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PubMed2006

RNA helicases and abiotic stress.

RNA helicases function as molecular motors that rearrange RNA secondary structure, potentially performing roles in any cellular process involving RNA metabolism. Although RNA helicase association with a range of cellular functions is well documented, their importance in response to abiotic stress is only beginning to emerge. This review summarizes the available data on the expression, biochemistry and physiological function(s) of RNA helicases regulated by abiotic stress. Examples originate primarily from non-mammalian organisms while instances from mammalian sources are restricted to post-translational regulation of helicase biochemical activity. Common emerging themes include the requirement of a cold-induced helicase in non-homeothermic organisms, association and regulation of helicase activity by stress-induced phosphorylation cascades, altered nuclear-cytoplasmic shuttling in eukaryotes, association with the transcriptional apparatus and the diversity of biochemical activities catalyzed by a subgroup of stress-induced helicases. The data are placed in the context of a mechanism for RNA helicase involvement in cellular response to abiotic stress. It is proposed that stress-regulated helicases can catalyze a nonlinear, reversible sequence of RNA secondary structure rearrangements which function in RNA maturation or RNA proofreading, providing a mechanism by which helicase activity alters the activation state of target RNAs through regulation of the reaction equilibrium.

Animals↗
PubMed2006

The RNA helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation.

MyoD regulates skeletal myogenesis. Since proteins associated with MyoD exert regulatory functions, their identification is expected to contribute important insights into the mechanisms governing gene expression in skeletal muscle. We have found that the RNA helicases p68/p72 are MyoD-associated proteins and that the noncoding RNA SRA also immunoprecipitates with MyoD. In vitro and in vivo experiments indicated that both p68/p72 and SRA are coactivators of MyoD. RNA interference toward either p68/p72 or SRA prevented proper activation of muscle gene expression and cell differentiation. Unexpectedly, reducing the levels of p68/p72 proteins impaired recruitment of the TATA binding protein TBP; RNA polymerase II; and the catalytic subunit of the ATPase SWI/SNF complex, Brg-1, and hindered chromatin remodeling. These findings reveal that p68/p72 play a critical role in promoting the assembly of proteins required for the formation of the transcription initiation complex and chromatin remodeling.

Amino Acid Sequence↗
PubMed1998

Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export.

The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae. It is shown here that Dbp5p is an ATP-dependent RNA helicase required for polyadenylated [poly(A)+] RNA export. Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope. This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation. The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.

Amino Acid Sequence↗
PubMed1996

A DEAD-box RNA helicase in the Escherichia coli RNA degradosome.

The Escherichia coli RNA degradosome is a multi-enzyme complex that contains the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. Both enzymes are important in RNA processing and messenger RNA degradation. Here we report that enolase and RhlB are two other major components of the degradosome. Enolase is a glycolytic enzyme with an unknown role in RNA metabolism. RhlB is a member of the DEAD-box family of ATP-dependent RNA helicases, which are found in both prokaryotes and eukaryotes. We show that the degradosome has an ATP-dependent activity that aids the degradation of structured RNA by PNPase. Incubation of the degradosome with affinity-purified antibody against RhlB inhibited the ATP-stimulated RNA degradation. These results suggest that RhlB acts by unwinding RNA structures that impede the processive activity of PNPase. RhlB is thus an important enzyme in mRNA turnover.

Adenosine Triphosphatases↗
PubMed2002

mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties.

Human melanoma cells can be reprogrammed to terminally differentiate and irreversibly lose proliferative capacity by appropriate pharmacological manipulation. Subtraction hybridization identified melanoma differentiation-associated gene-5 (mda-5) as a gene induced during differentiation, cancer reversion, and programmed cell death (apoptosis). This gene contains both a caspase recruitment domain and putative DExH group RNA helicase domains. Atypical helicase motifs of MDA-5 deviate from consensus sequences but are well conserved in a potentially new group of cloned and hypothetical proteins. mda-5 is an early response gene inducible by IFN and tumor necrosis factor-alpha, responding predominantly to IFN-beta. Protein kinase C activation by mezerein further augments mda-5 expression induced by IFN-beta. Expression of mda-5 is controlled transcriptionally by IFN-beta, and the MDA-5 protein localizes in the cytoplasm. mda-5 displays RNA-dependent ATPase activity, and ectopic expression of mda-5 in human melanoma cells inhibits colony formation. In these contexts, mda-5 may function as a mediator of IFN-induced growth inhibition and/or apoptosis. MDA-5 is a double-stranded RNA-dependent ATPase that contains both a caspase recruitment domain and RNA helicase motifs, with a confirmed association with growth and differentiation in human melanoma cells.

