ISSN 00268933, Molecular Biology, 2014, Vol. 48, No. 1, pp. 16–28. © Pleiades Publishing, Inc., 2014.

Original Russian Text © A.A. Kotov, N.V. Akulenko, M.V. Kibanov, L.V. Olenina, 2014, published in Molekulyarnaya Biologiya, 2014, Vol. 48, No. 1, pp. 22–35.

REVEIWS
UDC 577.218

DEADBox RNA Helicases in Animal Gametogenesis

A. A. Kotov, N. V. Akulenko, M. V. Kibanov, and L. V. Olenina
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182 Russia;
email: olenina_ludmila@mail.ru
Received August 23, 2013; in final form, August 26, 2013

Abstract—The review summarizes a current knowledge about a role of RNA helicases in the development
and maintenance of gamenogenesis in eukaryotes. We focused on three RNA helicase family members—
Vasa/DDX4, Belle/DDX3, and SpindleE/TDRD9—that contain characteristic amino acid sequence
motifs (DEAD box) and perform substantial conserved functions in the germinal tissues of various species
from Drosophila to human. These enzymes are involved in a broad range of activities associated with the reg
ulation of transcription, splicing, nuclear export and, especially, with translation initiation. Expression of
genes required for gametogenesis is regulated mainly at the transcriptional level. RNA helicases are involved
in the formation of cytoplasmic ribonucleoprotein (RNP) granules and RNA silencing. A highly conserved
central domain is characteristic of DEADbox RNA helicases and determines their basic biological activity
in ATPdependent unwinding of short RNA duplexes.
DOI: 10.1134/S0026893314010063
Keywords: DEADbox RNA helicases, DDX3, Vasa, SpindleE, oogenesis, spermatogenesis, RNA silencing

INTRODUCTION DEADBOX RNA HELICASES:

Proteins carrying a motif known as DEAD box
(where DEAD is the oneletter code of the amino acid
residues constituting the motif) belong to helicase
superfamily 2 (SF2) [1] and are found in both prokary
otes and eukaryotes [2, 3]. These proteins are involved
in all aspects of RNA metabolism. Almost identical in
all organisms their central region possess a biochemi
cal activity although functions of these proteins differ
in various cells and tissues. The specificity of their
interactions with RNA and proteins is determined by
the N and Cterminal domains of polypeptide chain.
DEADbox RNA helicases (hereafter referred to as
RNA helicases) realizes ATPdependent RNA
unwinding; likewise they carried out the formation of
RNA duplexes and act as chaperones in the formation
of large ribonucleoprotein (RNP) complexes. RNA
helicases that exhibited particular interest are impli
cated in the maintenance of germinal tissues, where a
fine posttranscriptional regulation is need for gene
expression and cellspecific RNP granules form. In
spite of the low intensity (processivity) of RNA
unwinding, the enzymes are involved in oogenesis and
spermatogenesis, silencing of mobile elements and
other repeats in the germinal tissues, and formation of
gonads during embryo development. Here we focused
on several DEADbox proteins, namely, Vasa/DDX4,
which was identified in Drosophila melanogaster more
than 25 years ago and now considered as a conserved
marker of germline in all eukaryotes [4–6];
DDX3/Belle/DED1 [7–10]; and SpindleE/TRDR9
[11, 12].
GENERAL INFORMATION
Structural Characteristics of Helicases
All members of the DEADbox enzyme family
have a highly conserved core helicase region, which
binds both ATP and RNA. The core region consists of
two nearly identical domains, which contain at least 12
characteristic motifs in conserved positions. The fam
ily owes its name to one of the motifs (motif II, Asp
GluAlaAsp, or DEAD) [2, 3]. The helicase core
forms a cleft, where ATP and RNA binding takes
place. All structural and biochemical functions of
RNA helicases are determined by the residues and
motifs involved in the process [3]. The helicase core
interacts with a short (5nt) RNA fragment, and the
interaction involves exclusively the RNA sugarphos
phate backbone. The RNA binding mode is highly
conserved among all known DEADbox proteins.
RNA forms a characteristic bend upon binding as a
result of the interaction with αhelical motif Ib of heli
case. This was demonstrated in studies of recombinant
protein Vasa in complex with oligo(U) RNA substrate
in the presence of ATP analog [13]. The bend is
incompatible with the doublestranded state of the
RNA region bound with the enzyme, and the region is
denatured [13, 14]. Both of the helicase domains are
structurally similar to the RecAlike domains of bacte
rial recombinase A. All RNA helicases analyzed to
date additionally have N and Cterminal uncon
served domains, which range from several to several
hundreds of residues in size and seem to play a role in
interactions with other proteins or RNA targets. The

