RNA localization mechanisms in oocytes

Abstract

In many animals, normal development depends on the asymmetric distribution of maternal determinants, including various coding and noncoding RNAs, within the oocyte. The temporal and spatial distribution of localized RNAs is determined by intricate mechanisms that regulate their movement and anchoring. These mechanisms involve cis-acting sequences within the RNA molecules and a multitude of trans-acting factors, as well as a polarized cytoskeleton, molecular motors and specific transporting organelles. The latest studies show that the fates of localized RNAs within the oocyte cytoplasm are predetermined in the nucleus and that nuclear proteins, some of them deposited on RNAs during splicing, together with the components of the RNA-silencing pathway, dictate the proper movement, targeting, anchoring and translatability of localized RNAs.

Full text

Introduction
The early stages of embryogenesis are dependent on the proper
spatial and temporal distribution within the oocyte (and
ultimately within the egg) of the vast stockpile of maternal
molecules accrued during the elaborate and lengthy process of
oogenesis. Among these molecules, various localized RNAs
are crucial players involved in the generation of oocyte polarity
and/or patterning of the embryo. In addition, certain RNAs
localized in germinal granules (also know as P-granules or
polar granules) of the oocyte are involved in the specification
of germ cell fate. Some of these localized RNAs are noncoding
structural or regulatory RNAs, whereas others become
translated into localized proteins. Here, we discuss recent work
that has provided insight into the multifaceted phenomenon of
RNA localization in oocytes.
Roles of localized RNAs
In many invertebrates and vertebrates, the position of the
anterior-posterior and dorsal-ventral axes of the embryo and
the formation of the germ line are controlled by the asymmetric
localization of various RNAs (and/or their translation products)
in the oocyte (Hashimoto et al., 2004; Jansen, 2001; King et
al., 1999; Kloc et al., 2001; Kloc et al., 2002a; Palacios and
Johnston, 2001; Riechmann and Ephrussi, 2001; Zhou and
King, 2004). In Drosophila, the localization of nanos and oskar
mRNAs to the posterior pole of the oocyte, and bicoid RNA to
the anterior pole, plays a role in the formation of the anterior-
posterior body axis, pole plasm assembly and germ cell
function. Mislocalization of oskar RNA in females that have a
fourfold increase in osk gene dosage or in females bearing the
osk bcd 3′UTR transgene (which contains the bicoid anterior
localization signal) results in misexpression of Oskar protein,
which in turn causes mislocalization of Nanos protein and
repression of bicoid. This results in a bicaudal embryo, its
anterior replaced by a mirror image of the posterior (Ephrussi
and Lehmann, 1992; Kim-Ha et al., 1991; Smith et al., 1992;
Wharton and Struhl, 1991). Another localized mRNA, gurken,
signals to the epithelial cells surrounding the oocyte for the
proper formation of the dorsal-ventral axis (Gonzales-Reyes et
al., 1995; Neuman-Silberberg and Shüpbach, 1993; Nilson and
Shüpbach, 1999).
In ascidians, the localized mRNAs macho-1 and pem
determine muscle fate and anterior and dorsal patterning,
respectively (Nishida and Sawada, 2001; Yoshida et al., 1996).
In sea urchin, the frog Xenopus laevis and zebrafish, subsets of
RNAs are also localized to the animal and vegetal poles of the
oocyte (Braat et al., 1999; Bruce et al., 2003; Di Carlo et al.,
2004; Howley and Ho, 2000; King et al., 1999; Kloc et al.,
2001; Kloc et al., 2002a). In Xenopus oocytes, the vegetally
localized mRNAs Vg1, VegT and Xwnt 11 are mesodermal and
endodermal determinants and determinants of the left-right
axis in the embryo (Hyatt and Yost, 1998; Joseph and Melton,
1998; Ku and Melton, 1993; Rebagliati et al., 1985; Thomsen
and Melton, 1993; Zhang et al., 1998; Zhang and King, 1996).
Other vegetally localized mRNAs in Xenopus, such as Xcat2
and Xdazl, are believed to play a role in germ cell
determination or migration (Houston et al., 1998; Mosquera et
al., 1993). In zebrafish, several mRNAs are localized either to
the animal or vegetal pole of the oocyte (Howley and Ho,
2000). Two of them: the mRNA encoding the DEAD-box RNA
helicase Vasa and the mRNA encoding the T-box gene product
eomesodermin are cortically localized in vitellogenic oocytes,
and might play a role in germ cell determination and the
specification of the organizer, respectively (Bruce et al., 2003;
Knaut et al., 2000; Knaut et al., 2002).
Mechanisms of RNA transport
RNA is exported from the oocyte nucleus through nuclear
pores. Once in the cytoplasm, RNA can be trapped by or
targeted to specialized transporting organelles or bind to
cytoskeletal elements that assist in transport of RNA to its
ultimate destination. Alternatively, asymmetrical distribution
269
In many animals, normal development depends on the
asymmetric distribution of maternal determinants,
including various coding and noncoding RNAs, within the
oocyte. The temporal and spatial distribution of localized
RNAs is determined by intricate mechanisms that regulate
their movement and anchoring. These mechanisms involve
cis-acting sequences within the RNA molecules and a
multitude of trans-acting factors, as well as a polarized
cytoskeleton, molecular motors and specific transporting
organelles. The latest studies show that the fates of localized
RNAs within the oocyte cytoplasm are predetermined in
the nucleus and that nuclear proteins, some of them
deposited on RNAs during splicing, together with the
components of the RNA-silencing pathway, dictate the
proper movement, targeting, anchoring and translatability
of localized RNAs.
Key words: RNA localization, Microtubules, Oocytes
Summary
RNA localization mechanisms in oocytes
Malgorzata Kloc and Laurence D. Etkin*
Department of Molecular Genetics, The University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA
*Author for correspondence (e-mail: lde@mdanderson.org)
Journal of Cell Science 118, 269-282 Published by The Company of Biologists 2005
doi:10.1242/jcs.01637
Commentary
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270
can be achieved through a combination of RNA destabilization/
degradation and local protection (Tadros et al., 2003). In
certain animals, the oocyte nucleus is transcriptionally
quiescent and most RNAs are synthesized in the accessory cells
(called nurse cells in insects), from which they are exported to
the oocyte. In this case, independent mechanisms are often
responsible for RNA transport to and within the oocyte.
Specialized transporting organelles
Two pathways of RNA localization to the oocyte vegetal cortex
have been identified in Xenopus – the early pathway and the
late pathway (Fig. 1). The early pathway, also known as the
messenger transport organizer (METRO) pathway, functions in
early oogenesis (stages I and II) and transports germinal
granules, various RNAs (some of them
involved in germ cell specification), and
germline-specific mitochondria in a
specialized organelle that in Xenopus is
called the mitochondrial cloud (also known
as the Balbiani body) (Heasman et al., 1984;
Forristall et al., 1995; Kloc and Etkin, 1995)
(Figs 1 and 2). The Balbiani body was
discovered by von Wittich in 1845 in oocytes
of spiders. Between 1864-1893, Balbiani
conducted comprehensive studies of this
organelle in oocytes of myriapods and
spiders; later, this organelle was discovered
in oocytes of various invertebrates and
vertebrates, including humans (de Smedt et
al., 2000; Guraya, 1979; Heasman et al.,
1984; Kloc et al., 2004b). Interestingly, the
existence of the Balbiani body in the oocytes
of marsupials and eutherian mammals
(including nonhuman primates and humans)
is not widely known. This situation arises
primarily from the fact that the mouse, the
dominant model organism in mammalian
studies, does not have a Balbiani body in its
oocytes. In this respect, the mouse is
probably an exception among mammals.
