maleless
Vertebrate helicases
The prototypic DEAD/DExH family member eIF-4A, has RNA-dependent NTPase activity as a monomer but functions as a helicase when dimerised 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 recognise 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).
The human RNA helicase II/Gu protein (RH-II/Gu) is a member of the D-E-A-D box protein family. It is a unique enzyme
that possesses an ATP-dependent RNA-unwinding activity and has an RNA-folding activity that introduces an intramolecular
secondary structure in single-stranded RNA. This report shows that these two enzymatic activities are distinct. ATP[S], GTP
and low concentrations of ATP enhance the RNA-folding activity of RH-II/Gu but not the RNA-helicase activity. High
concentrations of ATP are required for the helicase activity but are inhibitory to the RNA-folding activity. Mg2+ is required for
the helicase activity but not for the RNA-folding reaction. Anti-RH-II/Gu antibody inhibits the
RNA-unwinding activity but not the folding activity. Mutations of the DEVD sequence, which corresponds to the DEAD box,
and the SAT motif enhances RNA-folding activity of RH-II/Gu but completely inhibits the RNA-helicase activity. A mutant
that lacks the COOH-terminal 76 amino acid residues, including the four FRGQR repeats, has unwinding activity but does not
catalyze the folding of a single-stranded RNA. The two enzymatic activities of RH-II/Gu reside in distinct domains. Amino
acids 1-650 are active in the RNA-unwinding reaction but lack RNA-folding activity. Amino acids 646-801 fold
single-stranded RNA but lack helicase activity. This report shows distinct RNA-unwinding and RNA-folding activities residing
in separate domains within the same protein (Valdez, 1998).
Full-length human nuclear DNA helicase II (NDH II) was cloned and overexpressed in a baculovirus-derived expression
system. Recombinant NDH II unwinds both DNA and RNA. Limited tryptic digestion produces active helicases with
molecular masses of 130 and 100 kDa. The 130-kDa helicase is missing a glycine-rich domain (RGG-box) at the carboxyl
terminus, while the 100-kDa form is missing both its double-stranded RNA binding domains (dsRBDs) at the amino terminus and
its RGG-box. Hence, the dsRBDs and the RGG-box are dispensable for unwinding. In contrast, the isolated DEXH
core alone can neither hydrolyze ATP nor unwind nucleic acids. These enzymatic activities are not regained by fusing a
complete COOH or NH2 terminus to the helicase core. Hence, an active helicase requires part of the NH2 terminus, the DEXH
core, and a C-terminal extension of the core. Both dsRBDs and the RGG-box were bacterially expressed as glutathione
S-transferase fusion proteins. The two dsRBDs have a strong affinity to double-stranded RNA and cooperated upon RNA
binding, while the RGG-box binds preferentially to single-stranded DNA. A model is suggested in which the flanking
domains influence and regulate the unwinding properties of NDH II (Zhang,1997).
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, it was of interest to identify its specific RNA
target sequence. To this end, it 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 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, it has been called HEL117
(for helicase of 117.4 kDa). Besides 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 not only in rat cells but
also in cells of other mammals as well as 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).
RNA helicase A is an enzyme that possesses both RNA and DNA helicase activities. In this report, the isolation of
a mouse cDNA encoding RNA helicase A is described. The deduced amino acid sequence derived from mouse RNA helicase A cDNA
exhibits 87% and 47% identity to its human and Drosophila homologs, respectively. Using Southern blot analysis employing a
mouse backcross panel, the mouse RNA helicase A gene has been assigned to chromosome 1, mapping near the D1Bir20 locus
at MGD position 67. Northern blot and primer extension analyses indicate that, although its level is variable, RNA helicase A
appears to be expressed from a single transcription start site in all tissues tested. Sequence analysis of the upstream genomic
DNA reveal that the promoter region lacks a TATA box and contains two high-affinity sites for Sp1, one ISRE, a binding
site for interferon regulatory factor, and three AP2-binding sites. These findings suggest that the transcriptional regulation of
the RNA helicase A gene is complex (Lee, 1998a).