16
 DEADBOX RNA HELICASES IN ANIMAL GAMETOGENESIS
domains are responsible for the polyfunctional char
acter and specificity of the proteins and often harbor
RNA recognition motifs (RRMs) [3].
Biochemical Activity of Helicases
In vivo DEADbox RNA helicases are involved in
a broad variety of interactions ranging from a remod
eling of RNP complexes to a fixation of specific tran
scripts in a translationally arrested state (so called
RNA clamping) [3, 14, 15]. Like the majority of pro
teins involved in RNA metabolism, DEADbox pro
teins usually function as participants of complexes
consisting from tens or even hundreds of components,
for instance as in the case of spliceosomes or function
ing ribosomes.
Local duplex unwinding. RNA helicases utilize the
energy of ATP to bind and to unwind an RNA duplex.
However, this is restricted only to the duplexes that
contain no more than two helix turns. Many DEAD
box RNA helicases unwind the duplexes of no more
than 10–12 bp [16]. The enzymes do not move along
the duplex with a certain polarity to unwind it [17], in
contrast to other RNA and DNA helicases, but
directly penetrate into a duplex region and separate its
strands. The mechanism is termed as local strand sep
aration [18, 19]. In spite of its low processivity, the
unwinding via local strand separation ensures a
remodeling of RNA or RNP complexes by RNA heli
cases in vivo without a largescale unfolding of the
RNA or RNP structure.
ATP utilization in duplex unwinding. Each RNA
unwinding event utilizes one ATP molecule regardless
of the duplex length, however, ATP binding and
hydrolysis event does not necessarily lead to duplex
unwinding [20, 21]. The process rate decreases with
the increasing length and stability of the RNA duplex.
Some RNA helicases were found to possess RNA
dependent ATPase activity, which can be stimulated in
vitro by poly(C) and poly(I) RNA substrates and ribo
somal RNA, as is in the case of eIF4a [14, 22]. The
majority of DEADbox RNA helicases require ATP
binding, but not ATP hydrolysis, for their activity. ATP
hydrolysis is necessary for the protein release from
RNA and for a rapid substrate turnover [20, 23].
Remodeling of RNP complexes. The RNA helicases
under discussion can remove proteins from structured
or nonstructured RNA, this process also requires ATP
hydrolysis. This means that duplex unwinding it not
essential for the separation of RNA and proteins
[24, 25]. In accordance with the nonprocessive action
of DEADbox RNA helicases, protein removing from
the complexes takes place at the RNA–protein con
tact site, which is no more than 8 nt. This property
limits the remodeling of RNP complexes, thus pre
venting their total dissociation [24, 26].
Role of helicases in the RNA renaturation. Many
helicases, including certain DEADbox proteins,
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
17
facilitate RNA renaturation at least in vitro in addition
to duplex unwinding [27–30]. Similar activity was
observed in vivo for some of these enzymes, such as
DED1 [29]. Renaturing activity does not seem to
require the additional energy of ATP hydrolysis and is
not a reverse of duplex unwinding; i.e., the annealing
involves another RNA region. As a result, RNA heli
cases that catalyze both ATPdependent duplex
unwinding and ATPindependent annealing facilitate
a rearrangement of the RNA secondary structure [29].
This capability is thought to be of principal impor
tance for the DEADbox proteins to function as RNA
chaperones in RNA remodeling.
RNA clamping. DEADbox proteins can function
as centers of RNP complex formation. It is thought
that RNA bound with helicase forms a characteristic
bend, but is not released from the complex in the pres
ence of additional protein factors, which inhibit
ATPase activity of RNA helicase. This clamp function
is characteristic of eIF4AIII, which is a component of
the splicing complex [31, 32].
Functional Features of DEADBox RNA Helicases
The majority of eukaryotic DEADbox RNA heli
cases perform certain functions in one or more pro
cesses, including transcription, ribosome biogenesis,
premRNA splicing, mRNA export, translation initi
ation, and mRNA degradation [3, 15]. However, the
molecular mechanisms whereby the DEADbox pro
teins play a role in these processes are only partly
understood. RNA helicase eIF4A is involved in trans
lation initiation [3, 33, 34]. The protein is considered
as a prototype of DEADbox RNA helicases because
their main biochemical properties were initially eluci
dated with eIF4A. The formation of the 43S preinitia
tion complex at 5' end of mRNA is a key event that
determines the translation rate in eukaryotes. The
multimeric eIF4F complex recognizes the cap struc
ture to ensure the subsequent association of 40S ribo
some subunit with mRNA. The eIF4F complex con
sists of the eIF4E capbinding protein, eIF4A RNA
helicase, and the scaffold protein eIF4G [35]. It is
known that eIF4A has only a low affinity for mRNA,
and its ATPase and helicase activities are stimulated in
the presence of the other components of the eIF4F
complex or upon the interaction with eIF4B and
eIF4H cofactors [22, 36, 37]. As mentioned above,
ATPase activity of eIF4A is stimulated by its interac
tion with an RNA substrate [22]. The specificity of
eIF4A and other RNA helicases is determined by the
sequences of the N and Cterminal unconserved
domains, posttranslational modification, interactions
with tissuespecific partners, and the temporal and
spatial specifics of partner expression.
18
Splicing?
Transcription
Translation
initiation
Translational
repression
Fig. 1. DDX3 involvement in various intracellular processes associated with RNA metabolism according to published data.
DDX3 HELICASES
Tissue Specificity of DDX3 and Its Role
in Germinal Tissue
The eukaryotic DDX3 subfamily includes many
DEADbox RNA helicases [38]. In humans, highly
homologous genes for DDX3 proteins were found on
the X chromosome (DDX3X) and in nonrecombining
region of the Y chromosome (DDX3Y, or DBY) [7].
DDX3X is expressed in various tissues, while DBY is
expressed exclusively in germ cells at the premeiotic
stage of spermatogenesis [39]. DBY is located on the
AZF Ychromosomal locus, which is responsible for
male fertility [40–44]. DBY deletions are associated
with a spermatogenesis defect known as Sertoly Cells
Only (SCO) syndrome [41, 43, 45], which leads to a
complete absence of germ cells. Somatic Sertoli cells,
which form a niche for germ cells, remain intact.
DDX3X and DBY have 92% amino acid sequence
similarity. DDX3X is also synthesized in the testis, but
its production occurs in postmeiotic cells, so that the
protein cannot compensate for a DBY loss [39]. The
molecular nature of spermatogenesis failures associ
ated with lack of DBY expression remains obscure.
belle gene is the only DDX3 homolog in D. mela
nogaster [10]. All of the known silent alleles of belle are
lethal. Normally, belle is expressed in germinal tissues
at a high level and maintains both female and male fer
tility [10]. Belle protein is accumulated in the cyto
plasm of nurse cells in germ cellspecific perinuclear
nuage (cloud) granules, where it is colocalized with
another RNA helicase, Vasa, a main nuage marker
[10]. Germinal granules were earlier considered as the
organelles that was associated with RNA processing
and translational regulation [46, 47]. Currently, they
are thought to play a key role in biogenesis of short
piRNAs, which are responsible for silencing trans
posons, causing genome instability, and for regulating
translation of certain RNAs [48]. Belle was also found
KOTOV et al.
mRNA maturation
mRNA export
DDX3
RNAmediated
silencing
Stress granule
formation
in germinal granules of spermatocytes [49]. Belle acts
as an important factor regulating Notch metabolic sig
naling pathway in ovarian follicular cells, which con
trol the late steps of oogenesis [50]. Mutations of belle
result in a massive loss of premeiotic germ cells in the
testis [49]. It seems germ cells lost during early sper
matogenesis in the testes of flies with belle mutations;
i.e., the situation resembles the human SCO syn
drome.
Functions of the DDX3 Proteins in RNA Metabolism
The functions of DDX3X (hereafter referred to as
DDX3) were studied better than the functions of DBY
in mammals. DDX3 is involved in the nuclear mRNA
export, RNA unwinding, translation initiation, tran
scriptional regulation, cellcycle control, and apopto
sis [51–55] (Fig. 1).
Nuclear export. Although a predominantly cyto
plasmic protein, DDX3 can shuttle between the
nucleus and cytoplasm with the help of CRM1 and
TAP nuclear export proteins [54, 56–58]. DDX3 is
recruited to spliced mRNAs, acting as a component of
TAPcontaining RNP complexes and accompanying
their export from the nucleus into the cytoplasm [54].
In addition, DDX3 acts as a component of the
CRM1dependent pathway to facilitate the nuclear
export of partly spliced HIV1 RNA [56]. It should be
noted that inhibition of the DDX3 function may be a
promising strategy for developing antivirus therapies.
DDX3 is not essential for general mRNA export, but
is involved in the export of certain mRNAs, although
the process is not completely understood. At the same
time, there is no convincing evidence that DDX3 plays
a role in the spliceosome function [59, 60]. It seems
more likely that DDX3 binds with spliced transcripts
as a part of RNP complexes in the nucleus.
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
 DEADBOX RNA HELICASES IN ANIMAL GAMETOGENESIS
Transcription. DDX3 is known to interact with
promoters of Ecadherin and IFNβ genes [58, 61, 62]
and transcription factor Sp1and to positively regulate
transcription of p21waf, which encoding cyclin
dependent kinase inhibitor [53, 63]. A lower level of
Dacapo (p21 homolog) was observed in belle mutants
of D. melanogaster [64]. It is interest that DDX3 effect
on p21waf transcription is mediated by ATPase, but
not unwinding, activity [53]. The role of DDX3 in reg
ulating transcription is far from fully understanding,
and the molecular basis of its interactions with pro
moters of specific genes remains unclear.
Translation. Mammalian helicase DDX3 does not
play essential role in regulating total cell translation
[54, 55, 65], in contrast to its homolog DED1 of