However, as a consequence of its absence in
the mouse, there is not a single study that
experimentally addresses the role of the
Balbiani body in mammalian oocytes.
The Balbiani body is a spherical
membraneless structure that forms in contact
with the oocyte nucleus in early oogenesis,
and fragments and disperses in late
oogenesis (Figs 1 and 2). Its constant
components are mitochondria, endoplasmic
reticulum (ER), membranous vesicles and
lipid droplets. In Xenopus stage I oocytes,
the Balbiani body is ~40 µm in diameter,
contains half a million mitochondria, which
differ in morphology and metabolism from
those of cytoplasmic mitochondria, and is
rich in membranous vesicles, and ER
cysternae. The vegetal apex of the Xenopus
Balbiani body (called the METRO region)
contains germinal granules and various
localized RNAs (Fig. 2) (Forristall et al., 1995; Heasman et al.,
1984; Kloc et al., 2004b). For nearly 150 years, the role of the
Balbiani body remained a complete mystery, although on the
basis of its biochemical and ultrastrucural composition, it was
commonly believed that this organelle participates either in the
formation of lipids or in the multiplication of mitochondria
(Guraya, 1979). In 1993, Kloc et al. showed that the Balbiani
body/mitochondrial cloud in Xenopus is a vehicle that
transports localized RNA to the vegetal pole of the oocyte.
Cis-acting elements directing RNA to the mitochondrial
cloud (mitochondrial cloud localization elements, MCLEs)
have been identified in the 3′UTR of Xcat2 mRNA and in the
noncoding Xlsirts RNA in Xenopus oocytes (Allen et al., 2003;
Kloc et al., 1993; Zhou and King, 1996). Xcat2 mRNA also
has a germinal granule localization element (GGLE) in its
Journal of Cell Science 118 (2)
A
B
Early (METRO) pathway
Late (Vg1) pathway
1
2
3
4
5
6
Fig. 1. Pathways of RNA localization in Xenopus oogenesis. (A) The early (METRO)
localization pathway operates in early oogenesis (stages I and II) and uses the
mitochondrial cloud (Balbiani body) to localize RNAs such as Xcat2 mRNA (red) and
noncoding Xlsirts RNA (blue) to the vegetal pole of the oocyte. (1) In stage I oocytes,
RNAs synthesized in the nucleus (yellow) enter (either via nuage or by the
diffusion/entrapment mechanism) the mitochondrial cloud (mitochondria shown in green),
which faces the vegetal pole of the oocyte. Xcat2 mRNA becomes localized to the
germinal granules (red spheres) and Xlsirts RNA is localized between the germinal
granules at the vegetal apex (METRO region) of the mitochondrial cloud. (2) In stage II
oocytes, the mitochondrial cloud moves to the vegetal cortex and starts to disperse. (3) In
stage III-VI oocytes, the mitochondrial cloud disperses, and germinal granules and
localized RNAs form a disc at the apex of the vegetal cortex. (B) The late (Vg1) pathway
localizes mRNAs such as Vg1 or VegT, using MTs, molecular motors and possibly the
ER. (4) In stage I oocytes, these RNAs (purple) are uniformly distributed within the
oocyte cytoplasm and are excluded from the mitochondrial cloud. (5) In late stage II
oocytes, RNAs concentrate, in a wedge, around the moving mitochondrial cloud and a
subdomain of ER, and translocate on MTs towards the vegetal pole. (6) Later in oogenesis
(stages III-VI), late-pathway RNAs localize and anchor at the cortex of the vegetal half of
the oocyte (for details, see Kloc and Etkin, 1995).
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271
RNA localization mechanisms
3′UTR (Kloc et al., 2000). Recently, Claussen et al. identified
a 300-nucleotide element in the 5′UTR of XNIF mRNA that
directs it to the mitochondrial cloud (Claussen et al., 2004).
The localization of RNAs into the mitochondrial cloud is
independent of microfilaments and MTs and probably involves
a diffusion and entrapment mechanism (Kloc et al., 1996;
Chang et al., 2004). The movement of the mitochondrial cloud
to the vegetal cortex of the oocyte is also independent of
MTs and microfilaments, and the mechanism underlying this
movement remains a mystery; one possibility is passive
translocation along the cytoplasm streaming towards the
vegetal cortex (Kloc et al., 2004b).
The Balbiani body has recently been discovered in the
oocytes of several insects (Fig. 2) (Bradley et al., 2001; Cox
and Spradling, 2003; Jaglarz et al., 2003; Kloc et al., 2004b).
In Drosophila, it contains germline-specific mitochondria,
forms in the oocyte in early oogenesis, and might facilitate
localization of oskar mRNA to the posterior pole of the oocyte
(Cox and Spradling, 2003). The Balbiani body differs from
Drosophila sponge bodies, which were previously thought to
be equivalent to the Xenopus Balbiani body. The sponge bodies
form at the surface of nurse cell nuclei later in oogenesis,
associate with Exuperantia (Exu), a novel protein that is a core
component of a protein complex involved in the localization of
mRNAs within the nurse cells and oocyte, and mediate
transport of bicoid RNA (Wilsch-Brauninger et al., 1997).
Another ooplasmic organelle involved in the localization of
RNAs in oocytes is the ER. In ascidians, the maternal mRNAs
pem and macho-1 are localized in the egg on the network of
cortical rough ER, which compacts after fertilization to form
the centrosome-attracting body (CAB), which is responsible
for the unequal cleavages (Sardet et al., 2003). Recently, Chang
et al. showed that, in Xenopus, the ER vesicles are involved in
the entrapment of Xcat2 and Xdazl mRNA within the
mitochondrial cloud (Chang et al., 2004). Since these
experiments used injected synthetic fluorescent RNA
synthesized from the Xcat2 3′UTR and Xdazl localization
element, it remains to be seen whether the same mechanism is
involved in the localization of endogenous Xcat2 and Xdazl
mRNAs to the mitochondrial cloud.
MT-dependent transport
Oocytes have a highly polarized system of MTs and molecular
motors that are responsible for the proper localization of most
localized RNAs in Drosophila and of late-pathway (also known
as the Vg1 pathway) RNAs in Xenopus (Fig. 1).