RNA helicase A (RHA) is the human homolog of the Drosophila Maleless protein, an essential factor for the development of male flies. Recently, it
was shown that RHA cooperates with the cAMP-responsive element in mediating the cAMP-dependent transcriptional activation of a number of genes.
Due to the participation of cAMP as a second messenger in a number of signaling pathways, the function of RHA was examined during mammalian
embryogenesis. To examine the role(s) of RHA in mammalian development, RHA knockout mice were generated by homologous recombination.
Homozygosity for the mutant RHA allele leads to early embryonic lethality. Histological analysis, combined with terminal
deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) reactions of RHA-null embryos, reveals marked apoptotic cell death specifically in
embryonic ectodermal cells during gastrulation. RNA in situ analyses of the expression of HNF-3beta and Brachyury, two molecular markers for
gastrulation, show that RHA-null embryos at days 7.5 and 8.5 express both HNF-3beta and Brachyury in a pattern similar to those of pre- and early-streak stage embryos, respectively.
Thus, although no embryonic mesodermal layer
is observed by histological analysis of RHA-deficient embryos, extraembryonic mesoderm is present and differentiates
into allantois, the mesodermal layer of the yolk sac, and blood island-like structures. These observations indicate that the early
onset of gastrulation, although delayed, is not affected by the loss of RHA function (Lee, 1998b).
The RHA mutant phenotype is first observed at E7.5, a stage
at which normal embryos exhibit elongated and characteristically shaped embryonic cups with distinct anterior/posterior and
dorsal/ventral polarity. In a normal E7.5 embryo, gastrulation is well advanced and a mesodermal layer surrounds
the embryonic ectoderm. Often the amnion is closed, the allantois is just forming, and the extraembryonic ectoderm is lifted up
into the two-layered chorion. The parietal and visceral endoderm form squamous sheets around the embryo proper. In contrast
to this, mutant embryos are smaller in size and significantly delayed in development. Morphologically,
they resemble embryos from E6.5 litters and contained a small two-layered egg cylinder. The parietal endodermal cells are
rounded, and a cuboidal layer of visceral endoderm surrounds the entire conceptus. No mesodermal layer can be
distinguished in sections and no amnion is present. However, the extraembryonic ectoderm forms a distinct ectoplacental
cone and, in some embryos, a thickening of the embryonic ectoderm is observed. Although few dying
cells are observed in the control embryos, numerous apoptotic bodies are present in the mutants,
localized predominantly in the embryonic ectoderm. The apoptotic bodies are frequently clustered and also present in the
proamniotic cavity.
More striking differences are noted between mutants and controls at E8.5. At this stage, normal littermates have already
initiated organogenesis, whereas RHA mutants are developmentally similar to the E7.5 mutants, manifesting
only a slight increase in size. Although the embryonic portion of the visceral endoderm appears flattened
and the embryonic ectoderm acquires a pseudostratified epithelial morphology, no typical mesoderm is detected. Apoptotic
cell death is profound in cells constituting the embryonic ectoderm.
To explore the possible contribution of genetic background to the observed effects of the RHA mutation, the mutant RHA
allele was backcrossed into the outbred strain Black Swiss. Unexpectedly, the observed phenotype is more severe. At
E7.5, the embryonic ectoderm is almost entirely absent and only a few dying cells remain. By E8.5, only the
extraembryonic portion remains and exhibits some degree of differentiation. Independent of the absence of
morphologically discernible embryonic mesoderm, extraembryonic mesoderm is generated and gives rise to allantois, the
lining of the exocoelom, and even to a few small yolk sac blood island-like structures. However, the ectoplacental cone
remains relatively undifferentiated and apoptotic cells are abundant. On the basis of these observations, it is concluded that
the deletion of the RHA affects the activity of genes critical for the differentiation of embryonic ectoderm and hence the normal progression of
gastrulation (Lee, 1998b).
