spindle E/homeless Viral and Vertebrate helicases
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. 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 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 (Shuman, 1992).
A human RNA helicase gene, DBP1, was cloned by PCR methods using degenerate oligonucleotide primers corresponding to highly conserved motifs among known members of the DEAH-box protein family. The full-length DBP1 contains 3028 nucleotides and codes for a protein of 813 amino acids with a calculated mol. wt. of 92723 daltons. The predicted amino acid sequence shares extensive homology with Prp2, Prp16, and Prp22 proteins, which are required to splice mRNA precursors in budding yeast. The protein encoded by DBP1 has RGD, RD, and HS(A/T) repeat motifs close to the N-terminus. Southern blot analysis suggests the presence of a homolog of the DBP1 genes in other species, and Northern blot analysis shows that DBP1 is expressed ubiquitously in the various human organs investigated. The DBP1 gene is found to be on chromosome 4p15.3 and encodes a putative nuclear ATP-dependent RNA helicase (Imamura, 1997).
To identify human homologs of the yeast helicase family, PCR primers were constructed that correspond to the highly conserved region of the DEAH box protein family; five cDNA fragments were successfully amplified, using HeLa poly(A)+ RNA as a substrate. One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which is involved in the release of spliced mRNAs from the spliceosomes. Expression of HRH1 in an S. cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype. These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein. Interestingly, it is HRH1 but not Prp22 that contains an arginine- and serine-rich domain (RS domain), characteristic of some splicing factors such as members of the SR protein family. HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain. It is speculated that HRH1 might be targeted to the spliceosome through this interaction (Ono, 1994).
The prototypic DEAD/DExH family member eIF-4A, has RNA-dependent NTPase activity as a monomer but functions as a helicase when dimerized with eIF-4B, which binds RNA through an RNP RNA-binding domain. It is currently thought that most DEAD/DExH proteins either require accessory proteins to function or else recognize a specific sequence in an unknown RNA substrate. One RNA binding domain, termed the double-stranded RNA-binding domain (dsRBD), was first identified as a 70 residue repeat motif in three proteins: dsRNA-dependent (DAI) protein kinase, Xenopus RNA-binding protein xlrbpa and Drosophila maternal effect protein Staufen. Searches with dsRNA-binding domain profiles detect two copies of the domain in each of RNA helicase A, Drosophila Maleless and C. elegans ORF T20G5-11 (of unknown function). RNA helicase A is unusual in being one of the few characterised DEAD/DExH helicases that are active as monomers. Other monomeric DEAD/DExH RNA helicases (p68, NPH-II) have domains that match another RNA-binding motif, the RGG repeat. The DEAD/DExH domain appears to be insufficient on its own to promote helicase activity and additional RNA-binding capacity must be supplied either as domains adjacent to the DEAD/DExH-box or by bound partners as in the eIF-4AB dimer. The presence or absence of extra RNA-binding domains should allow classification of DEAD/DExH proteins as monomeric or multimeric helicases (Gibson, 1994).
Translation initiation factor elF-4B is an RNA-binding protein that promotes the association of the mRNA to the eukaryotic 40S ribosomal subunit. One of its better characterized features is the ability to stimulate the activity of the DEAD box RNA helicase elF-4A. In addition to an RNA recognition motif (RRM) located near its amino-terminus, elF-4B contains an RNA-binding region in its carboxy-terminal half. The elF-4A helicase stimulatory activity resides in the carboxy-terminal half of elF-4B, and the RRM has little impact on this function. To better understand the role of the elF-4B RRM, attempts were made to identify its specific RNA target sequence. To this end, in vitro RNA selection/amplifications were performed using various portions of elF-4B. These experiments were designed to test the RNA recognition specificity of the two elF-4B regions implicated in RNA binding and to assess the influence of elF-4A on the RNA-binding specificity. The RRM was shown to bind with high affinity to an RNA stem-loop structure with conserved primary sequence elements. Discrete point mutations in an in vitro-selected RNA identified residues critical for RNA binding. Neither the carboxy-terminal RNA-interaction region, nor elF-4A, influence the structure of the high-affinity RNA ligands selected by elF-4B, and elF-4A by itself does not select any specific RNA target. Previous studies have demonstrated an interaction of elF-4B with ribosomes, and it has been suggested that this association is mediated through binding to ribosomal RNA. The RRM of elF-4B interacts directly with 18S rRNA and this interaction is inhibited by an excess of the elF-4B in vitro-selected RNA. ElF-4B can bind simultaneously to two different RNA molecules, supporting a model whereby elF-4B promotes ribosome binding to the 5' untranslated region of an mRNA by bridging it to 18S rRNA (Methot, 1996).