Schizosaccaromyces cerevisiae [8, 9]. However, the
helicase is necessary for initiating translation of cer
tain cellular and viral transcripts with highly struc
tured 5'untranslated regions (5'UTRs) [54, 55, 65].
DDX3 physically interacts with certain factors
involved in translation initiation, including eIF4E,
eIF4G, PABP, eIF4A, eIF2, and eIF3 [52, 54, 65, 66].
Interestingly, DDX3 can inhibit capdependent trans
lation by interacting with eIF4E through the con
served eIF4Ebinding motif YxxxxLϕ (38YIPPHLR44
in humans), which is located on the Nterminal
region, and preventing eIF4E from interaction with
the scaffold protein eIF4G [66]. Likewise, Belle inter
acts with eIF4E in D. melanogaster oocytes [67].
DDX3 is essential for the recruitment of the eIF4F
complex to 5' ends of certain mRNAs, ensuring a local
unwinding of 5' end, but is not necessary for subse
quent ribosome scanning [65]. In addition, DDX3
activates IRESdependent translation of hepatitis C
virus RNA [66]. There is evidence that DDX3 inter
acts with the GCrich, highly structured 5'UTR of
cyclin E1 mRNA [55]. The list of mRNAs that have a
structured 5'UTR and are specifically regulated by
DDX3 at the stage of translation initiation is incom
plete to date. DED1, a Schizosaccaromyces pombe
homolog of DDX3, is involved in the translational
control of mRNAs of Btype cyclins (Cig2 and Cdc13),
which have extended and, most likely, highly struc
tured 5'UTRs [68, 69]. Accordingly, mutations of
ded1 gene inhibit cyclin B translation and arrest the
cell cycle at G2/M [69].
It is thought that other transcripts associated with
the cell cycle regulation also compose RNA targets of
DDX3 because a DDX3 knockdown delays the enter
to S phase for HeLa cells [55]. A temperaturesensitive
mutation of DDX3 gene leads to a cell cycle arrest in
G1 in cultured golden hamster cells at a nonpermissive
temperature, which is accompanied by a drop in cyclin
A mRNA [70] and rather suggests a transcriptional
level for regulation.
The formation of RNP granules. DDX3 occurs pre
dominantly in the cytoplasm, in accordance with its
role in translation initiation, and operates as essential
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
19
component of cytoplasmic stress granules, which are
generated after cell exposure to external stress and
contain stalled preinitiation complexes [54, 71, 72].
The role of DDX3 in the formation of stress granules
is independent of its helicase and ATPase activities
and is determined by its Nterminal domain, which is
enriched in arginine and glycine (RG motifs) [72].
The RG motifs may be involved in interactions with
RNA and additionally provide potential sites for argi
nine methylation, which facilitates the formation of
large RNP complexes. However, such methylation of
RG motifs was not still demonstrated for DDX3. Heli
case DDX3 directly interacts with the HIV1 RNA
and is necessary for its translation, along with the
translation initiation factors eIF4G and PABP [65]. In
addition, DDX3 substitutes for eIF4E in recognizing
cap structure of the viral RNA and promotes the for
mation of the DDX3–eIF4G–PABP ternary com
plex. HIV1 RNA is consequently compartmentalized
in large cytoplasmic RNP granules, which contain the
ternary complex and provide a platform for ultimate
formation of translation initiation complexes [73].
DDX3 and RNA interference. Experiments per
formed independently with human HeLa and D. mel
anogaster S2 cells allowed to identify DDX3 and its
homolog Belle as components of RNA interference
pathways, which depend on short RNAs [74–76].
Belle is shown to be coimmunoprecipitated with the
AGO2, FMR1, VIG, and AGO1 components of
siRNA and microRNA silencing complexes [75].
Other authors believe that Belle regulates the Notch
signaling pathway in ovarian follicular cells, then a
Belle knockdown phenotype reproducing the Dicer1
knockdown phenotype [50]. Dicer1 is a key nuclease
that is essential for microRNA biogenesis in Droso
phila. Loss of function of Belle or Dicer1 provides an
delayed activation of the Notch signaling and, in par
ticular, a dereppression of the regulatory protein
Delta. This leads to delay of differentiation and cell
cycle progression in ovarian follicular cells and even
tually results in loss of anteroposterior polarization of
developing oocyte [50]. The findings allow to assume
that Belle contributes in regulating the Notch signal
ing pathway with the involvement of microRNAs. A
role in piRNAmediated silencing, which is specific
for germinal tissues, was not observed for Belle [49].
Further studies are necessary for a better understand
ing of the molecular role of DDX3 in RNA silencing.
Taken together, the data on the involvement of
DDX3 in the transcriptional regulation, nuclear export,
translation initiation, and RNA silencing (Fig. 1) dem
onstrate that the protein performs essential functions
in these processes, which are associated with metabo
lism of specific RNAs. Numerous data implicate
DDX3 in the cell cycle control, carcinogenesis, and
germ cell differentiation. The impact of DDX3 on the
maintenance and differentiation of male germ cells at
early stages is critical for spermatogenesis in both
20 KOTOV et al.
human and Drosophila. Extremely high functional
pleiotropy of DDX3 complicates significantly its
investigation. The identification and analysis of
mRNAs associated with DDX3 in germinal tissues will
enable to study in more details the conserved functions
of the protein in maintaining gametogenesis in
eukaryotes.
SPINDLEE HELICASE
SpindleE Helicase Is Required for Oogenesis
and Spermatogenesis Promotion in Drosophila
SpindleE (or homeless) gene encoding another
RNA helicase that is specific to germ cells and belongs
to the DExH subfamily of DEADbox family was
identified in a genetic screening for female sterility
mutations in D. melanogaster [11]. This gene encodes
RNA helicase containing an additional Tudor
domain, whose specific function is to recognize meth
ylated arginine and lysine residues [77]. Loss of func
tion mutations of spindleE cause multiple defects in
oogenesis. They often lead to incorrect position of the
oocyte, which normally occurs at the posterior pole of
the egg chamber, owing to that initial name of gene
(homeless) emerges [11, 12]. Helicase SpindleE is
need for the formation of the anteroposterior and dor
soventral axes of the oocyte and, consequently,
oncoming embryo [11, 12, 78]. The above defects in
oogenesis arise owing to the disturbances of transport
and localization of key mRNAs (K10, bicoid, and
oskar), and decreasing expression level of Gurken, a
ligand of the receptor for the epidermal growth factor
that responsible for the dorsoventral oocyte polariza
tion in late oogenesis [11, 12, 78]. gurken mRNA level
remains unchanged in spindleE mutants, suggesting a
control of translational regulation [12].
Role of SpindleE in piRNAMediated Silencing
spindleE mutations also cause defects of sper
matogenesis, which include meiotic chromosome
nondisjunction, chromosome bridges in anaphase,
chromosome fragmentation, loss of chromosome
material, and tripolar meiosis. Simultaneously it is
found that Stellate repeats are derepressed, and nee
dlelike Stellate crystals accumulate in primary sper
matocytes [79]. The meiotic defects observed in sper
matogenesis in the lack of SpindleE are similar to
those characteristics of a deletion of Suppressor of Stel
late (Su(Ste)) locus. The Stellate–Su(Ste) genetic sys
tem consists of two components, tandem Stellate
genes, which are located on the X chromosome, and
homologous Su(Ste) repeats, which are placed on the
Y chromosome. When the Y chromosome is missing
protein crystals accumulate in primary spermatocytes
[80, 81]. Actually overexpression of Stellate protein in
the absence of Su(Ste) locus, leads to the crystal for
mation and the observed meiotic defects [82]. It
remained obscure for many years how Su(Ste) locus
regulated expression of Stellate repeats. After the dis
covery of RNA interference and a germlinespecific
piRNAmediated silencing pathway it has been found
that, SpindleE is essential for biogenesis of short
piRNAs and silencing the mobile genetic elements in
the ovary and Stellate repeats in the testis [83–85].
Stellate repression by Su(Ste) repeats is based on bidi
rectional transcription of Su(Ste) locus and generation
of short Su(Ste) antisense piRNAs, which are comple
mentary to Stellate sequence [83]. The mechanism
whereby SpindleE affects the piRNA production is
still unknown. Considering that SpindleE is accumu
lated in germinal RNP nuage granules [48, 49], a spe
cific structure that provides compartment for both
piRNAmediated silencing and generation of new
piRNAs, one can assume that SpindleE contributes
as RNA helicase to both processes. However, there is
no experimental evidence to support this assumption,
nor a direct interaction is demonstrated for SpindleE
and the key piRNAmediated silencing proteins Aub
ergine and AGO3 [86].
Mammalian Homologs of SpindleE
SpindleE homologs (TDRD9) are found in
human, mouse, and other vertebrates. Mouse RNA
helicase TDRD9 is essential for normal spermatogen
esis and is involved in silencing of mobile genetic ele
ments, in particular, Line1 retrotransposon [87]. In
mouse embryonic prospermatogonia (gonocytes),
TDRD9 functions in complex with MIWI2 (a func
tional analog of Drosophila Aubergine), and these two
proteins is found together in cytoplasmic granules
known as the piPbodies (Fig. 2) [87, 88]. The pheno
typic manifestation of Tdrd9 mutations is similar to
that of Miwi2 mutations, which result in derepression
of Line1 transposon, demethylation of the corre
sponding DNA sequences, and disturbances in late
spermatogenesis [89, 90]. Males homozygous for
Tdrd9 mutation are found to be sterile [89, 91]. Tdrd9
is expressed in the mouse embryonic ovaries as well,
but its mutations do not cause considerable distur
bances in oogenesis, and mutant females are fertile. It
seems that another pathway, which involves Dicer and
siRNAs, is responsible for mobile element silencing in
the oocytes [92, 93]. As already mentioned, TDRD9
and MIWI2 function within the same cytoplasmic
piPbodies, and these granules additionally contain
GW182, DCP1a, DDX6/p54, and XRN1 proteins,