Adenosine Triphosphatases↗
PubMed1999

A cold shock-induced cyanobacterial RNA helicase.

The ability to modify RNA secondary structure is crucial for numerous cellular processes. We have characterized two RNA helicase genes, crhB and crhC, which are differentially expressed in the cyanobacterium Anabaena sp. strain PCC 7120. crhC transcription is limited specifically to cold shock conditions while crhB is expressed under a variety of conditions, including enhanced expression in the cold. This implies that both RNA helicases are involved in the cold acclimation process in cyanobacteria; however, they presumably perform different roles in this adaptation. Although both CrhB and CrhC belong to the DEAD box subfamily of RNA helicases, CrhC encodes a novel RNA helicase, as the highly conserved SAT motif is modified to FAT. This alteration may affect CrhC function and its association with specific RNA targets and/or accessory proteins, interactions required for cold acclimation. Primer extension and analysis of the 5' untranslated region of crhC revealed the transcriptional start site, as well as a number of putative cold shock-responsive elements. The potential role(s) performed by RNA helicases in the acclimation of cyanobacteria to cold shock is discussed.

Amino Acid Sequence↗
PubMed2006

The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin.

Terminating a gene's activity requires that pre-existing transcripts be matured or destroyed and that the local chromatin structure be returned to an inactive configuration. Here we show that the Drosophila homolog of the mammalian P68 RNA helicase plays a novel role in RNA export and gene deactivation. p68 mutations phenotypically resemble mutations in small bristles (sbr), the Drosophila homolog of the human mRNA export factor NXF1. Full-length hsp70 mRNA accumulates in the nucleus near its sites of transcription following heat shock of p68 homozygotes, and hsp70 gene shutdown is delayed. Unstressed mutant larvae show similar defects in transcript accumulation and gene repression at diverse loci, and we find that p68 mutations are allelic to Lighten-up, a known suppressor of position effect variegation. Our observations reveal a strong connection between transcript clearance and gene repression. P68 may be needed to rapidly remove transcripts from a gene before its activity can be shut down and its chromatin reset to an inactive state.

Blotting, Northern↗
PubMed2002

An RNA helicase, DDX1, interacting with poly(A) RNA and heterogeneous nuclear ribonucleoprotein K.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional protein known to be involved in the regulation of transcription, translation, nuclear transport, and signal transduction. To systematically obtain insight into mechanisms of hnRNP K activities, we set out to identify protein factors that interact with hnRNP K by using glutathione S-transferase-hnRNP K affinity chromatography followed by liquid chromatography/mass spectrometry/mass spectrometry analysis. Several partner proteins in the K562 cell lysates were identified through this method. One of them is a DEAD box-containing putative RNA helicase, DDX1. In vitro binding and co-immunoprecipitation studies confirmed the protein-protein interaction between hnRNP K with DDX1, and the region spanning amino acids 1-276 of hnRNP K is apparently responsible for its physical interaction with DDX1. Interestingly, their interaction was disrupted by the addition of poly(C), poly(A), and poly(U) RNA substrates. We found that DDX1 was a homopolymeric poly(A) RNA-binding protein. On the other hand, the ATPase activity of the purified recombinant DDX1 protein was stimulated by these homopolymeric RNAs and yeast total RNA but not by DNA. Moreover, the immunoprecipitated DDX1 complex but not purified DDX1 can unwind double-stranded RNA having single-stranded poly(A) overhangs.

Adenosine Triphosphatases↗
PubMed2000

Structural domains involved in the RNA folding activity of RNA helicase II/Gu protein.

RNA helicase II/Gu (RH II/Gu) is a nucleolar protein that unwinds dsRNA in a 5' to 3' direction, and introduces a secondary structure into a ssRNA. The helicase domain is at the N-terminal three-quarters of the molecule and the foldase domain is at the C-terminal quarter. The RNA folding activity of RH II/Gu is not a mere artifact of its binding to RNA. This study narrows down the RNA foldase domain to amino acids 749-801 at the C-terminus of the protein. Dissection of this region by deletion and site-directed mutagenesis shows that the four FRGQR repeats, as well as the C-terminal end bind RNA independently. These juxtaposed subdomains are both important for the RNA foldase activity of RH II/Gu. Mutation of either repeat 2 or repeat 4, or simultaneous mutation of Lys792, Arg793 and Lys797 at the C-terminal end of RH II/Gu to alanines inhibits RNA foldase activity. The last 17 amino acids of RH II/Gu can be replaced by an RNA binding motif from nucleolar protein p120 without a deleterious effect on its foldase activity. A model is proposed to explain how RH II/Gu binds and folds an RNA substrate.