In typical somatic cells, MT minus-ends are arranged around
a MT-organizing center (MTOC) close to the nucleus, and MT
plus-ends occupy the cell periphery. This polarity is recognized
by molecular motors, which are typically unidirectional –
either minus-end directed or plus-end directed (Cohen, 2002;
Lopez de Heredia and Jansen, 2004; Welte, 2004). However,
traffic along the MTs in oocytes is much more complex than
previously thought. As a consequence, different experiments
on the same subject often yield contradictory results and
conclusions, depending on the method used by the researchers
(Stephenson, 2004). Thus, some of the original papers and even
some of the review articles on MT trafficking are extremely
confusing.
Injection of exogenous RNAs and analysis of mutants
GV
GV
C
GV
GV
A
B
*
*
*
*
*
Fig. 2. Balbiani body in the oocytes of insects and Xenopus.
(A) Fragment of the ovariole from the ovary of the cricket Acheta
domesticus seen with a Nomarski contrast microscope. This is a
panoistic type of ovary. In this type of ovary (unlike in the meroistic
type found in Drosophila), there are no nurse cells, and the oocyte
nucleus (the germinal vesicle, GV) is large and transcriptionally
active. In the previtellogenic oocytes (on the left), there is one
Balbiani body (arrow) located at the anterior pole of the oocyte. In
older oocytes, there are two Balbiani bodies, one at the anterior and
another at the posterior pole (marked by a star) (for details, see
Bradley et al., 2001). (B) Balbiani bodies (mitochondrial clouds) in
stage I and stage II Xenopus oocytes. Whole mount in situ
hybridization with antisense Xcat2 probe shows that Xcat2 mRNA is
localized in the mitochondrial cloud (arrow), which always faces the
vegetal pole (star) of the oocyte. In stage I oocytes, the mitochondrial
cloud is located close to the GV and, in stage II oocytes, the
mitochondrial cloud translocates to the vegetal pole.
(C) Mitochondrial cloud from stage I Xenopus oocyte. Three-
dimensional reconstruction from 21 serial electron microscopic
sections of an oocyte hybridized to the Xcat2 RNA probe, artificially
colored. Germinal granules (arrows indicate the individual granules),
labeled with Xcat2 mRNA (red), are located between the
mitochondria and concentrated in the METRO region at the vegetal
apex (star) of the cloud (for details, see Kloc et al., 2002). Bar, 60
µm in A, 120 µm in B, and 6 µm in C.
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272
indicate that, in Drosophila, oskar mRNA is localized to the
posterior pole by plus-end-directed motors (kinesins), whereas
bicoid mRNA is localized to the anterior pole and gurken
mRNA is localized to the anterior-dorsal region by minus-end-
directed motors (dyneins) (Brendza et al., 2000; Cohen, 2002;
Lopez de Heredia and Jansen, 2004). These findings raise a
fascinating question: how can the same molecular motor direct
bicoid and gurken RNAs to different cellular locations?
Recently, MacDougall et al. used time-lapse photography to
track the localization of fluorescent gurken RNA in living
Drosophila oocytes (MacDougall et al., 2003). They showed
that the movement of gurken RNA is dependent on dynein and
MTs but occurs in two distinct steps using distinct arrays of
MTs. First, gurken particles move to the oocyte anterior; later,
they turn dorsally towards the nucleus. The authors suggested
that, in this instance, as well as in others, the use of distinct
MT networks might be responsible for transport by the same
motor of different cargos to different destinations. Recent
experiments showing that kinesin heavy chain (Khc) mutants
that exhibit impaired oskar localization also, unexpectedly,
exhibit impaired localization of gurken and bicoid mRNA
indicate that anterior and anterior-dorsal transport also require
kinesin (Duncan and Warrior, 2002; Welte, 2004). Several
models for cooperation between minus-end-directed and plus-
end-directed motors in the oocyte have been proposed. For
example, kinesin might indirectly activate dynein (through an
adaptor protein) or these two motors might cooperate by
recycling one another – i.e. kinesin could transport dynein to
MT plus-ends, where dynein would bind RNA cargo and
transport it to the minus-ends, and vice versa (Cohen, 2002;
Duncan and Warrior, 2002; Januschke et al., 2002; Welte,
2004).
Further complications arise because the polarity of the
Drosophila oocyte cytoskeleton changes during oogenesis
(which is divided into 14 stages) (Theurkauf et al., 1992). Just
after formation of the oocyte (stage 1), the MTOC assembles
at the anterior pole of the oocyte and MTs extend from it to
the nurse cells. The oocyte then grows by acquiring molecules
and organelles (such as centrosomes and mitochondria) from
the nurse cells by actin-based and minus-end-directed, MT-
based transport (Swan et al., 1999). In early oogenesis (stages
2-6), the MTOC shifts from the anterior to the posterior pole,
resulting in localization of oskar mRNA and Gurken protein as
a crescent at the oocyte posterior. Subsequently, Gurken signals
to overlying follicle cells and they adopt a posterior fate. The
genes required for the MTOC shift from the anterior to the
posterior pole in early oogenesis include the armitage gene,
which encodes a homolog of SDE3, an RNA helicase involved
in post-transcriptional gene silencing, and probably other genes
whose products are involved in RNA silencing (see below),
such as spindle-E, aubergine and maelstrom. In armitage
mutants, the MTOC does not switch to the posterior pole,
which impairs posterior localization of oskar mRNA and
causes its premature translation (Chekulaeva and Ephrussi,
2004; Cook et al., 2004; Tomari et al., 2004). It is possible that
this defect in MOTC switching is caused not by the mutation
in armitage but, directly or indirectly, by the premature
synthesis of Oskar protein. However, the fact that oocytes
overexpressing Oskar from the transgene have normal MT
organization (Riechmann et al., 2002) makes it very unlikely
that MT polarization and oskar silencing are co-dependent.
At the beginning (stage 7) of mid oogenesis (stages 7-10),
the posterior follicular cells send an unknown signal back to
the oocyte, causing a re-polarization of the oocyte MTs; the
MTOC at the oocyte posterior disassembles, and new MTs are
nucleated from the anterior and lateral oocyte cortex
(Schnorrer et al., 2002). At this stage, localized oskar mRNA
becomes translated at the posterior of the oocyte, bicoid mRNA
is localized at the oocyte anterior, and gurken mRNA and
protein become located at the anterior-dorsal corner of the
oocyte, which sets up the final polarity of the oocyte.
The polarization of the Drosophila cytoskeleton seems to
require more than just a simple switch in the position of the
MTOC; it also depends on the partitioning-defective (PAR)
proteins (Huynh and St Johnston, 2004). Evolutionary
conserved PAR proteins are involved in the establishment and
maintenance of cell polarity during development and in
somatic cells in various species. These proteins include PAR-
1, a serine/threonine kinase; PAR-2, a RING finger protein;
PAR-3, which has a PDZ domain [PDZ domains are
homologous in diverse signaling proteins including
postsynaptic density protein-95 (PSD-95), a protein involved
in signaling at the post-synaptic structures; DLG, the
Drosophila Discs Large protein; and ZO-1, the zonula
occludens 1 protein]; PAR-4, a homolog of mammalian kinase
LKB1; and PAR-5, a homolog of 14-3-3 (which are highly
conserved proteins involved in the regulation of protein
phosphorylation and mitogen-activated protein kinase
pathways). In Drosophila oocytes, PAR-1 regulates posterior
patterning through phosphorylation and thus stabilization of
Oskar (Riechmann et al., 2002) and, together with other PAR
proteins, PAR-1 might control the dynamics of MT assembly.
