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First published online 27 July 2004
doi: 10.1242/dev.01286
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Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montréal, Québec H3A 1B1, Canada
* Author for correspondence (e-mail: paul.lasko{at}mcgill.ca)
Accepted 27 May 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Translation, Germ cells, DEAD-box, Axis-patterning, Gurken, Drosophila, cIF2, Vasa
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). During oogenesis, the TGF
signaling molecule
Gurken (Grk) establishes polarity within the oocyte along both the
anteroposterior and dorsoventral axes
(Neuman-Silberberg and Schüpbach,
1993; González-Reyes et
al., 1995; Roth et al.,
1995) (reviewed by Nilson and
Schüpbach, 1999). Subsequently, at the posterior end of the
oocyte and early embryo, a specialized region of cytoplasm, called pole plasm,
accumulates RNAs and proteins required for germline development (reviewed by
Mahowald, 2001). Pole plasm
assembly is initiated through the localized translation of oskar
(osk) mRNA (Ephrussi et al.,
1991; Kim-Ha et al.,
1991). Vasa (Vas) protein and nanos (nos) mRNA
are among several molecules that accumulate in the pole plasm downstream of
Osk. Localized translation of nos mRNA in the pole plasm produces a
Nos protein gradient that is essential to determine abdominal fate, thus
directly linking germ cell development to posterior somatic patterning in the
embryo (Lehmann and Nüsslein-Volhard,
1991; Wang and Lehmann,
1991).
osk, grk and nos RNAs are all under complex translational
regulation in the developing oocyte, mediated through cis-acting elements in
their untranslated regions (UTRs). Genetic and biochemical analyses in
Drosophila ovaries and embryos have identified several translational
regulatory proteins. Bruno (Bru) is involved in repressing translation of
osk and grk (Kim-Ha et
al., 1995; Webster et al.,
1997; Filardo and Ephrussi,
2003; Nakamura et al.,
2004), and Smaug (Smg) is involved in repressing translation of
nos (Smibert et al.,
1996; Dahanukar et al.,
1999; Nelson et al.,
2004). Although recent work has linked both Bru and Smg to the
cap-binding step of translation initiation
(Wilhelm et al., 2003;
Nakamura et al., 2004;
Nelson et al., 2004),
translational repression of osk, grk and nos probably
targets multiple steps of translation (Lie
and Macdonald, 1999; Clark et
al., 2000; Nakamura et al.,
2004). In general, details of the mechanisms of translational
derepression and activation for specific transcripts remain obscure. In
several organisms, translational activation of maternal mRNAs involves
cytoplasmic polyadenylation. The activity of the Drosophila
cytoplasmic polyadenylation element-binding protein, called oo18 RNA-binding
protein or Orb (Lantz et al.,
1992) is implicated in activating translation of osk and
possibly of other mRNAs (Chang et al.,
1999; Castagnetti and Ephrussi,
2003).
Our work addresses the function of the highly conserved DEAD-box RNA
helicase Vas, which is required for the progression of oogenesis and for pole
plasm assembly (Schüpbach and
Wieschaus, 1986; Hay et al.,
1988; Lasko and Ashburner,
1988; Liang et al.,
1994). Based on sequence similarity with yeast Ded1p (reviewed by
Linder, 2003), and the finding
that expression of several proteins is reduced in vas mutants, Vas
has been suggested to function in translational regulation (reviewed by
Johnstone and Lasko, 2001).
Females bearing hypomorphic vas mutations complete oogenesis but
produce embryos lacking germ cells and lacking posterior segments, indicating
an essential role for vas in both processes
(Schüpbach and Wieschaus,
1986). An earlier function for Vas, during oogenesis, was also
demonstrated through the study of null mutations that are viable but produce
no embryos (Styhler et al.,
1998; Tomancak et al.,
1998). Although vas-null oocytes display minimal
disruption of grk RNA accumulation, Grk protein levels are severely
reduced, leading to the hypothesis that Vas could play a role in grk
translational control (Styhler et al.,
1998; Tomancak et al.,
1998). Vas also appears to represent an important link between
meiotic cell cycle progression and developmental events such as establishment
of polarity. In response to a meiotic checkpoint, activated by a delay in DNA
double-strand break (DSB) repair during oogenesis, Vas is post-translationally
modified, and this corresponds to a downregulation in Grk protein accumulation
(Ghabrial and Schüpbach,
1999).