known as components of processing (P) bodies identi
fied initially in somatic tissues [87, 88]. The socalled
pibodies (also known as intermitochondrial cement)
are granules of another type that are found in the
immediate vicinity of the piPbodies in the cytoplasm
of embryonic gonocytes and contain MILI, which is a
functional analog of Drosophila AGO3, and TDRD1,
which is another Tudor domaincontaining protein
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
 DEADBOX RNA HELICASES IN ANIMAL GAMETOGENESIS
Gonocytes Spermatogonia Late
and early spermatocytes
spermatocytes
Birth
piPbodies, pibodies pibodies,
pibodies CB
Designations:
piPbodies piPbodies (IMC)
Fig. 2. Germinal granules at various stages of mouse spermatogensis. Consecutive steps of germ cell differentiation are shown.
Embryonic gonocytes contain two types of granules differing in protein composition, piP and pibodies (the latter also known
as intermitochondrial cement, IMC). Postnatal spermatogonia and early spermatocytes contain pibodies as germinal granules.
Chromatoid body (CB) precursors start to form in mature spermatocytes. The ultimate CB forms in round spermatids and grad
ually degenerates in elongated spermatids. Modified from [94].
(Fig. 2) [88]. A similar compartmentalization of the
components of the piRNAmediated silencing path
way is observed in Drosophila primary spermatocytes
due to the formation of piNGbodies [49, 94]. The
piNGbody is a relatively large granule and consists of
one central and several peripheral lobes. SpindleE,
Aubergine, and DCP1 are accumulated in the periph
eral lobes, while AGO3 occurs at the center of the
granule. Thus, the components of the piRNAmedi
ated silencing pathway and proteins involved in
mRNA degradation (e.g., decapping protein 1,
DCP1) are compartmentalized in both insect and
mammal germ cells to ensure a high efficiency of
piRNA biogenesis. SpindleE, Aubergine, and DCP1
in Drosophila and TDRD9, MIWI2, and DCP1a in
mouse are components of functionally similar germi
nal granules. However, the functional significance of
this association is still unclear.
The multiplicity of SpindleE functions (the trans
lational control of specific mRNAs, including mRNA
gurken, and the role in the biogenesis of short piRNA
and piRNAmediated silencing) is determined by two
different domains, the helicase/ATPase and Tudor
domains. The helicase domain allows SpindleE to
unwind the secondary and tertiary RNA structures
and to remodel RNP complexes, utilizing the energy
of ATP hydrolysis. Tudor domaincontaining proteins
are considered to be important structural components
of piRNAmediated silencing complexes in germinal
nuage granules because the Tudor domain is capable of
interacting with symmetrically methylated arginine
residues in proteins of the PIWI subfamily, which
includes Aubergine and AGO3 [86–88, 95, 96]. How
ever, the role of RNA helicase SpindleE in the
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
21
Round Elongated Sperm
spermatids spermatids
Meiosis
CB CB –
Chromatoid body (CB)
piRNAmediated silencing pathway remains obscure.
The enzyme could protect Aubergine and AGO3 from
premature degradation or could activate translation of
their mRNAs.
VASA (DDX4) HELICASE
Role of Maternal Vasa in Embryonic Gonad
Development
vasa gene was initially identified in D. melanogaster
in a genetic screening for maternaleffect lethal muta
tions [4–6]. In Drosophila, germline precursor pole
cells specialize early, at the syncytial stage of embryo
cleavage, and their specialization is determined by
maternal factors accumulated in polar granules at the
posterior pole of the oocyte. Mutations of genes
encoding polar granule components impair the
gonadal development and cause sterility of the next
generation (the socalled grandchildless effect). The
products of many grandchildless genes are necessary
for pole plasm assembly. The most essential roles in the
cascade are played by oskar and vasa products because
polar granules do not form in their absence [6]. Both
Vasa and Oskar are necessary for the localization and
translation of specific mRNAs, such as nanos, gurken,
pumilio, cyclinB, mtlrRNA, Hsp83, and orb, in polar
granules [97–100]. The proteins encoded by these
mRNAs are essential for embryo development. Trans
lation of nanos mRNA in the pole plasm at the poste
rior pole of the embryo generates a gradient of the
Nanos morphogen, while lack of the maternal nanos
mRNA in the pole plasm causes defects in abdominal
segmentation of the developing embryo and prevents
pole cell migration into the developing gonads [99].
22 KOTOV et al.
Thus, maternal Vasa is necessary for embryonic gonad
development and abdominal segmentation, but the
target mRNAs directly regulated by Vasa in the process
are still unidentified.
The fate of prospective germ cells is determined in
early embryo development by the maternal factors
including Vasa in polar granules in Drosophila, Cae
norhabditis elegans, and Xenopus laevis. In contrast,
maternal factors were not found to play a role in the
process in mammals. Germline cell precursors arise
from the pluripotent epiblast cell population, and their
development is determined by surrounding somatic
cells via celltocell signaling [99, 101].
Vasa Functions in Gametogenesis
RNA helicase Vasa in Drosophila and its homologs
in mammals, birds (Gallus gallus domesticus), nema
tode (Caenorhabditis elegans), fish (Brachydanio
rerio), and amphibians (Xenopus laevis) are specific
markers of germline cells [102]. Deficit of vasa prod
uct violates many aspects of oogenesis in Drosophila.
Only a minor portion of oocytes complete oogenesis,
while the others degenerate [6, 103]. The molecular
mechanism of pathogenesis associated with lack of
Vasa function is unknown, but it is showed that similar
oogenesis defects often correspond to disturbances of
SpindleE expression, which also functions as a RNA
helicase and a component of nuage (see above). In
oogenesis Vasa acts as a translational activator of spe
cific mRNAs, such as gurken and meiP26 mRNAs
[104, 105]. Vasa positively regulates the production of
MeiP26 in vivo by interacting with an Urich element
in 3'UTR of meiP26 mRNA. MeiP26 is necessary
for limiting the growth and proliferation of germ cells
in early oogenesis. Its synthesis is low in germ stem
cells and extremely intense in 16cell cysts. Lack of
MeiP26 expression stimulates the intense germ cell
growth and proliferation, leading to ovarian and tes
ticular tumor [106, 107]. It is thought that Vasa physi
cally interacts with the translation initiation factor
eIF5B (also known as dIF2) to positively regulate
translation of the mRNAs essential for oogenesis [104,
105, 108]. A distortion of Vasa–eIF5B interaction by a
mutation substantially reduces the level of gurken
translation, leads to female sterility, and determines a
phenotype identical to that of the null mutations of
vasa in Drosophila [104].
Zygotic expression of Vasa is not found to be essen
tial in maintaining spermatogenesis in Drosophila, and
males with any known silent vasa alleles are fertile
[6, 103]. It is unclear whether this circumstance may
point to a principal difference in germ cellmaintain
ing mechanism between males and females. The idea
that Vasa is necessary for germ cell maintenance and
development in oogenesis, but not in spermatogenesis,
seems paradoxical in view of the fact that Vasa regu
lates translation of meiP26 mRNA. Deficiencies in
MeiP26 expression results in an accumulation of non
differentiated multicellular cysts in the ovaries and tes
tes [107]. In addition, Vasa was recently found to play an
important role in piRNAmediated silencing in both
male and female germinal tissues of Drosophila.
Role of Vasa in piRNAMediated Silencing
Elevated expression of mobile elements in germinal
tissues increases the transposition rate and generates
potentially harmful insertions and doublestrand
breaks in genetic material to be passed to the next gen
eration. As discovered in the early 21th century,
piRNAmediated silencing serves to protect the
genome integrity from active endogenous elements
(transposons and repeats) in the germline [83–85,
109]. A major pool of piRNAs originate from long
transcripts of the socalled piRNA clusters, which are
particular genome regions that lack genes and contain
fragments of mobile elements [110]. The generation of
piRNAs and piRNAmediated silencing occur mostly
in cytoplasmic germinal nuage granules. When Vasa
expression is absent, nuage granules become undetect
able (Fig. 3) and piRNAmediated silencing is dis
torted in the Drosophila ovaries and testes, resulting in
mobilization of transposons and Stellate genes overex
pression [49, 85, 109, 111]. Vasa is a basal architectural
element of nuage granules because the occurrence of
all cytoplasmic nuage components identified to date in
nuage appears to be Vasadependent [48, 49, 85]. In
ovarian nurse cells, Vasa is associated with the cyto
plasmic side of nuclear pores, which mediate the
export of long piRNA precursor transcripts produced
from a large piRNA cluster. The export of the tran
scripts through the pores involves both nuclear heli
case UAP56 and the HP1 homolog Rhino, whose
expression is specific to the ovaries [112]. It can be
assumed that the function of Vasa in nuage granules is
to clamp the transcripts transported from the nucleus
and to present them to factors responsible for their fur
ther processing.
Mammalian Homologs of Vasa
The protein known as mouse Vasa homolog
(MVH) is expressed in mouse germ cells since they
colonize the embryonic gonads, and its synthesis con
tinues up to the stage of postmeiotic spermatids and
firstorder oocytes [113]. In the mouse testes, MVH is
found in cytoplasmic granules, including piP and pi
bodies of fetal gonocytes [87, 88], and the chromatoid
body, which ultimately forms in round spermatids
(Fig. 2) [114]. Although MVH is produced in both
male and female germinal tissues, alterations of its
synthesis exert an adverse effect on spermatogenesis,
but not on female fertility. Male mice with Mvh muta
tions fail to produce the sperm because their sper
matogenesis is arrested in meiosis between leptotene
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
 DEADBOX RNA HELICASES IN ANIMAL GAMETOGENESIS 23