In par mutants, the MT minus-ends do not switch from the
anterior to the posterior oocyte cortex in early oogenesis
(Vaccari and Ephrussi, 2002), and there is a disorganization of
the MT cytoskeleton in mid oogenesis in which MT minus-
ends are located around the oocyte cortex and MT plus-ends
are directed towards the oocyte center (Benton et al., 2002;
Shulman et al., 2000). Although the molecular targets and
mode of action of PAR proteins within the oocyte cytoskeleton
are unknown, it is possible that PAR kinases, like the
mammalian PAR-1 homolog, MARK, regulate MT stability by
phosphorylation of MT-associated proteins (Benton et al.,
2002; Drewes et al., 1997).
In Xenopus oocytes, several mRNAs, including Vg1 mRNA,
localize to the vegetal cortex during mid and late oogenesis
(oocyte stages III-V) via the MT-dependent, late (or Vg1)
pathway (Fig. 1) (Kloc and Etkin, 1995; Melton, 1987; Weeks
and Melton, 1987; Yisraeli et al., 1990; Zhou and King, 1996;
Zhou and King, 2004). The MT-dependent localization of Vg1
mRNA starts with the ‘streaming’ of RNA towards the vegetal
cortex around the so-called wedge region of the oocyte, which
contains a subdomain of ER organized on top of the
mitochondrial cloud. Either the ER serves as a matrix for the
formation of polarized MT tracks that transport Vg1 mRNA,
or Vg1 binds to the ER vesicles (possibly through the
Vg1RBP/Vera protein), and the ER-Vg1 mRNA complexes are
transported on the MTs to the oocyte vegetal cortex (Deshler
et al., 1997; Kloc and Etkin, 1998).
There is some evidence that the movement of Vg1 mRNA
to the Xenopus oocyte vegetal cortex is mediated by the plus-
end-directed motor kinesin II (Betley et al., 2004) and possibly
Journal of Cell Science 118 (2)
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RNA localization mechanisms
by kinesin I (Yoon and Mowry, 2004). Paradoxically, the
majority (around 96%) of MTs in Xenopus stage III and VI
oocytes have their minus-ends directed towards the cortex,
which is the reverse of what is typical for eukaryotic cells
(Pfeiffer and Gard, 1999). The majority of the molecules
transported to the cortex should therefore use minus-end-
directed motors, such as cytoplasmic dyneins or minus-end-
directed kinesins. Possible explanations for these apparently
contradictory findings (i.e. that movement of Vg1 to the vegetal
cortex is mediated by plus-end-directed motors but that most
MTs have minus-ends directed towards the cortex) are that, in
Xenopus, as in Drosophila, the polarity of the MTs shifts
during oogenesis and/or that the RNA-binding factors decide
the directionality of the RNA transport. Rat neurons provide
an example of this: the ribonucleoprotein granules of the RNA-
binding protein Staufen fused to green fluorescent protein
(GFP) are able to associate with both kinesin and dynein, and
occasionally reverse the direction of their movement on MTs
(Köhrmann et al., 1999; Villace et al., 2004). Although we do
not understand the molecular events that lead to the changes in
the direction of movement along MTs, the novel Halo-like
proteins discovered in mutants that exhibit defective transport
of lipid droplets might be involved. Halo proteins might affect
the duration of the particular run of the cargo by altering the
binding strength between a particular motor and MTs, which
would change the frequency of dissociation of motor proteins
from MTs, or by controlling the number of motors that bind to
MTs. Another possibility is that Halo proteins act in trans,
changing the net direction of the cargo translocation: in the
presence of Halo, the net movement of the cargo is plus-end
directed whereas, in the absence of Halo, the net movement of
cargo is minus-end directed (Fig. 3) (Gross et al., 2003; Cohen,
2003; Welte, 2004).
RNA localization via MTs depends on the presence of
cis-acting localization elements (LEs) located in the 3′UTR
(common), the 5′UTR (rare), or both (Table 1) (Betley et al.,
2002; Bubunenko et al., 2002; Claussen et al., 2004; Kloc
et al., 2002a; Sasakura and Makabe, 2002; Zhou and King,
1996). Proper secondary structure is often important for the
functioning of LEs, and several LEs can act redundantly. The
best characterized are those of: Vg1, VegT, fatvg, Xvelo1 and
XNIF mRNAs and noncoding Xlsirts RNA in Xenopus (Chan
et al., 1999; Claussen and Pieler, 2004; Claussen et al., 2004;
Kloc et al., 1993; Kwon et al., 2002; Mowry and Cote, 1999;
Yaniv and Yisraeli, 2001); oskar, nanos, gurken and bicoid
mRNAs in Drosophila (Bergsten and Gavis, 1999; Brunel and
Ehresmann, 2004; Bullock and Ish-Horowicz, 2001; Crucs et
al., 2000; Kim-Ha et al., 1993; Macdonald and Kerr, 1997;
Macdonald and Kerr, 1998; Macdonald and Struhl, 1988; Thio
et al., 2000; Wagner et al., 2004); and a group of maternal
mRNAs in zebrafish and ascidians (Knaut et al., 2002;
Sasakura and Makabe, 2002).
Multiple RNA-binding proteins that interact with these
elements, and thus link RNA, MTs and molecular motors, have
been identified (Table 1), primarily in Drosophila and Xenopus.
For example, Staufen, Mago nashi, Y14 (also known as
Tsunagi), Barentsz (Btz) and Hrp48 are components of the
oskar mRNA transport complex (Hachet and Ephrussi, 2001;
Huynh et al., 2004; Mohr et al., 2001; St Johnston et al., 1991;
van Eeden et al., 2001), and Egalitarian (Egl) and Bicaudal D
(BicD) are components of the dynein motor complex in
Drosophila oocytes (Navarro et al., 2004). Staufen, a double-
stranded-RNA-binding protein is essential for the localization
of oskar and bicoid mRNA in oocytes and prospero mRNA in
neuroblasts (Li et al., 1997; Zhou and King, 2004). Mago nashi
and Y14, a putative RNA-binding protein, are highly conserved
from Schizosaccharomyces pombe to humans, and in
Drosophila they are required for the correct localization of
oskar mRNA at the posterior pole. Mago nashi and Y14
interact both in vitro and in vivo. They also interact with the
mRNA export factor TAP, and are associated with mRNA
during its export from the nucleus to the cytoplasm (Hachet
and Ephrussi, 2001; Le Hir et al., 2001a; Le Hir et al., 2001b).
Btz, which shares some sequence similarity with the human
nucleo-cytoplasmic shuttling protein malignant lymph node 51
(MLN51), is essential for the localization of oskar mRNA to
the posterior pole of the Drosophila oocyte. When expressed
in Drosophila oocytes, the mammalian homolog of Btz
interacts with mammalian Staufen in an RNA-dependent
manner (Macchi et al., 2003; van Eeden et al., 2001).