Amino Acid Sequence↗
PubMed2001

Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis.

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse-chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A(1) and A(2) are inhibited and delayed at site A(0). Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.

Amino Acid Sequence↗
PubMed1991

The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells.

An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nucleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3' to 5' and the 5' to 3' directions. The properties of the RNA helicase described here differ from the deaminase RNA unwinding activity described in Xenopus oocytes (Bass, B.L., and Weintraub, H. (1987) Cell 48, 607-613) and from the p68 HeLa RNA helicase (Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339, 562-564).

Adenosine Triphosphate↗
PubMed1997

An3 protein encoded by a localized maternal mRNA in Xenopus laevis is an ATPase with substrate-specific RNA helicase activity.

ATP-dependent RNA helicases from the DEAD box family of proteins are involved in a number of RNA processing and utilization events. An3 protein from Xenopus laevis is an RNA helicase of the DEAD box family of proteins. An3 is synthesized by a mRNA that is localized to one end of Xenopus laevis oocytes. An3 protein is found in the nucleus of ooctes, and more specifically, during the middle stages of oocyte development, with extra nucleoli that contain amplified copies of rRNA genes in the nucleolus. By expressing glutathione-S-transferase:An3 fusion proteins in E. coli, sufficient amounts of An3 protein were isolated to examine its enzymatic activities. ATPase activity, NTP substrate range and RNA helicase activity were tested. An3 protein ATPase activity was evident but not stimulated by any of a variety of RNA tested. An3 protein was able to resolve the duplex formed by an in vitro substrate, in the presence of ATP or dATP. An3 required both 3' and 5' single-stranded regions of RNA flanking the RNA duplex it resolves.

Adenosine Triphosphatases↗
PubMed1997

The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication.

The plum pox potyvirus (PPV) protein CI is an RNA helicase whose function in the viral life cycle is still unknown. The CI protein contains seven conserved sequence motifs typical of RNA helicases of the superfamily SF2. We have introduced several individual point mutations into the region coding for motif V of the PPV CI protein and expressed these proteins in Escherichia coli as maltose binding protein fusions. Mutations that abolished RNA helicase activity also disturbed NTP hydrolysis. No mutations affected the RNA binding capacity of the CI protein. These mutations were also introduced in the PPV genome making use of a full-length cDNA clone. Mutant viruses carrying CI proteins with reduced RNA helicase activity replicated very poorly in protoplasts and were unable to infect whole plants without rapid pseudoreversion to wild-type. These results indicate that motif V is involved in the NTP hydrolysis step required for potyvirus RNA helicase activity, and that this activity plays an essential role in virus RNA replication inside the infected cell.

Adenosine Triphosphatases↗
PubMed1995

Poly(U) binding activity of hepatitis C virus NS3 protein, a putative RNA helicase.

A non-structural protein of the hepatitis C virus (HCV), NS3, contains amino acid sequence motifs characteristic of serine-proteinases and RNA helicases. RNA binding activity of the NS3 protein with an apparent dissociation constant of 2 x 10(-7) M was detected using a poly(U)-Sepharose resin. Competitive RNA binding analysis suggested that the NS3 protein binds preferentially to the poly(U) sequence, which is located at the 3' end of HCV RNA. Mutational analysis of NS3 protein revealed the possibility that both the RNA helicase region and the serine-proteinase region were necessary for full RNA binding activity.

Amino Acid Sequence↗
PubMed2005

Signaling to the DEAD box--regulation of DEAD-box p68 RNA helicase by protein phosphorylations.

P68 nuclear RNA helicase is essential for normal cell growth. The protein plays a very important role in cell development and proliferation. However, the molecular mechanism by which the p68 functions in cell developmental program is not clear. We previously observed that bacterially expressed his-p68 was phosphorylated at multiple sites including serine/threonine and tyrosine [L. Yang, Z.R. Liu, Protein Expr. Purif., 35: 327]. Here we report that p68 RNA helicase is phosphorylated at tyrosine residue(s) in HeLa cells. Phosphorylation of p68 at threonine or tyrosine residues responds differently to tumor necrosis factor alpha (TNF-alpha)induced cell signal. Kinase inhibition and in vitro kinase assays demonstrate that p68 RNA helicase is a cellular target of p38 MAP kinase. Phosphorylation of p68 affects the ATPase and RNA unwinding activities of the protein. In addition, we demonstrate here that phosphorylation of p68 RNA helicase controls the function of the protein in the pre-mRNA splicing process. Interestingly, phosphorylation at different amino acid residues exhibits different regulatory effects. The data suggest that function(s) of p68 RNA helicase may be subjected to the regulation of multiple cell signal pathways.