(a)
Vasa Aubergine DAPI Superimposition

vasa
mutation

Norm

(b)
Vasa AGO3 DAPI Superimposition

vasa
mutation

Norm

Fig. 3. Nuage granules are disrupted in the background of vasa mutation. Confocal microscopy sections of spermatocyte nuclei
were obtained for D. melanogaster males with vasa mutation and their heterozygous siblings used as a wildtype control. The prep
arations were stained using antibodies against (a) Vasa and Aubergine or (b) Vasa and AGO3, with DAPI staining of chromatin.
Signals from (a) Vasa and Aubergine or (b) Vasa and AGO3 are in norm colocalized in small nuage granules and large piNGbody
(arrow). Scale bar, 5 µm. Single large nuage granules, piNG–bodies indicated by arrows, and small nuage granules visible as rings
around the nuclei are clearly detectable in spermatocytes from control testes. Granules of both types disappear in the background
of vasa mutation, and Aubergine and AGO3 replace from nuage to the cytoplasm [49].

and zygotene. Premeiotic spermatocytes display a high
rate of apoptosis. As a result, pachytene spermatocytes
and postmeiotic germ cells are completely absent from
mice homozygous for a mutation disrupting Mvh
expression. MVH is necessary for de novo methylation
of DNA loci harboring the Line1 and IAP retrotrans
posons. MVH is important for the formation of germi
nal granules, production of piRNAs, and piRNA
mediated silencing of transposons in the fetal testes
[115]. Spermatogenetic defects observed in mice with
altered MVH expression are similar to those in MILI
or MIWI2deficient mice [90]. Although MVH was
not observed to directly interact with piRNA, the pro
duction of MILIassociated piRNAs is substantially
decreased in the MVHdeficient fetal testes [115].
Thus, the Vasa homolog acts as an essential factor of
piRNA silencing in mice, as in Drosophila. A total of
858 MVHassociated mRNAs were identified in the
adult mouse testes, and some of them are important
for spermatogenesis [116]. These include the mRNAs
of RNA helicase DDX25, carnitine acetyltransferase,
and translation initiation factor eIF4B. It was also
shown that histone acetyltransferase Hat1 is a compo
nent of the chromatoid body and acetylates MVH at
Lys405 at a certain developmental stage. This modifi
cation abolishes RNAbinding, but not ATPase activ
ity of MVH [116]. Presumably, mRNAs in complex
with MVH are stored in a translationally inactive state
in the chromatoid body and that MVH acetylation
facilitates their release and transfer into polysomes in
the cytoplasm.
It should be noted that a human homolog of Vasa is
similarly synthesized in germ cells after their migra
tion into the fetal gonads [117] and that its ectopic
expression in pluripotent stem cells leads to their dif
ferentiation to germ cells [118]. A decreased produc