Nuclear DNA helicase II (NDH II) unwinds both DNA and RNA. NDHII cDNA is 4,528 bases in length, which corresponds well with a 4.5-4.7-kilobase-long mRNA as detected by Northern blot analysis. The open reading frame of NDH II cDNA predicts a polypeptide of 1287 amino acids and a calculated molecular mass of 141,854 daltons. NDH II is related to a group of nucleic acid helicases from the DEAD/H box family II, with the signature motif DEIH in domain II. Two further proteins of this family, i.e. human RNA helicase A and Drosophila Maleless (Mle) protein, are found to be highly homologous to NDH II. With RNA helicase A, there is 91.5% identity and 95.5% similarity between the amino acid residues; with Mle protein, a 50% identity and an 85% similarity is observed. Antibodies against human RNA helicase A cross-react with NDH II, further supporting evidence that NDH II is the bovine homolog of human RNA helicase A. Immunofluorescence studies reveal a mainly nuclear localization of NDH II. A role for NDH II in nuclear DNA and RNA metabolism is suggested (Zhang, 1995).
Human p68 RNA helicase is a nuclear RNA-dependent ATPase that belongs to a family of putative helicases known as the DEAD box proteins. These proteins have been implicated in aspects of RNA function including translation initiation, splicing, and ribosome assembly in a variety of organisms ranging from Escherichia coli to humans. While members of this family are believed to function in the manipulation of RNA secondary structure, little is known about the regulation of these enzymes. p68 possesses a region of sequence similarity to the conserved protein kinase C phosphorylation site and calmodulin binding domain (also known as the IQ domain) of the neural-specific proteins neuromodulin (GAP-43) and neurogranin (RC3). p68 is phosphorylated by protein kinase C in vitro and binds calmodulin in a Ca(2+)-dependent manner. Both phosphorylation and calmodulin binding inhibit p68 ATPase activity, suggesting that the RNA unwinding activity of p68 may be regulated by dual Ca2+ signal transduction pathways through its IQ domain (Buelt, 1994).
RNA helicase A is an abundant nuclear enzyme found in HeLa cells that unwinds double-stranded RNA in a 3' to 5' direction. A complementary DNA (cDNA) clone expressing RNA helicase A was isolated by screening a human cDNA library with polyclonal antibodies produced against the purified protein. The deduced amino acid sequence from this clone shows that RNA helicase A is a member of the DEAH family of proteins and thought to be helicases. Sequence comparison among all known proteins of the DEAH family reveals that the highest homology is between RNA helicase A and the Maleless protein (Mle) of Drosophila. There is 49% identity and 85% similarity throughout the overall primary sequences of both proteins, suggesting that RNA helicase A is the human counterpart of Drosophila Mle. Polyclonal antibodies against Drosophila Mle recognize RNA helicase A in crude nuclear extracts of HeLa cells as well as the purified protein. A recombinant RNA helicase A containing 6 histidine residues at the NH2 terminus was expressed in Sf9 cells using a baculovirus vector. The protein isolated from insect cells and the enzyme purified from HeLa cells both exhibit identical RNA helicase and RNA-dependent ATPase activities (Lee, 1993).
The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor Mle. In transient transfection assays, RHA is found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB (see Drosophila dCREB2). Since a mutation in RHA that compromises its helicase activity correspondingly reduces CREB-dependent transcription, it is proposed that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters. The involvement of a DNA helicase such as RHA in signal-dependent transcription is intriguing because it suggests that recruitment of CBP complexes may promote local unwinding of promoter DNA via RHA and thereby permit engagement of the transcriptional apparatus (Nakajima, 1997).
A rat cDNA of 117.4 kDa contains RNA helicase consensus motifs, among them a "DEAD" box, has been called HEL117 (for helicase of 117.4 kDa). In addition to the helicase consensus motifs, HEL117 contains an arginine-serine (RS)-rich domain, which occurs in some proteins involved in RNA splicing. The COOH-terminal region of 78 residues of HEL117 is 38.5% identical and 59% similar to the COOH-terminal region of a yeast PRP5 protein that is involved in RNA splicing. Rabbit antibodies identify a single polypeptide in rat cells, in the cells of other mammals, and in the chicken. The antibodies reveal a finely punctate and speckled intranuclear staining in immunofluorescence microscopy. A monoclonal antibody against a human splicing factor containing an RS domain (SC35) shows (in double immunofluorescence microscopy) largely overlapping staining consistent with HEL117 being involved in RNA splicing (Sukegawa, 1995).
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