DEAD-box RNA Helicases↗
PubMed1999

Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway.

Human DNA helicase VIII (HDH VIII) was isolated in the course of a systematic study of the DNA unwinding enzymes present in human cells. From a HeLa cell nuclear extract a protein with an Mrof 68 kDa in SDS-PAGE was isolated, characterised and micro-sequenced. The enzyme shows ATP- and Mg2+-dependent activity is not stimulated by RPA, prefers partially unwound 3'-tailed substrates and moves along the bound strand in the 5' to 3' direction. HDH VIII can also unwind partial RNA/DNA and RNA/RNA duplexes. Microsequencing of the polypeptide showed that this enzyme corresponds to G3BP, an element of the Ras pathway which binds specifically to the GTPase-activating protein. HDH VIII/G3BP is analogous to the heterogeneous nuclear ribonucleoproteins and contains a sequence rich in RGG boxes similar to the C-terminal domain of HDH IV/nucleolin, another DNA and RNA helicase.

Adenosine Triphosphatases↗
PubMed1992

Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases.

Three distinct nucleic acid-dependent ATPases are packaged within infectious vaccinia virus particles; one of these enzymes (nucleoside triphosphate phosphohydrolase II or NPH-II) is activated by single-stranded RNA. Purified NPH-II is now shown to be an NTP-dependent RNA helicase. RNA unwinding requires a divalent cation and any one of the eight common ribo- or deoxyribonucleoside triphosphates. The enzyme acts catalytically to displace an estimated 10-fold molar excess of duplex RNA under in vitro reaction conditions. NPH-II binds to single-stranded RNA. Turnover of the bound enzyme is stimulated by and coupled to hydrolysis of NTP. Photocrosslinking of radiolabeled RNA to NPH-II results in label transfer to a single 73-kDa polypeptide. The sedimentation properties of the helicase are consistent with NPH-II being a monomer of this protein. Immunoblotting experiments identify NPH-II as the product of the vaccinia virus I8 gene. The I8-encoded protein displays extensive sequence similarity to members of the DE-H family of RNA-dependent NTPases. Mutations in the NPH-II gene [Fathi, Z. & Condit, R.C. (1991) Virology 181, 258-272] define the vaccinia helicase as essential for virus replication in vivo. Encapsidation of NPH-II in the virus particle suggests a role for the enzyme in synthesis of early messenger RNAs by the virion-associated transcription machinery.

Amino Acid Sequence↗
PubMed2004

The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer.

Previously, we reported that in clam oocytes, cytoplasmic polyadenylation element-binding protein (CPEB) co-immunoprecipitates with p47, a member of the highly conserved RCK family of RNA helicases which includes Drosophila Me31B and Saccharomyces cerevisiae Dhh1. Xp54, the Xenopus homologue, with helicase activity, is a component of stored mRNP. In tethered function assays in Xenopus oocytes, we showed that MS2-Xp54 represses the translation of non-adenylated firefly luciferase mRNAs and that mutations in two core helicase motifs, DEAD and HRIGR, surprisingly, activated translation. Here we show that wild-type MS2-Xp54 tethered to the reporter mRNA 3'-untranslated region (UTR) represses translation in both oocytes and eggs in an RNA-dependent complex with endogenous Xp54. Injection of mutant helicases or adenylated reporter mRNA abrogates this association. Thus Xp54 oligomerization is a hallmark of translational repression. Xp54 complexes, which also contain CPEB and eIF4E in oocytes, change during meiotic maturation. In eggs, CPEB is degraded and, while eIF4E still interacts with Xp54, this interaction becomes RNA dependent. Supporting evidence for RNA-mediated oligomerization of endogenous Xp54, and RNA-independent association with CPEB and eIF4E in oocytes was obtained by gel filtration. Altogether, our data are consistent with a model in which the active form of the Xp54 RNA helicase is an oligomer in vivo which, when tethered, via either MS2 or CPEB to the 3'UTR, represses mRNA translation, possibly by sequestering eIF4E from the translational machinery.

Animals↗