Hrp48 is a member of the heterogeneous nuclear
ribonucleoprotein A/B (hnRNPA/B) family of RNA-binding
proteins that bind 5′ and 3′ regulatory regions of oskar mRNA.
Because the Hrp48 mutant exhibits defective formation of
GFP-Staufen particles, it is believed that HRP48 plays a role
in the assembly of Staufen–oskar mRNA transport particles
(Huynch et al., 2004). BicD is essential for the organization
and maintenance of the polarized MT network and the
plus-end motor
minus-end motor
coordination machinery
HALO-like protein
-
-
A
B
RNA cargo
+
+
Fig. 3. Model of the action of Halo-like proteins in bidirectional
transport on MTs. (A) In the absence of Halo proteins, plus-end and
minus-end motors alternate their binding to coordination machinery
(possibly containing dynactin) and move RNA cargo towards either
plus-ends or minus-ends of MTs. (B) Halo proteins change the steric
properties of the coordination machinery, which weakens its binding
to the minus-end motor and increases its binding to the plus-end
motor, and results in the movement of the cargo towards the MT
plus-end [adapted from Gross (Gross, 2003) and Gross et al. (Gross
et al., 2003)].
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274 Journal of Cell Science 118 (2)
Localization elements (LEs) and trans-acting factors
RNA LE Trans-acting factor Motor References
Drosophila bicoid mRNA Five independent
domains (I-V) in
3′UTR
Staufen, BicD, Egl, Swa Dynein-dynactin
complex and
kinesin I
Brunel and, Ehresmann, 2004; Duncan and
Warrior, 2002; Januschke et al., 2002;
Macdonald and Struhl, 1988; Schnorrer
et al., 2000
Drosophila gurken mRNA GLE2 in 5′UTR BicD, Egl, Hrb27C, Sqd, Otu Dynein-dynactin
complex and
kinesin I
Duncan and Warrior, 2002; Filardo and
Ephrussi, 2003; Goodrich et al., 2004;
Januschke et al., 2002; Saunders and
Cohen, 1999
Drosophila nanos mRNA
(localization is mainly
based on
diffusion/entrapment, and
a small portion localizes to
the germ plasm, possibly
through a LE in the
3′UTR)
Multiple redundant
subelements in 3′UTR
p75 Unknown Bergsten and Gavis, 1999; Bergsten et al.,
2001; Forrest and Gavis, 2003
Drosophila oskar mRNA Multiple subelements in
3′UTR
EJC, Staufen, Barentsz Mago
nashi, Y14 (Tsunagi), Hrp48,
Tropomyosin II (TmII),
Bruno (regulation of
translation)
Kinesin I Brendza et al., 2000; Filardo and Ephrussi,
2003; Hachet and Ephrussi, 2001; Huynh
et al., 2004; Januschke et al., 2002; Kim-
Ha et al., 1993; Mohr et al., 2001; St
Johnston et al., 1991; van Eeden et al.,
2001
Xenopus late-pathway RNAs:
Vg1 mRNA
VegT mRNA
Xvelo1 mRNA
FatVg mRNA
Multiple redundant
subelements in 3′UTR
Reiterated VM1 and E2
elements in 3′UTR
Multiple subelements in
3′UTR
Multiple redundant
subelements in 3′UTR
VgRBP/Vera
VgRBP60/hnRNPI
VgRBP71 (probable translation
activator not involved in
transport)
Prrp
Strong binding to Prrp, and
weak to VgRBP/Vera
Unknown
Kinesin I and/or
II through
Staufen
Unknown
Unknown
Bubunenko et al., 2002; Cote et al., 1999;
Deshler et al., 1997; Deshler et al., 1998;
Gautreau et al., 1997; Havin et al., 1998;
Kolev and Huber, 2003; Kroll et al.,
2002; Kwon et al., 2002; Mowry, 1996;
Mowry and Melton, 1992; Yaniv and
Ysraeli, 2001; Yoon and Mowry, 2004
Claussen and Pieler, 2004
Chan et al., 1999
Xenopus early-pathway RNAs when using late pathway:
Xcat2 mRNA, Xpat mRNA
and Xwnt11
XNIF mRNA
Xlsirts noncoding RNA
LE in 3′UTR
LE in 5′UTR
Repeat element
VegRBP/Vera
VgRBP71
VegRBP/Vera
VgRBP71
Prrp
Unknown
Kinesin II
Unknown
Unknown
Betley et al., 2004
Yoon and Mowry, 2004
Claussen et al., 2004
Allen et al., 2003; Kloc et al., 1993
Xenopus early-pathway RNAs:
Xcat2 mRNA
XNIF mRNA
Xlsirts noncoding RNA
MCLE and GGLE
within LE
MCLE within LE
Repeat element
Unknown
62 and 64 kDa proteins
Unknown
Unknown
Unknown
Unknown
Kloc et al., 2000; Zhou and King, 1996
Claussen et al., 2004;
Allen et al., 2003; Kloc et al., 1993
Xenopus early- and late-
pathway RNAs
CAC motif in LEs Unknown Unknown Betley et al., 2002
Zebrafish vasa mRNA
(vegetal localization after
injection into Xenopus
oocytes)
LE in 3′UTR Unknown Unknown Knaut et al., 2002
Ascidians type I and type II
mRNAs: HrPOPK-1,
HrPet-1, Pet-2 and Pet-3,
HrWnt5, Hr2F-1
(posterior localization
after injection into eggs)
LE in 3′UTR, different
subelements for type I
and type II RNAs
Unknown Unknown Sasakura and Makabe, 2002; Sasakura et
al., 2000
Journal of Cell Science

275
RNA localization mechanisms
structural integrity of Drosophila oocytes and nurse cells. BicD
protein contains four heptad-repeat domains typical of
intermediate filament proteins and might be an integral part of
the cytoskeleton (Oh and Steward, 2001). BicD forms a
complex with Egl, which interacts directly with dynein light
chain (Dlc) through an Egl domain distinct from that
responsible for the binding to BicD (Navarro et al., 2004).
In Xenopus, Vg1RBP (also known as Vera) binds to various
subelements of LEs in Vg1, VegT and Xvelo1 mRNAs
(Claussen and Pieler, 2004; Git and Standart, 2002; Kwon et
al., 2002). The Xenopus homolog of Drosophila Staufen
associates with kinesin I and the vegetally localized RNAs Vg1
and VegT in oocytes. The function of the Vg1 localization
element (VLE) in VglRNA is blocked by the expression in
oocytes of dominant-negative XStau234 (a mutant form
of XStau containing only a double-stranded-RNA-binding
domain) (Yoon and Mowry, 2004). The presence of Staufen in
RNA-transporting complexes in Drosophila and Xenopus
indicates common elements in localization mechanisms in
invertebrates and vertebrates.