MOLECULAR BIOLOGY Vol. 48 No. 1 2014

24 KOTOV et al.
tion of the human Vasa homolog may result from
hypermethylation of promoter region in its gene and
leads to strong oligospermia [119].
Thus Vasa is a conserved protein of germinal tissues
in multicellular eukaryotes. Like other RNA helicases
Vasa performs a variety of functions, playing an archi
tectonic and functional roles in RNP complexes and
being involved in RNP remodeling, the regulation of
mRNA translation, and piRNAmediated silencing.
CONCLUSIONS
According to many structural and functional stud
ies DEADbox RNA helicases play key roles in
eukaryotic development and gametogenesis. Three
RNA helicases considered in this review perform
essential conserved functions during the development
and maintenance of germinal tissues and are involved
in several different processes in vivo, including trans
lational regulation, RNA transport, RNP complex
formation, RNAmediated silencing, etc. The ques
tion arises whether the functions of these three
enzymes are redundant or overlap to a certain extent.
Studies performed in Drosophila in this century shed
light on the problem. The roles of SpindleE and Vasa
in piRNAmediated silencing are not redundant
because expression of either protein does not compen
sate for a deficit of the other, in spite of the phenotypic
similarity of the corresponding mutants. Structurally
close Vasa and Belle seem to regulate different meta
bolic pathways. Further studies directed to identify
specific RNAs associated with these proteins will pro
vide for a better understanding of their functions in
germinal tissues.
ACKNOWLEDGMENTS
We are grateful to V.A. Gvozdev for helpful discus
sion and valuable comments.
This work was supported by the program “Molecu
lar and Cell Biology” of the Presidium of the Russian
Academy of Sciences and the Russian Foundation for
Basic Research (project nos. 120431155, 120433093,
and 130400699).
REFERENCES
1. Gorbalenya A.E., Koonin E.V. 1993. Helicases:
Amino acid comparisons and structure–function rela
tionships. Curr. Opin. Struct. Biol. 3, 419–429.
2. Linder P., Lasko P.F., Ashburner M., Leroy P.,
Nielsen P.J., Nishi K., Schnier J., Slonimski P.P. 1989.
Birth of the DEAD box. Nature. 337, 121–122.
3. Linder P., Jankowsky E. 2011. From unwinding to
clamping: The DEAD box RNA helicase family.
Nature Rev. Mol. Cell Biol. 12, 505–516.
4. Schupbach T., Wieschaus E. 1986. Germline auton
omy of maternaleffect mutations altering the embry
onic body pattern of Drosophila. Dev. Biol. 113, 443–
448.
5. Hay B., Jan L.Y., Jan Y.N. 1988. A protein component
of Drosophila polar granules is encoded by vasa and has
extensive sequence similarity to ATPdependent heli
cases. Cell. 55, 577–587.
6. Lasko P.F., Ashburner M. 1990. Posterior localization
of vasa protein correlates with, but is not sufficient for,
pole cell development. Genes Dev. 4, 905–921.
7. Lahn B.T., Page D.C. 1997. Functional coherence of
the human Y chromosome. Science. 278, 675–680.
8. Chuang R.Y., Weaver P. L., Liu Z., Chang T.H. 1997.
Requirement of the DEADbox protein Ded1p for
messenger RNA translation. Science. 275, 1468–1471.
9. De la Cruz J., Iost I., Kressler D., Linder P. 1997. The
p20 and Ded1 proteins have antagonistic roles in
eIF4Edependent translation in Saccharomyces cere
visiae. Proc. Natl. Acad. Sci. U. S. A. 94, 5201–5206.
10. Johnstone O., Deuring R., Bock R., Linder P.,
Fuller M.T., Lasko P. 2005. Belle is a Drosophila
DEADbox protein required for viability and in the
germ line. Dev. Biol. 277, 92–101.
11. Gillespie D.E., Berg C.A. 1995. Homeless is required
for RNA localization in Drosophila oogenesis and
encodes a new member of the DEH family of RNA
dependent ATPases. Genes Dev. 9, 2495–2508.
12. GonzálezReyes A., Elliott H., St. Johnston D. 1997.
Oocyte determination and the origin of polarity in
Drosophila: The role of the spindle genes. Development.
124, 4927–4937.
13. Sengoku T., Nureki O., Nakamura A., Kobayashi S.,
Yokoyama S. 2006. Structural basis for RNA unwind
ing by the DEADbox protein Drosophila Vasa. Cell.
125, 287–300.
14. Linder P., FullerPace F.V. 2013. Looking back on the
birth of DEADbox RNA helicases. Biochim. Biophys.
Acta. 1829, 750–755.
15. Jankowsky E. 2011. RNA helicases at work: Binding
and rearranging. Trends Biochem. Sci. 36, 19–29.
16. Jarmoskaite I., Russell R. 2011. DEADbox proteins
as RNA helicases and chaperones. Wiley Interdiscip.
Rev. RNA. 2, 135–152.
17. Pyle A.M. 2008. Translocation and unwinding mecha
nisms of RNA and DNA helicases. Ann. Rev. Biophys.
37, 317–336.
18. Yang Q., Jankowsky, E. 2006. The DEADbox protein
Ded1 unwinds RNA duplexes by a mode distinct from
translocating helicases. Nature Struct. Mol. Biol. 13,
981–986.
19. Yang Q., Del Campo M., Lambowitz A.M., Jan
kowsky E. 2007. DEADbox proteins unwind duplexes
by local strand separation. Mol. Cell. 28, 253–263.
20. Chen Y., Potratz J.P., Tijerina P., Del Campo M.,
Lambowitz A.M., Russell R. 2008. DEADbox pro
teins can completely separate an RNA duplex using a
single ATP. Proc. Natl. Acad. Sci. U. S. A. 105, 20203–
20208.
21. Henn A., Cao W., Licciardello N., Heitkamp S.E.,
Hackney D.D., De La Cruz E.M. 2010. Pathway of
ATP utilization and duplex rRNA unwinding by the
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
 DEADBOX RNA HELICASES IN ANIMAL GAMETOGENESIS
DEADbox helicase, DbpA. Proc. Natl. Acad. Sci.
U. S. A. 107, 4046–4050.
22. Grifo J.A., Abramson R.D., Satler C.A., Merrick W.C.
1984. RNAstimulated ATPase activity of eukaryotic
initiation factors. J. Biol. Chem. 259, 8648–8654.
23. Liu F., Putnam A., Jankowsky E. 2008. ATP hydrolysis
is required for DEADbox protein recycling but not for
duplex unwinding. Proc. Natl. Acad. Sci. U. S. A. 105,
20209–20214.
24. Fairman M.E., Maroney P.A., Wang W., Bowers H.A.,
Gollnick P., Nilsen T.W., Jankowsky E. 2004. Protein
displacement by DExH/D “RNA helicases” without
duplex unwinding. Science. 304, 730–734.
25. Lund M.K., Guthrie C. 2005. The DEADbox protein
Dbp5p is required to dissociate Mex67p from exported
mRNPs at the nuclear rim. Mol. Cell. 20, 645–651.
26. Tran E.J., Zhou Y., Corbett A.H., Wente S.R. 2007.
The DEADbox protein Dbp5 controls mRNA export
by triggering specific RNA : protein remodeling
events. Mol. Cell. 28, 850–859.
27. Rossler O.G., Straka A., Stahl H. 2001. Rearrange
ment of structured RNA via branch migration struc
tures catalysed by the highly related DEADbox pro
teins p68 and p72. Nucleic Acids Res. 29, 2088–2096.
28. Chamot D., Colvin K.R., KujatChoy S.L., Owttrim G.W.
2005. RNA structural rearrangement via unwinding
and annealing by the cyanobacterial RNA helicase,
CrhR. J. Biol. Chem. 280, 2036–2044.
29. Yang Q., Jankowsky E. 2005. ATP and ADPdepen
dent modulation of RNA unwinding and strand
annealing activities by the DEADbox protein DED1.
Biochemistry. 44, 13591–13601.
30. UhlmannSchiffler H., Jalal C., Stahl H. 2006.
Ddx42p, a human DEAD box protein with RNA
chaperone activities. Nucleic Acids Res. 34, 10–22.
31. Bono F., Ebert J., Lorentzen E., Conti E. 2006. The
crystal structure of the exon junction complex reveals
how it maintains a stable grip on mRNA. Cell. 126,
713–725.
32. Andersen C.B., Ballut L., Johansen J.S., Chamieh H.,
Nielsen K.H., Oliveira C.L., Pedersen J.S., Seraphin B.,
Le Hir H., Andersen G.R. 2006. Structure of the exon
junction core complex with a trapped DEADbox
ATPase bound to RNA. Science. 313, 1968–1972.
33. Sonenberg N., Hinnebusch A.G. 2009. Regulation of
translation initiation in eukaryotes: Mechanisms and
biological targets. Cell. 136, 731–745.
34. Parsyan A., Svitkin Y., Shahbazian D., Gkogkas C.,
Lasko P., Merrick W.C., Sonenberg N. 2011. mRNA
helicases: The tacticians of translational control.
Nature Rev. Mol. Cell Biol. 12, 235–245.
35. Jackson R.J., Hellen C.U., Pestova T.V. 2010. The
mechanism of eukaryotic translation initiation and
principles of its regulation. Nature Rev. Mol. Cell Biol.
11, 113–127.
36. Rozovsky N., Butterworth A.C., Moore M.J. 2008.
Interactions between eIF4AI and its accessory factors
eIF4B and eIF4H. RNA. 14, 2136–2148.
37. Rogers G.W.Jr., Richter N.J., Lima W.F., Merrick W.C.
2001. Modulation of the helicase activity of eIF4A by
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
