screening, we identified a DEAD box RNA helicase protein from Zea mays that interacted with MA16, which we named Z. maysDEAD box RNA helicase 1 (ZmDRH1). The sequence of ZmDRH1 includes the eight RNA helicase motifs and two glycine-rich regions with arginine,glycine-rich (RGG) boxes at the amino (N)- and carboxy (C)-termini of the protein. Both MA16 and ZmDRH1 were located in the nucleus and nucleolus, and analysis of the sequence determinants for their cellular localization revealed that the region containing the RGG motifs in both proteins was necessary for nuclear/nucleolar localization The two domains of MA16, the RNA recognition motif (RRM) and the RGG, were tested for molecular interaction with ZmDRH1. MA16 specifically interacted with ZmDRH1 through the RRM domain. A number of plant proteins and vertebrate p68/p72 RNA helicases showed evolutionary proximity to ZmDRH1. In addition, like p68, ZmDRH1 was able to interact with fibrillarin. Our data suggest that MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA (rRNA) metabolism. [source] Characterisation of gene expression in bovine adipose tissue before and after fatteningANIMAL GENETICS, Issue 3 2000M Oishi Summary It has been reported that fattening causes bovine adipose tissue development associated with an enlargement in adipocyte cell size. As a first study to elucidate mechanisms of bovine adipose tissue development during fattening, our experiment was designed to characterise gene expression in bovine adipose tissue before and after fattening. We randomly isolated a large number of cDNA clones derived from bovine adipose tissue before and after fattening. Sequence analysis of the isolated clones showed that 3 and 10 clones from before and after fattening, respectively, correspond to genes related to adipocyte development and/or function in the adipose tissue. In addition, we isolated cDNA clones that possess negative signal by hybridising the cDNA population from the adipose tissue after fattening with that before fattening as a probe. As a result, we identified five types of transcripts observed in the adipose tissue after fattening but not before fattening. Two of the five are likely to encode bovine orthologs of phospholipase A2 and RNA helicase p68, while the other three represent unknown genes. Further functional investigation of the identified genes might lead to elucidation of mechanisms of bovine adipose tissue development during fattening. [source] RNA helicase encoded by melanoma differentiation,associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease,ARTHRITIS & RHEUMATISM, Issue 7 2009Shinji Sato Objective To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods An anti,CADM-140 antibody,positive prototype serum sample was used to screen a HeLa cell,derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro,transcribed and in vitro,translated products using anti,CADM-140 antibody,positive and anti-CADM-140 antibody,negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti,CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases. Results By cDNA library screening and immunoprecipitation of in vitro,transcribed and in vitro,translated products, we obtained a cDNA clone encoding melanoma differentiation,associated gene 5 (MDA-5). The anti,CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti,CADM-140 antibody routinely available. [source] Crystallization and preliminary characterization of the Thermus thermophilus RNA helicase Hera C-terminal domainACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Markus G. Rudolph Heat-resistant RNA-dependent ATPase (Hera) from Thermus thermophilus is a DEAD-box RNA helicase. Two constructs encompassing the second RecA-like domain and the C-terminal domain of Hera were overproduced in Escherichia coli and purified to homogeneity. Single crystals of both Hera constructs were obtained in three crystal forms. A tetragonal crystal form belonged to space group P41212, with unit-cell parameters a = 65.5, c = 153.0,Å, and contained one molecule per asymmetric unit. Two orthorhombic forms belonged to space group P212121, with unit-cell parameters a = 62.8, b = 70.9, c = 102.3,Å (form I) and a = 41.6, b = 67.6, c = 183.5,Å (form II). Both orthorhombic forms contained two molecules per asymmetric unit. All crystals diffracted X-rays to beyond 3,Å resolution, but the tetragonal data sets displayed high Wilson B values and high mean |E2, 1| values, indicating potential disorder and anisotropy. The tetragonal crystal was phased by MAD using a single selenium site. [source] Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domainsFEBS JOURNAL, Issue 2 2007Anne R. Karow RNA helicases mediate structural rearrangements of RNA or RNA,protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis,Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication. [source] The DEAD box