Another example of the commonality of RNA localization
mechanisms between different organisms comes from the
study of zebrafish Vasa, whose RNA is a component of the
germ plasm in the embryo. In zebrafish stage I oocytes, vasa
mRNA is uniformly distributed within the cytoplasm. In stage
II oocytes, it is localized to the cortical cup; in stage III oocytes,
it is enriched in the animal cortex. In late-stage oocytes
(starting from stage IV), vasa mRNA remains cortical and is
localized to germ plasm granules. In cleaving embryos, vasa
mRNA segregates asymmetrically to the germ plasm and
subsequently to the founder population of primordial germ cell
(PGCs). Although nothing is known about the mechanisms
responsible for the localization of vasa mRNA in zebrafish
oocytes, the asymmetrical segregation of vasa mRNA to the
germ plasm in the embryos depends on intact MTs and also on
the presence of the product of the maternal-effect gene nebel
(Howley and Ho, 2000; Knaut et al., 2000). Recently, Knaut et
al. showed that when vasa mRNA 3′UTR (which directs its
localization to the germ plasm in zebrafish embryos) is fused
to a GFP reporter and injected into Xenopus oocytes, it directs
reporter to Xenopus germ plasm (Knaut et al., 2002). Although
there is no obvious similarity between the 3′UTR of zebrafish
vasa mRNA and the 3′UTR of the Xenopus vasa-like
DEADSouth mRNA, both are able to direct transcripts to germ
plasm in either species (Knaut et al., 2002). The experiments
discussed above show not only the universality of the
localization mechanisms between oocytes and embryos and
between different species, but also the intrinsic interspecies
similarity of the germ plasm.
Anchoring RNA at its destination
Asymmetrically localized RNAs in the oocyte must be
anchored at their final destinations. RNA can be transported
directly to its anchor, it can be captured as it diffuses by, or a
combination of these two mechanisms can be involved.
Drosophila oocytes contain an actin-filament-based anchor at
the posterior pole that requires the F-actin-binding protein
Bifocal, tropomyosin II, Dmoesin and Cap for proper
organization (Babu et al., 2004; Baum, 2002). Dmoesin is
required for the crosslinking of F-actin to the membrane of the
posterior pole. In Dmoesin-deficient flies, which have defects
in localization of oskar mRNA, the actin filament network is
loose and detached from the plasma membrane (Jankovics et
al., 2002; Polesello et al., 2002). Recently, Cha et al., studying
the role of Khc in oskar mRNA localization in Drosophila, re-
evaluated the distribution and polarity of the MTs in the
Drosophila oocyte, and the role of the posterior anchor in the
localization of oskar mRNA (Cha et al., 2002). They concluded
that, during late oogenesis, the MT minus-ends are uniformly
distributed over the oocyte cortex, the lowest MT density being
at the posterior. In stage 8, the plus-end-directed movement
mediated by kinesin transports oskar mRNA towards the
oocyte interior, preventing its association with unwanted
cortical sites. In late stage 8 and early stage 9, the majority of
MTs at the posterior pole depolymerize, unmasking (making
available) the cortical actin anchor that tethers oskar mRNA
(Fig. 4). Entrapment at the posterior pole also mediates
localization of nanos mRNA in late oogenesis and possibly also
cyclin B, gcl (germ-cell-less) and pgc (noncoding polar granule
component) RNAs (Dalby and Glover, 1992; Forrest and
Gavis, 2003; Jongens et al., 1992; Nakamura et al., 1996).
Similarly, the microfilament-rich oocyte cortex is necessary for
the anchoring of vegetally localized RNAs in Xenopus oocytes
(Elinson et al., 1993; Forristall et al., 1995; Kloc and Etkin,
1995; Klymkowsky et al., 1991; Pondel and King, 1988;
Yisraeli et al., 1990). The disruption of actin and/or cytokeratin
filaments releases late-pathway RNAs from the vegetal cortex
(Alarcon, and Elinson, 2001). Interestingly, two vegetally
localized RNAs – early-pathway noncoding Xlsirts and late-
pathway VegT mRNA – are necessary for the anchoring of
other late-pathway RNAs to the oocyte cortex (Heasman et al.,
2001; Kloc and Etkin, 1994). Indeed, we and others have
shown that Xlsirts and VegT function as structural RNAs in the
organization of the cytokeratin cytoskeleton at the vegetal
cortex of Xenopus oocytes (M.K., K. Wilk, M. Bilinski and
L.D.E., unpublished).
The fate of a localized RNA depends on its nuclear
and/or cytoplasmic history
The latest theme in the RNA localization saga concerns how
the events preceding the export of RNA into the oocyte
cytoplasm – either events in the oocyte nucleus or events in the
nuclei and cytoplasm of the accessory/nurse cells – determine
the fate of RNA, including its stability, translatability, mode of
transport and destination. The most striking finding is the
discovery that the polarity of RNA transport depends on the
source of RNA and its binding partners. Fluorescent bicoid
RNA injected directly into Drosophila oocyte cytoplasm
moves on MTs to the closest cortical surface. However, if first
injected into the nurse cell cytoplasm and then withdrawn and
re-injected into the oocyte, the bicoid RNA moves to the
anterior. Transport to the proper destination depends on the
ability of Exu protein and bicoid mRNA to be assembled into
complexes in nurse cell cytoplasm. Moreover, this finding
indicates that the direction of transport along the oocyte
microtubular system in Drosophila depends on the identity of
the proteins bound to the RNA cargo (Cha et al., 2001).
Drosophila oskar provides another example. The hybrid
RNA lacZ/oskar-3′UTR is efficiently transported to the
posterior pole of the oocyte (Kim-Ha et al., 1993), which
Journal of Cell Science

276
implies that the oskar RNA 3′UTR contains all signals
necessary and sufficient to direct RNA to the posterior pole.
However, the hybrid RNA cannot move to the posterior pole
in the absence of endogenous, properly spliced oskar mRNA.
This leads to the conclusion that the hybrid molecule
piggybacks on the endogenous oskar mRNA and suggests that
the oskar RNA 3′UTR, although necessary, is not sufficient for
transport to the posterior pole and that the splicing events in
the nucleus regulate cytoplasmic localization of mRNA
(Hachet and Ephrussi, 2004).
Splicing not only removes introns from RNA, but also
deposits a stable protein complex called the exon-exon
junction complex (EJC) upstream of mRNA exon-exon
junctions. In Drosophila and Xenopus, the EJC, besides
containing proteins directly involved in splicing, contains the
Y14-Mago heterodimer, which binds to the nucleo-
cytoplasmic shuttling complex TAP/p15, which transports
spliced RNA through the nuclear pores. In addition, the EJC
serves as an anchoring point for the factors responsible for the
nonsense-mediated decay (NMD) of mRNA containing
premature termination codons (le Hir et al., 2001a; le Hir et
al., 2001b; Wagner and Lykke-Andersen, 2002). Hachet and
Ephrussi showed that the Drosophila EJC, deposited on oskar
mRNA in a splicing-dependent manner in the nucleus, is
necessary for the oskar mRNA 3′UTR to assemble into a
functional localization complex (Hachet and Ephrussi, 2004).
The authors suggested that the first EJC landmark on oskar
mRNA mediates subsequent interactions with other factors
and specifies the structure of the oskar mRNA localization
complex.
In Xenopus oocytes, the fate of localized RNA also
probably depends on its nuclear or cytoplasmic history.