In Drosophila, MLE plays a critical role(s) in the development of male flies. However, at the post-developmental stage,
as-yet-unidentified sets of factors present in both sexes may recruit the function of MLE to increase the expression of certain
genes including para, an X-linked gene coding for a sodium channel. The higher genetic complexity of mammals
makes it possible to envision more diverse roles for RHA than MLE in transcriptional regulation processes. The phenotypes
observed in RHA mutant mouse embryos may indicate the earliest developmental stage when RHA begins to act in
conjunction with its interacting transcription factor(s), functionally resembling the male-specific lethal proteins of Drosophila. It is presently unknown which transcriptional activation processes require the function of RHA during development
and differentiation. The cAMP signaling pathway is currently the only demonstrated process that requires RHA to provoke
target gene activation (Lee, 1998b and references).
Unlike cellular mRNA, retroviral mRNA bypasses the tight coupling of the
splicing and nuclear export steps to allow the export of intron-containing viral
RNA transcripts to the cytoplasm. Two distinct nuclear export pathways for
retroviral mRNA have been described: a CRM-1 dependent pathway mediated by the
HIV-1 Rev protein and the Rev Response Element (RRE), and a CRM-1 independent
pathway mediated by the Constitutive Transport Element (CTE) of type D
retroviruses. Two CTE-binding proteins, RNA helicase A (RHA) and Tap, have been
implicated in the nuclear export of CTE-containing RNA. Expression of
RRE-containing RNA can also be mediated by a cellular
protein, Sam68, independently of Rev. Sam68, RHA
and Tap cooperate in the nuclear export of both CTE- and RRE-containing RNA. RHA
binds to Sam68 and to Tap both in vivo and in vitro. Over-expression of Sam68
activates both RRE- and CTE-regulated reporter gene expression in human cells
and in quail cells in the presence of human Tap. This activation is
competitively inhibited by the nuclear transport domain (NTD) of RHA and a
transdominant negative mutant of Tap. Conversely, the activation of CTE by Tap
in quail cells is inhibited by a transdominant mutant of Sam68 and NTD. It is
proposed that both HIV and type D retroviruses may access the same constitutive
RNA nuclear export pathway involving RHA, Tap and Sam68, even though HIV also
utilizes the Rev protein for more efficient nuclear export. it is likely that
this constitutive export pathway is also used by cellular mRNA, but at a
different interface with the splicing process (Reddy, 2000).
Beyond their role at nuclear pore complexes, some nucleoporins function in
the nucleoplasm. One such nucleoporin, Nup98
(see Drosophila Nup98-96),
binds chromatin and regulates gene expression. To gain insight into how
Nup98 contributes to this process, this study focused on identifying novel
binding partners and understanding the significance of these interactions.
The DExH/D-box helicase DHX9
(see Drosophila mle) as
an intranuclear Nup98 binding partner (see Nup98 protein-protein interaction network). Various results, including in vitro
assays, show that the FG/GLFG region of Nup98 binds to N- and C-terminal
regions of DHX9 in an RNA facilitated manner. Importantly, binding of
Nup98 stimulates the ATPase activity of DHX9, and a transcriptional
reporter assay suggests Nup98 supports DHX9-stimulated transcription.
Consistent with these observations, it was found that Nup98 and DHX9 bind
interdependently to similar gene loci and their transcripts. Based on
these data, the study proposes that Nup98 functions as a co-factor that
regulates DHX9 and, potentially, other RNA helicases (Capitanio, 2017).
ENY2 protein of Drosophila melanogaster is a subunit of several multiprotein complexes and connects various stages of gene expression. This work is devoted to studying the interaction of ENY2 with RNA helicase MLE. This interaction was confirmed by independent methods. Data indicating that this interaction is conserved in evolution and is important for the functioning of MLE in both sexes were obtained (Nikolenko, 2019).
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