25
eIF4B, eIF4H, and eIF4F. J. Biol. Chem. 276, 30914–
30922.
38. Rosner A., Rinkevich B. 2007. The DDX3 subfamily
of the DEAD box helicases: Divergent roles as
unveiled by studying different organisms and in vitro
assays. Curr. Med. Chem. 14, 2517–2525.
39. Ditton H.J., Zimmer J., Kamp C., RajpertDe Meyts E.,
Vogt P.H. 2004. The AZFa gene DBY (DDX3Y) is
widely transcribed but the protein is limited to the
male germ cells by translation control. Hum. Mol.
Genet. 13, 2333–2341.
40. Tiepolo L., Zuffardi O. 1976. Localization of factors
controlling spermatogenesis in the nonfluorescent
portion of the human Y chromosome long arm. Hum.
Genet. 34, 119–124.
41. Foresta C., Ferlin A., Moro E. 2000. Deletion and
expression analysis of AZFa genes on the human Y
chromosome revealed a major role for DBY in male
infertility. Hum. Mol. Genet. 9, 1161–1169.
42. Foresta C., Moro E., Ferlin A. 2001. Y chromosome
microdeletions and alterations of spermatogenesis.
Endocr. Rev. 22, 226–239.
43. Lardone M.C., Parodi D.A., Valdevenito R.,
Ebensperger M., Piottante A., Madariaga M., Smith R.,
Pommer R., Zambrano N., Castro A. 2007. Quantifi
cation of DDX3Y, RBMY1, DAZ, and TSPY mRNAs in
testes of patients with severe impairment of spermato
genesis. Mol. Hum. Reprod. 13, 705–712.
44. NavarroCosta P., Plancha C.E., Gonçalves J. 2010.
Genetic dissection of the AZF regions of the human
Y chromosome: Thriller or filler for male (in)fertility?
J. Biomed. Biotechnol. 2010, 936569.
45. Kamp C., Huellen K., Fernandes S., Sousa M., et al.
2001. High deletion frequency of the complete AZFa
sequence in men with Sertolicellonly syndrome.
Mol. Hum. Reprod. 7, 987–994.
46. Findley S.D., Tamanaha M., Clegg N.J., Ruohola
Baker H. 2003. Maelstrom, a Drosophila spindleclass
gene, encodes a protein that colocalizes with Vasa and
RDE1/AGO1 homolog, Aubergine, in nuage. Devel
opment. 130, 859–871.
47. Snee M.J., Macdonald P.M. 2004. Live imaging of
nuage and polar granules: Evidence against a precur
sor–product relationship and a novel role for Oskar in
stabilization of polar granule components. J. Cell. Sci.
117, 2109–2120.
48. Lim A.K., Kai T. 2007. A unique germline organelle,
Nuage, functions to repress selfish genetic elements in
Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A.
104, 6714–6719.
49. Kibanov M.V., Egorova K.S., Ryazansky S.S.,
Sokolova O.A., Kotov A.A., Olenkina O.M., Stol
yarenko A.D., Gvozdev V.A., Olenina L.V. 2011. A
novel organelle, the piNGbody, in the nuage of
Drosophila male germ cells is associated with piRNA
mediated gene silencing. Mol. Biol. Cell. 22, 3410–
3419.
50. Poulton J.S., Huang Y.C., Smith L., Sun J., Leake N.,
Schleede J., Stevens L.M., Deng W.M. 2011. The
microRNA pathway regulates the temporal pattern of
26 KOTOV et al.
Notch signaling in Drosophila follicle cells. Develop
ment. 138, 1737–1745.
51. Schröder M. 2010. Human DEADbox protein 3 has
multiple functions in gene regulation and cell cycle
control and is a prime target for viral manipulation.
Biochem. Pharmacol. 79, 297–306.
52. Lee C.S., Dias A.P., Jedrychowski M., Patel A.H.,
Hsu J.L., Reed R. 2008. Human DDX3 functions in
translation and interacts with the translation initiation
factor eIF3. Nucleic Acids Res. 36, 4708–4718.
53. Chao C.H., Chen C.M., Cheng P.L., Shih J.W.,
Tsou A.P., Lee Y.H. 2006. DDX3, a DEAD box RNA
helicase with tumor growthsuppressive property and
transcriptional regulation activity of the p21waf1/cip1
promoter, is a candidate tumor suppressor. Cancer Res.
66, 6579–6588.
54. Lai M.C., Lee Y.H., Tarn W.Y. 2008. The DEADbox
RNA helicase DDX3 associates with export messenger
ribonucleoproteins as well as tipassociated protein
and participates in translational control. Mol. Biol.
Cell. 19, 3847–3858.
55. Lai M.C., Chang W.C., Shieh S.Y., Tarn W.Y. 2010.
DDX3 regulates cell growth through translational con
trol of cyclin E1. Mol. Cell. Biol. 30, 5444–5453.
56. Yedavalli V.S., Neuveut C., Chi Y.H., Kleiman L.,
Jeang K.T. 2004. Requirement of DDX3 DEAD box
RNA helicase for HIV1 RevRRE export function.
Cell. 119, 381–392.
57. Sekiguchi T., Iida H., Fukumura J., Nishimoto T.
2004. Human DDX3Y, the Yencoded isoform of
RNA helicase DDX3, rescues a hamster temperature
sensitive ET24 mutant cell line with a DDX3X muta
tion. Exp. Cell Res. 300, 213–222.
58. Schröder M., Baran M., Bowie A.G. 2008. Viral tar
geting of DEAD box protein 3 reveals its role in
TBK1/IKKepsilonmediated IRF activation. EMBO J.
27, 2147–2157.
59. Zhou Z., Licklider L.J., Gygi S.P., Reed R. 2002.
Comprehensive proteomic analysis of the human spli
ceosome. Nature. 419, 182–185.
60. Merz C., Urlaub H., Will C.L., Luhrmann R. 2007.
Protein composition of human mRNPs spliced in vitro
and differential requirements for mRNP protein
recruitment. RNA. 13, 116–128.
61. Soulat D., Bürckstümmer T., Westermayer S., Gon
calves A., Bauch A., Stefanovic A., Hantschel O., Ben
nett K.L., Decker T., SupertiFurga G. 2008. The
DEADbox helicase DDX3X is a critical component
of the TANKbinding kinase 1dependent innate
immune response. EMBO J. 27, 2135–2146.
62. Botlagunta M., Vesuna F., Mironchik Y., et al., 2008.
Oncogenic role of DDX3 in breast cancer biogenesis.
Oncogene. 27, 3912–3922.
63. Wu D.W., Liu W.S., Wang J., Chen C.Y., Cheng Y.W.,
Lee H. 2011. Reduced p21 (WAF1/CIP1) via alter
ation of p53DDX3 pathway is associated with poor
relapsefree survival in earlystage human papilloma
virusassociated lung cancer. Clin. Cancer Res. 17,
1895–1905.
64. Ambrus A.M., Frolov M.V. 2010. Mutation of the
DEADbox helicase belle downregulates the cyclin
dependent kinase inhibitor Dacapo. Cell Cycle. 9,
1016–1020.
65. SotoRifo R., Rubilar P.S., Limousin T., de Breyne S.,
Décimo D., Ohlmann T. 2012. DEADbox protein
DDX3 associates with eIF4F to promote translation of
selected mRNAs. EMBO J. 31, 3745–3756.
66. Shih J.W., Tsai T.Y., Chao C.H.,Wu Lee Y.H. 2008.
Candidate tumor suppressor DDX3 RNA helicase
specifically represses capdependent translation by
acting as an eIF4E inhibitory protein. Oncogene. 27,
700–714.
67. Yarunin A., Harris R.E., Ashe M.P., Ashe H.L. 2011.
Patterning of the Drosophila oocyte by a sequential
translation repression program involving the d4EHP
and Belle translational repressors. RNA Biol. 8, 904–
912.
68. Daga R.R., Jimenez J. 1999. Translational control of
the cdc25 cell cycle phosphatase: A molecular mecha
nism coupling mitosis to cell growth. J. Cell Sci. 112,
3137–3146.
69. Grallert B., Kearsey S.E., Lenhard M., Carlson C.R.,
Nurse P., Boye E., Labib K. 2000. A fission yeast gen
eral translation factor reveals links between protein
synthesis and cell cycle controls. J. Cell Sci. 113,
1447–1458.
70. Fukumura J., Noguchi E., Sekiguchi T., Nishimoto T.
2003. A temperaturesensitive mutant of the mamma
lian RNA helicase, DEADBOX X isoform, DBX,
defective in the transition from G1 to S phase. J. Bio
chem. 134, 71–82.
71. Goulet I., Boisvenue S., Mokas S., Mazroui R.,
Côté J. 2008. TDRD3, a novel Tudor domaincontain
ing protein, localizes to cytoplasmic stress granules.
Hum. Mol. Genet. 17, 3055–3074.
72. Shih J.W., Wang W.T., Tsai T.Y., Kuo C.Y., Li H.K., Wu
Lee Y.H. 2012. Critical roles of RNA helicase DDX3
and its interactions with eIF4E/PABP1 in stress gran
ule assembly and stress response. Biochem. J. 441,
119–129.
73. SotoRifo R., Rubilar P.S., Ohlmann T. 2013. The
DEADbox helicase DDX3 substitutes for the cap
binding protein eIF4E to promote compartmentalized
translation initiation of the HIV1 genomic RNA.
Nucleic Acids Res. 41, 6286–6299.
74. Ulvila J., Parikka M., Kleino A., Sormunen R.,
Ezekowitz R.A., Kocks C., Rämet M. 2006. Double
stranded RNA is internalized by scavenger receptor
mediated endocytosis in Drosophila S2 cells. J. Biol.
Chem. 281, 14370–14375.
75. Zhou R., Hotta I., Denli A.M., Hong P., Perrimon N.,
Hannon G.J. 2008. Comparative analysis of argo
nautedependent small RNA pathways in Drosophila.
Mol. Cell. 32, 592–599.
76. Kasim V., Wu S., Taira K., Miyagishi M. 2013. Deter
mination of the role of DDX3 a factor involved in
mammalian RNAi pathway using an shRNAexpres
sion library. PLoS ONE. 8, e59445.
77. Siomi M.C., Mannen T., Siomi H. 2010. How does the
royal family of Tudor rule the PIWIinteracting RNA
pathway? Genes Dev. 24, 636–646.