When synthetic Xcat2 mRNA is injected into the nucleus
of stage I or early stage II Xenopus oocytes, it behaves
like endogenous Xcat2 mRNA, following the
mitochondrial cloud pathway and entering germinal
granules (Fig. 5 and Table 1) (Kloc et al., 1996; Kloc et
al., 2000). However, when synthetic Xcat2 RNA is
injected into the nuclei or the cytoplasm of older oocytes
(stage III/IV), it follows the MT-dependent Vg1 pathway
and probably uses a kinesin motor (Fig. 5 and Table 1)
(Betley et al., 2004; Kloc et al., 1996; Zhou and King,
1996). Similarly, when an early-pathway XNIF mRNA
is injected into stage I oocytes it moves to the
mitochondrial cloud, but when the same RNA is injected
into stage III oocytes it follows the late (Vg1) pathway,
and its LE binds to the same proteins that bind the Vg1-
LE (Table 1) (Claussen et al., 2004).
The findings discussed above suggest that the mode of
transport in Xenopus oocytes and Drosophila oocytes
depends not only on the cis-acting elements but also on
the identity and availability of the factors with which they
associate. Indeed, modification of the localized RNA
both in the nucleus and in the cytoplasm has recently
been observed in Xenopus. Modification of the late-
pathway RNAs Vg1 and VegT in stage III/IV oocytes
begins in the nucleus, in which hnRNP1 and Vg1RBP/
Vera bind to the RNA, forming a core complex. After
export into the cytoplasm, this complex undergoes
remodeling and recruitment of additional components
(the proline-rich, RNA-binding protein Prrp and XStau)
that lead to the formation of an RNP-transporting/anchoring
complex (Kress et al., 2004; Zhao et al., 2001).
Translational regulation of localized RNAs
Another process directed by the events in the nucleus is the
regulation of translation. To produce proteins at the right time
and place within the oocyte, localized mRNAs must be
translationally silent not only during their transport but also at
the final destination until their protein product is required. In
Drosophila oocytes, Oskar protein does not accumulate before
its mRNA becomes localized to the posterior pole in mid
oogenesis (Markussen et al., 1995; Rongo et al., 1995). The
fact that unlocalized oskar mRNA is associated with
polysomes indicates that either regulated protein degradation
or inhibition of translation is involved (Benton and St Johnston,
2002; Braat et al., 2004; Riechmann et al., 2002). The Bruno
protein binds to Bruno-response elements (BREs) in the oskar
mRNA 3′UTR, and mutation of these elements causes
premature translation of oskar (Kim-Ha et al., 1995). Bruno
also binds to the gurken mRNA 3′UTR and plays a role in its
translational regulation (Filardo and Ephrussi, 2003).
At least eight distinct transacting factors, including Btz,
Y14-Mago (a component of the EJC; see above), Yps and Cup,
assemble with oskar mRNA into RNP particles and participate
in oskar mRNA localization and its translational repression
(Hachet and Ephrussi, 2001; Palacios et al., 2004; van Eeden
et al., 2001; Wilhelm et al., 2000). Wilhelm et al. have
proposed that an interaction between Cup and eukaryotic
initiation factor 4E (eIF4E) blocks the initiation of oskar
Journal of Cell Science 118 (2)
12 3
Ring Canal
Kinesin
Oskar mRNA
Microtubule
Cortical actin anchor
Fig. 4. Transport and anchoring of oskar mRNA in Drosophila oogenesis.
(1) In early oogenesis (stage 2-6), the MTs extend from the oocyte posterior
to the nurse cells. Then, oskar mRNA is transported from the nurse cells, on
the MTs, via ring canals, to the posterior cortex of the oocyte. (2) In mid
oogenesis (stage 8), there is a re-polarization of the oocyte MTs, the MTOC
at the oocyte posterior disassembles, and new MTs are nucleated from most
of the oocyte cortex. The plus-end-directed motor kinesin transports oskar
mRNA away from the cortex and towards the oocyte interior. (3) Subsequent
destabilization of MTs at the oocyte posterior (late stage 8 and early stage 9)
uncovers the posterior actin anchor, leading to the entrapment and
concentration of oskar mRNA at the posterior pole [adapted from Cha et al.
(Cha et al., 2004)].
Journal of Cell Science

277
RNA localization mechanisms
mRNA translation in early oogenesis (Wilhelm et
al., 2003). In late oogenesis, after oskar becomes
localized at the posterior, Cup no longer
associates with eIF4E, and this de-represses
translation (Lasko, 2003; Nakamura et al., 2004;
Wilhelm et al., 2003). The translational regulation
of oskar and gurken mRNAs also depends on the
members of the hnRNP family, such as Hrp48/p50/Hrb27 and
Squid (Goodrich et al., 2004; Gunkel et al., 1998; Yano et al.,
2004).
Little is known about translational regulation of localized
RNA in Xenopus oocytes. Vg1 mRNA undergoes 3′UTR-
dependent translational repression before being localized in
stage IV oocytes (Dale et al., 1989; King et al., 1999; Tannahill
and Melton, 1989). VgRBP71, a KH-domain protein that
interacts with the Vg1 mRNA LE, acts as a translational
activator of Vg1 mRNA by promoting the removal of its
translational repressor element. VgRBP71, which has RNA-
strand-separating activity, binds to the 3′ end of Vg1LE,
inducing the cleavage at an adjacent polyadenylation signal. As
a consequence, the Vg1 mRNA becomes polyadenylated at this
site and the downstream translational repressor element is
removed (Kolev and Huber, 2003). Xcat2 and Xdazl mRNAs
are probably silent throughout oogenesis. However, exogenous
Xcat2 mRNA is translated upon injection into stage IV oocytes
(Zhou and King, 1996). Since endogenous Xcat2 mRNA is
sequestered within germinal granules (Kloc et al., 1998; Kloc
et al., 2002a; Kloc et al., 2002b), it might be inaccessible to
the components of the translation machinery. By contrast,
exogenous Xcat2 RNA injected into stage III/IV oocytes and
unable to enter germinal granules is readily available to
undergo translation.
The translational silencing in Xenopus oocytes might also
depend on Y-box proteins such as mRNP3 and FRGY2/
mRNP4, which are present in mRNP storage particles and
probably mask or inhibit translation (Bouvet and Wolfe,
1994; Darnbrough and Ford, 1981; Murray et al., 1992).
34
A
B
?? ??
??
Vg1RBP/Vera
hnRNP
Prrp
Staufen & motor
Sm proteins ?
Late-pathway RNA
Injected late pathway RNA
Injected early pathway RNA
?? ?? ??