RNA helicase VBH-1 is required for germ cell function in C. elegansGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 9 2007L. Silvia Salinas Abstract Vasa and Belle are conserved DEAD box RNA helicases required for germ cell function. Homologs of this group of proteins in several species, including mammals, are able to complement a mutation in yeast (DED1) suggesting that their function is highly conserved. It has been proposed that these proteins are required for mRNA translation regulation, but their specific mechanism of action is still unknown. Here we describe functions of VBH-1, a C. elegans protein closely related to Belle and Vasa. VBH-1 is expressed specifically in the C. elegans germline, where it is associated with P granules, the C. elegans germ plasm counterpart. vbh-1(RNAi) animals produce fewer offspring than wild type because of defects in oocyte and sperm production, and embryonic lethality. We also find that VBH-1 participates in the sperm/oocyte switch in the hermaphrodite gonad. We conclude that VBH-1 and its orthologs may perform conserved roles in fertility and development. genesis 45:533,546, 2007. © 2007 Wiley-Liss, Inc. [source] The DEAD-box RNA helicase DDX1 interacts with RelA and enhances nuclear factor kappaB-mediated transcriptionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Musarat Ishaq Abstract DEAD-box RNA helicases constitute the largest family of RNA helicases and are involved in many aspects of RNA metabolism. In this study, we identified RelA (p65), a subunit of nuclear factor-kappaB (NF-,B), as a cellular co-factor of DEAD-box RNA helicase DDX1, through mammalian two hybrid system and co-immunoprecipitation assay. Additionally, confocal microscopy and chromatin immunoprecipitation assays confirmed this interaction. In NF-,B dependent reporter gene assay, DDX1 acted as a co-activator to enhance NF-,B-mediated transcription activation. The functional domains involved were mapped to the carboxy terminal transactivation domain of RelA and the amino terminal ATPase/helicase domain of DDX1. The DDX1 trans-dominant negative mutant lacking ATP-dependent RNA helicase activity lost it transcriptional inducer activity. Moreover, depletion of endogenous DDX1 by specific small interfering RNAs significantly reduced NF-,B-dependent transcription. Taken together, the results suggest that DDX1 may play an important role in NF-,B-mediated transactivation, and revelation of this regulatory pathway may help to explore the novel mechanisms for regulating NF-,B transcriptional activity. J. Cell. Biochem. 106: 296,305, 2009. © 2008 Wiley-Liss, Inc. [source] The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complexMOLECULAR MICROBIOLOGY, Issue 4 2010Martin Lehnik-Habrink Summary In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram-positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein,protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA-like domains that are found in all DEAD box RNA helicases and a C-terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C-terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome. [source] mRNA Encoding a Putative RNA Helicase of the DEAD-Box Gene Family is Up-Regulated in Trypomastigotes of Trypanosoma cruziTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2000ALBERTO M. DÍAZ AÑEL ABSTRACT. Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands. [source] Interaction of the plant glycine-rich RNA-binding protein MA16 with a novel nucleolar DEAD box RNA helicase protein from Zea maysTHE PLANT JOURNAL, Issue 6 2004Elisenda Gendra Summary The maize RNA-binding MA16 protein is a developmentally and environmentally regulated nucleolar protein that interacts with RNAs through complex association with several proteins. By using yeast two-hybrid screening, we identified a DEAD box RNA helicase protein from Zea mays that interacted with MA16, which we named Z. maysDEAD box RNA helicase 1 (ZmDRH1). The sequence of ZmDRH1 includes the eight RNA helicase motifs and two glycine-rich regions with arginine,glycine-rich (RGG) boxes at the amino (N)- and carboxy (C)-termini of the protein. Both MA16 and ZmDRH1 were located in the nucleus and nucleolus, and analysis of the sequence determinants for their cellular localization revealed that the region containing the RGG motifs in both proteins was necessary for nuclear/nucleolar localization The two domains of MA16, the RNA recognition motif (RRM) and the RGG, were tested for molecular interaction with ZmDRH1. MA16 specifically interacted with ZmDRH1 through the RRM domain. A number of plant proteins and vertebrate p68/p72 RNA helicases showed evolutionary proximity to ZmDRH1. In addition, like p68, ZmDRH1 was able to interact with fibrillarin. Our data suggest that MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA (rRNA) metabolism. [source] | |||||||||||||