In previous work, we identified a translation factor dIF2, now called
eIF5B, as a Vas-binding protein (Carrera et
al., 2000). A genetic interaction between null alleles of
dIF2 and vas suggested a functional link between these two
proteins. eIF5B/dIF2 has since been demonstrated in mammalian systems to be
required for all cellular translation and to act at the 60S ribosomal subunit
joining step of translation initiation
(Pestova et al., 2000b).
Subsequent work has indicated that translation can be regulated at the stage
of subunit joining (Ostareck et al.,
2001; Searfoss et al.,
2001). These results suggest that Vas could function as a
translational regulator of specific mRNAs through interaction with eIF5B.
To test this hypothesis, we created specific vas mutations that severely reduce its interaction with eIF5B. These mutant forms of Vas still localize correctly, allowing us to investigate which developmental functions of Vas require an interaction with eIF5B, and are therefore likely to involve a translational regulatory role. We found that the Vas-eIF5B interaction was essential for the progression of oogenesis, and for normal expression of Grk. In addition, we found that the interaction between Vas and eIF5B was crucial for germ cell specification, but we observed a much less stringent requirement for this interaction in posterior somatic segmentation. We conclude that interaction with eIF5B is essential for Vas function, and propose that Vas achieves translational regulation in the germline through eIF5B binding. The Vas-eIF5B interaction represents a significant opportunity to investigate how a tissue-specific regulator may control the ribosomal subunit joining step of translation initiation to activate the translation of specific transcripts.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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),
and inserted into XhoI/NotI digested pBluescript to use as a
template for mutagenesis. Specific deletions in vas were generated
using PCR-mediated mutagenesis, using the following primers: 
616-618,
5' TTTCTACGCACCTGTGGTGCC and 3' AGTCTGGCCAGATCCCTCCAAG;
616, 5' CCGGACTTTCTACGCACCTGTG and 3' (same as for
616-618); 617, 5' GACTTTCTACGCACCTGTGGTG and 3'
AACAGTCTGGCCAGATCCCTC; 618, 5' (same as for 616-618) and
3' CGGAACAGTCTGGCCAGATCC. Mutagenesis was followed by blunt-end
ligation, and was verified by sequencing. Each vas-coding region was
then digested out of pBluescript using XhoI and NotI, and
subcloned into a XhoI/NotI digested plasmid
P[w+ Pvas-gfp]
(Nakamura et al., 2001),
derived from pCaSpeR2. In addition, each vas-coding region was PCR
amplified out of pBluescript and subcloned into a NcoI/XhoI
digested pEG202 vector, used for expression in yeast.
Yeast interaction trap assays
The yeast strain EGY48 was co-transformed with `bait' constructs cloned in
pEG202, `prey' constructs cloned in pJG4-5, and a lacZ reporter
plasmid pSH18-34. ß-Galactosidase activity was monitored using a
plate-based assay as described previously
(Golemis et al., 1997) and in
liquid culture (Reynolds et al.,
1997).
Protein expression and western blotting
Preparation of yeast protein extracts was performed according to the Yeast
Protocols Handbook (Clontech). For every transformed strain, a 5 ml overnight
culture in selective media was used to inoculate a 50 ml culture in YPD media,
incubated at 30°C with shaking (220-250 rpm) until
OD600=0.4-0.6. Cells were centrifuged at 1000 g for
5 minutes at 4°C, resuspended in 50 ml cold H2O, centrifuged at
1000 g for 5 minutes at 4°C and frozen in liquid nitrogen.