12
Early-pathway RNA
Fig. 5. Model illustrating the dependence of the fate of
localized endogenous and exogenous RNAs in
Xenopus oogenesis on their nuclear/cytoplasmic
history. Endogenous (A) and exogenous (B) early- and
late-pathway RNAs. (1) In stage I oocytes, the early-
pathway RNAs, such as Xcat2 mRNA, bind nuclear
proteins (possibly Sm proteins) that facilitate transport
(via nuclear pores) to the mitochondrial cloud (yellow)
and germinal granules (red spheres). The late-pathway
RNAs, such as Vg1 mRNA, bind to Vg1RPB/Vera and
hnRNP, which form a core complex facilitating export
from the nucleus, and diffuse uniformly within the
oocyte cytoplasm. (2) Later in oogenesis (starting
from late stage II or early stage III), Staufen and Prrp
proteins are added to the core complex assembled on
late-pathway RNAs. These bind to a molecular motor,
such as kinesin I and/or II, that transports RNAs on the
MT tracks that form a wedge around the remnants of
the mitochondrial cloud. (3) Early- and late-pathway
RNAs injected into the nuclei of stage I/early stage II
oocytes that bind the appropriate nuclear factors and
mimic the localization pattern of their endogenous
counterparts. There is no information on the fate of
early- or late-pathway RNAs injected into the
cytoplasm of stage I oocytes. (4) Early-pathway RNAs
injected into the nucleus or cytoplasm of stage III, or
older, oocytes behave like late-pathway RNAs
migrating on the MTs towards the vegetal cortex. This
indicates that the early-pathway binding factors
present in the nuclei of stage I/early stage II oocytes
are either absent or unavailable in older oocytes.
However, the early-pathway RNAs can bind some of
the cytoplasmic factors of the late-pathway machinery,
and they either mimic the movement of late-pathway
RNAs or piggyback on late-pathway RNAs. Late-
pathway RNAs injected into the nuclei or cytoplasm of
older oocytes bind the appropriate factors and after
export into the cytoplasm behave like their
endogenous counterparts – either assembling their
own transport complexes or piggybacking on
endogenous RNAs.
Journal of Cell Science

278
Interestingly, the Y-box protein Yps, related to Xenopus FrgY2
(implicated in translational silencing), was discovered in an
RNP complex containing oskar mRNA (see above) (Wilhelm
et al., 2000). Recently, Tanaka et al. suggested that, in
ascidians, translational repression of the localized mRNA
cipem also depends on the formation of an RNP complex
containing the Y-box protein CiYB (Tanaka et al., 2004).
Components of the RNA-silencing pathways as
possible regulators of RNA localization and
translation
RNA silencing regulates a plethora of vital functions in a
variety of organisms, acting through small noncoding RNAs
[short interfering (si)RNAs and/or micro (mi)RNAs] that are
produced by DICER-mediated cleavage of long double-
stranded or hairpin RNA precursors, respectively. Single-
stranded miRNA or siRNA in RNA-induced silencing
complexes (RISCs) recognize complementary target mRNA.
Perfect complementarity results in target degradation, and
partial complementarity results in translational repression
(Bartel, 2004; Dykxhoorn et al., 2003; Finnegan and Matzke,
2003; Nolan and Cogoni, 2004).
Recent observations raise the exciting possibility that
components of the RNA-silencing pathway regulate localized
RNAs in oocytes. In Drosophila, the armitage gene, which
encodes a component of the RNA-silencing pathway that is
also involved in MT polarization (Cook et al., 2004; Tomari et
al., 2004), is required for oskar mRNA silencing. Armitage is
thought to participate in the assembly of the RISC and, by
colocalizing with the oskar mRNA, it can spatially restrict
RNA-silencing activity to its particular location (Cook et al.,
2004).
Another gene required for RNA silencing in Drosophila and
efficient translation of oskar mRNA is aubergine, which
encodes a member of the RNAi-defective/Argonaute1
(RDE1/AGO1) protein family (Wilson et al., 1996). In
addition, aubergine is involved in the localization of
Maelstrom, a member of the Drosophila spindle-class family,
to the nuage (Findley et al., 2003). The nuage is a specialized,
perinuclear, electron-dense structure present in germ cells
across the animal kingdom (Kloc et al., 2004b). It is believed
to be a precursor of germinal (polar/P) granules, and recent
studies indicate that it participates in the exchange of
components between the nucleus and the germinal granules
(Findley et al., 2003; Kloc et al., 2004a; Kloc et al., 2002b). In
animals ranging from nematodes to mammals, the conserved
component of nuage is the DEAD-box RNA helicase Vasa
(Raz, 2000; Snee and Macdonald, 2004), which is probably
involved in translational control of RNAs localized in the
nuage and also, indirectly, in RNA silencing (Findley et al.,
2003). In Drosophila, Maelstrom shuttles between the nucleus
and cytoplasm, possibly via the nuage and sponge bodies
(Findley et al., 2003). Maelstrom mutants also exhibit defective
processing of Vasa and cause the mislocalization of two
proteins involved in RNA silencing, Dicer and Argonaute2,
which suggests a possible connection between the nuage and
the RNA-silencing pathway in Drosophila oocytes (Findley et
al., 2003).
Recently, a Vasa-like DEAD-box helicase and components
of the splicing machinery, including Sm proteins, have been
found in the nuage in Xenopus oocytes (Bilinski et al., 2004).
This suggests that a subset of Sm proteins have a splicing-
independent role. The nuage thus might serve as a platform for
the shuttling of proteins and RNAs between the nucleus and
cytoplasm and for the assembly of RNP particles necessary for
the correct localization of various mRNAs into the germinal
granules. These results also link the nuage and the RNA-
silencing pathway (Barbee et al., 2002; Bilinski et al., 2004;
Findley et al., 2003). Another possibility is that, as in
Drosophila, there is a connection in Xenopus oocytes between
splicing events and the fate of mRNAs localized in the nuage
and germinal granules.
Perspective
The mechanisms governing RNA localization in oocytes are
clearly extremely complex. For example, the mechanisms
governing the localization of endogenous RNAs are very often
different from those governing the localization of exogenously
introduced counterparts. The development of new imaging
technologies and in vivo techniques allowing monitoring of the
transport of endogenous RNA in real time (Bratu et al., 2003;
Cha et al., 2001; Famulok, 2004; Forrest and Gavis, 2003; Snee
and Macdonald, 2004; Stephenson, 2004) should alleviate the
problems arising from the current heterogeneity of research
techniques.
The recent findings that the fate of a localized RNA depends
on its nuclear and/or cytoplasmic history and its connection to
the RNA-silencing pathways, and the discovery that coding
and noncoding localized RNAs can play a structural role in the
organization of the oocyte cytoskeleton, should open
completely new avenues in the study of RNA localization in
oocytes. The majority of RNA localization studies in oocytes
are limited to model organisms such as Drosophila and
Xenopus, which are not necessarily reflective of other dipterans
(Bullock et al., 2004) or amphibians. For example, in directly
developing frog Eleutherodactylus coqui, in contrast to
Xenopus, the mRNAs of the Vg1 and VegT orthologs are
localized at the animal pole of the oocyte (Beckham et al.,
2003). Findings like this indicate the necessity of widening
studies to other, lesser-known species and taxa. Such studies
could reveal which phenomena and mechanisms operating in
RNA localization are fundamental to a given group of
organisms and which evolved to accommodate the specific
developmental needs of particular species.
This work was supported by grants from NSF (L.D.E.).
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Journal of Cell Science