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
 DEADBOX RNA HELICASES IN ANIMAL GAMETOGENESIS
78. GonzálezReyes A., St. Johnston D. 1994. Role of
oocyte position in establishment of anteriorposterior
polarity in Drosophila. Science. 266, 639–642.
79. Stapleton W., Das S., McKee B.D. 2001. A role of the
Drosophila homeless gene in repression of Stellate in
male meiosis. Chromosoma. 110, 228–240.
80. Hardy R.W., Lindsley D.L., Livak K.J., Lewis B.,
Siversten A.V., Joslyn G.L., Edwards J., Bonaccorsi S.
1984. Cytogenetic analysis of a segment of the Y chro
mosome of Drosophila melanogaster. Genetics. 107,
591–610.
81. Livak K.J. 1984. Organization and mapping of a
sequence on the Drosophila melanogaster X and Y
chromosomes that is transcribed during spermatogen
esis. Genetics. 107, 611–634.
82. Bozzetti M.P., Massari S., Finelli P., et al. 1995. The
Ste locus, a component of the parasitic crySte system
of Drosophila melanogaster, encodes a protein that
forms crystals in primary spermatocytes and mimics
properties of the βsubunit of casein kinase 2. Proc.
Natl. Acad. Sci. U. S. A. 92, 6067–6071.
83. Aravin A.A., Naumova N.M., Tulin A.V., Vagin V.V.,
Rozovsky Y.M., Gvozdev V.A. 2001. Doublestranded
RNAmediated silencing of genomic tandem repeats
and transposable elements in the D. melanogaster ger
mline. Curr. Biol. 11, 1017–1027.
84. Vagin V.V., Sigova A., Li C., Seitz H., Gvozdev V.,
Zamore P.D. 2006. A distinct small RNA pathway
silences selfish genetic elements in the germline. Sci
ence. 313, 320–324.
85. Malone C.D., Brennecke J., Dus M., Stark A.,
McCombie W.R., Sachidanandam R., Hannon G.J.
2009. Specialized piRNA pathways act in germline and
somatic tissues of the Drosophila ovary. Cell. 137, 522–
535.
86. Nishida K.M., Okada T.N., Kawamura T., et al. 2009.
Functional involvement of Tudor and dPRMT5 in the
piRNA processing pathway in Drosophila germlines.
EMBO J. 28, 3820–3831.
87. Shoji M., Tanaka T., Hosokawa M., et al. 2009. The
TDRD9–MIWI2 complex is essential for piRNA
mediated retrotransposon silencing in the mouse male
germline. Dev. Cell. 17, 775–787.
88. Aravin A.A., van der Heijden G.W., Castañeda J.,
Vagin V.V., Hannon G.J., Bortvin A. 2009. Cytoplas
mic compartmentalization of the fetal piRNA pathway
in mice. PLoS Genet. 5, e1000764.
89. Carmell M.A., Girard A., van de Kant H.J., Bourc’his D.,
Bestor T.H., de Rooij D.G., Hannon G.J. 2007.
MIWI2 is essential for spermatogenesis and repression
of transposons in the mouse male germline. Dev. Cell.
12, 503–514.
90. KuramochiMiyagawa S., Watanabe T., Gotoh K.,
et al. 2008. DNA methylation of retrotransposon
genes is regulated by Piwi family members MILI and
MIWI2 in murine fetal testes. Genes Dev. 22, 908–917.
91. KuramochiMiyagawa S., Kimura T., Ijiri T.W., et al.
2004. Mili, a mammalian member of piwi family gene,
is essential for spermatogenesis. Development. 131,
839–849.
MOLECULAR BIOLOGY Vol. 48 No. 1 2014
27
92. Tam O.H., Aravin A.A., Stein P., Girard A., et al. 2008.
Pseudogenederived small interfering RNAs regulate
gene expression in mouse oocytes. Nature. 453, 534–
538.
93. Watanabe T., Totoki Y., Toyoda A., et al. 2008. Endog
enous siRNAs from naturally formed dsRNAs regulate
transcripts in mouse oocytes. Nature. 453, 539–543.
94. Kibanov M.V., Gvozdev V.A., Olenina L.V. 2012.
Germ granules in spermatogenesis of Drosophila: Evi
dences of contribution to the piRNA silencing. Com
mun. Integr. Biol. 5, 130–133.
95. Kirino Y., Kim N., de PlanellSaguer M., Khandros E.,
Chiorean S., Klein P.S. Jongens TA, Mourelatos Z.
2009. Arginine methylation of Piwi proteins catalysed
by dPRMT is required for Ago3 and Aub stability.
Nature Cell Biol. 11, 652–658.
96. Kirino, Y., Vourekas, A., Sayed, N., de Lima Alves, F.,
Thomson, T., Lasko, P. Rappsilber J., Jongens T.A.,
Mourelatos Z. 2010. Arginine methylation of Aub
ergine mediates Tudor binding and germ plasm local
ization. RNA. 16, 70–78.
97. Lehmann R., NüssleinVolhard C. 1991. The maternal
gene nanos has a central role in posterior pattern for
mation of the Drosophila embryo. Development. 112,
679–691.
98. Mahowald A.P. 2001. Assembly of the Drosophila germ
plasm. Int. Rev. Cytol. 203, 187–213.
99. Santos A.C., Lehmann R. 2004. Germ cell specifica
tion and migration in Drosophila and beyond. Curr.
Biol. 14, R578–R 589.
100. Rangan P., De Gennaro M., JaimeBustamante K.,
Coux R.X., Martinho R.G., Lehmann R. 2009. Tem
poral and spatial control of germplasm RNAs. Curr.
Biol. 19, 72–77.
101. Hayashi K., de Sousa Lopes S.M., Surani M.A. 2007.
Germ cell specification in mice. Science. 316, 394–
396.
102. Lasko P. 2013. The DEADbox helicase Vasa: Evi
dence for a multiplicity of functions in RNA processes
and developmental biology. Biochim. Biophys. Acta.
1829, 810–816.
103. Styhler S., Nakamura A., Swan A., Suter B., Lasko P.
1998. vasa is required for GURKEN accumulation in
the oocyte, and is involved in oocyte differentiation
and germline cyst development. Development. 125,
1569–1578.
104. Johnstone O., Lasko P. 2004. Interaction with eIF5B is
essential for Vasa function during development. Devel
opment. 131, 4167–4178.
105. Liu N., Han H., Lasko P. 2009. Vasa promotes Droso
phila germline stem cell differentiation by activating
meiP26 translation by directly interacting with a (U)
rich motif in its 3' UTR. Genes Dev. 23, 2742–2752.
106. Neumüller R.A., Betschinger J., Fischer A., Bushati N.,
Poernbacher I., Mechtler K., Cohen S.M., Knoblich J.A.
2008. MeiP26 regulates microRNAs and cell growth
in the Drosophila ovarian stem cell lineage. Nature.
454, 241–245.
107. Page S.L., McKim K.S., Deneen B., van Hook T.L.,
Hawley R.S. 2000. Genetic studies of meiP26 reveal a
link between the processes that control germ cell pro
28 KOTOV et al.
liferation in both sexes and those that control meiotic
exchange in Drosophila. Genetics. 155, 1757–1772.
108. Carrera P., Johnstone O., Nakamura A., Casanova J.,
Jäckle H., Lasko P. 2000. VASA mediates translation
through interaction with a Drosophila yIF2 homolog.
Mol. Cell. 5, 181–187.
109. Nagao A., Mituyama T, Huang H., Chen D., Siomi M.C.,
Siomi H. 2010. Biogenesis pathways of piRNAs loaded
onto AGO3 in the Drosophila testis. RNA. 16, 2503–
2515.
110. Olovnikov I.A., Kalmykova A.I. 2013. piRNA clusters
as a main source of small RNAs in the animal germ
line. Biochemistry (Moscow). 78, 572–584.
111. Vagin V.V., Klenov M.S., Kalmykova A.I., Stolya
renko A.D., Kotelnikov R.N., Gvozdev V.A. 2004.
The RNA interference proteins and vasa locus are
involved in the silencing of retrotransposons in the
female germline of Drosophila melanogaster. RNA Biol.
1, 54–58.
112. Zhang F., Wang J., Xu J., et al. 2012. UAP56 couples
piRNA clusters to the perinuclear transposon silencing
machinery. Cell. 151, 871–884.
113. Tanaka S.S., Toyooka Y., Akasu R., KatohFukui Y.,
Nakahara Y., Suzuki R., Yokoyama M., Noce T. 2000.
The mouse homolog of Drosophila Vasa is required for
the development of male germ cells. Genes Dev. 14,
841–853.
114. Toyooka Y., Tsunekawa N., Takahashi Y., Matsui Y.,
Satoh M., Noce T. 2000. Expression and intracellular
localization of mouse Vasahomologue protein during
germ cell development. Mech. Dev. 93, 139–149.
115. KuramochiMiyagawa S., Watanabe T., Gotoh K.,
et al. 2010. MVH in piRNA processing and gene
silencing of retrotransposons. Genes Dev. 24, 887–892.
116. Nagamori I., Cruickshank V.A., SassoneCorsi P.
2011.Regulation of an RNA granule during spermato
genesis: Acetylation of MVH in the chromatoid body
of germ cells. J. Cell Sci. 124, 4346–4355.
117. Castrillon D.H., Quade B.J., Wang T.Y., Quigley C.,
Crum C.P. 2000. The human VASA gene is specifically
expressed in the germ cell lineage. Proc. Natl. Acad.
Sci. U. S. A. 97, 9585–9590.
118. Medrano J.V., Ramathal C., Nguyen H.N., Simon C.,
Reijo Pera R.A. 2012. Divergent RNAbinding pro
teins, DAZL and VASA, induce meiotic progression in
human germ cells derived in vitro. Stem Cells. 30, 441–
451.
119. Sugimoto K., Koh E., Sin H.S., et al. 2009. Tissue
specific differentially methylated regions of the human
VASA gene are potentially associated with maturation
arrest phenotype in the testis. J. Hum. Genet. 54, 450–
456.
Translated by T. Tkacheva
MOLECULAR BIOLOGY Vol. 48 No. 1 2014