Pellets were thawed in pre-warmed Cracking Buffer (8 M Urea, 5% SDS, 40 mM
Tris-HCl [pH6.8], 0.1 mM EDTA, 0.4 mg/ml Bromophenol Blue), supplemented with
1 mM PMSF, 1 x protease inhibitor cocktail (Roche Diagnostics) and 10
µl ß-mercaptoethanol/ml buffer. Samples were transferred into
microcentrifuge tubes containing glass beads, and heated at 70°C for 10
minutes. They were then vortexed for 1 minute, and centrifuged at 20,000
g for 5 minutes at 4°C. Supernatants were kept on ice
while pellets were boiled at 100°C for 3-5 minutes, vortexed for 1 minute
and centrifuged at 20,000 g for 5 minutes at 4°C, and then
combined with the first supernatants. Drosophila ovarian proteins
were extracted by homogenization in phosphate-buffered saline (PBS)/1 mM
PMSF/1 x protease inhibitor cocktail (Roche Diagnostics). Samples were
centrifuged at 18,000 g for 15 minutes at 4°C, and
supernatants were combined with SDS loading buffer. For western blotting,
proteins were resolved on SDS-PAGE gels and transferred onto nitrocellulose
membranes, blocked overnight at 4°C in PBS/2% skim milk/0.05% Tween-20
(PBSTM). Membranes were incubated for 1 hour at room temperature with primary
antibodies diluted in PBSTM, washed with PBSTM, then incubated for 1 hour at
room temperature with HRP-conjugated secondary antibodies (Amersham Pharmacia)
diluted 1:5000 in PBSTM. Membranes were washed with PBSTM and proteins were
detected by chemiluminescence (NEN). Rabbit anti-Vas was used at 1:5000. Mouse
anti-actin (ICN Biomedicals) was used at 1:5000. Mouse anti--Tubulin
(Sigma) was used at 1:5000. Rabbit anti-4EBP was used at 1:2000.
Immunohistochemistry and in situ hybridization
Immunostaining of ovaries and embryos with rabbit anti-Nos (1:1000), rabbit
anti-Osk (1:500), rabbit anti-Tud (1:250) and rat anti-Vas (1:2000) was
performed as described previously
(Kobayashi et al., 1999).
Fluorescent antibody staining was detected using goat anti-rabbit
Alexa546nm, anti-rat Alexa633nm and anti-mouse
Alexa568nm secondary antibodies (Molecular Probes). Immunostaining
with mouse anti-Grk (1:10) in ovaries was performed as follows: ovaries were
dissected in PBST (PBS/0.3% Triton) and fixed for 20 minutes in 200 µl of
4% formaldehyde/PBS + 600 µl heptane. Samples were rinsed with PBST, and
blocked for 1 hour in PBS/1.0% Triton/3% BSA. Samples were incubated with
primary antibodies diluted in PBST for 1 hour at room temperature, rinsed and
then washed overnight in PBST. Samples were then incubated with secondary
antibodies for 1 hour at room temperature, washed in PBST, incubated for 20
minutes in 0.5 µg/ml DAPI, washed in PBST and then mounted in 70%
glycerol/PBS. In situ hybridization was performed as described previously
(Kobayashi et al., 1999),
except that DMSO was omitted during fixation, and PBS/0.1% Tween-20 was used
instead of MAB throughout the protocol.
Fly strains and techniques
yw flies were used for P element-mediated germline transformation.
vas alleles used for subsequent analyses were
vasPD (Schüpbach
and Wieschaus, 1986) and vasPH165
(Styhler et al., 1998). To
visualize GFP in ovaries, they were dissected in PBS and fixed in 4%
formaldehyde/PBS/0.2% Tween-20. Samples were washed in PBS and mounted in 70%
glycerol/PBS. For live GFP visualization in embryos, they were collected on a
sieve and washed with H2O, then dechorionated with bleach and
washed again with H2O. Embryos were then mounted in Halocarbon oil
(series 400) and examined immediately. For visualization of dorsal appendages,
eggs were collected on a sieve and washed with H2O, then mounted in
Hoyer's medium and incubated overnight at 60°C.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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).
Previous work has shown that interaction with eIF5B involved residues
C-terminal to amino acid 310 of Vas
(Carrera et al., 2000). In
order to map more precisely the region of Vas required for interaction with
eIF5B, we created a series of large deletions within Vas and tested these in
the yeast two-hybrid system against the clone of eIF5B that we previously
isolated in a yeast two-hybrid screen (Fig.
1B). We confirmed that the C-terminal half of Vas was required for
interaction with eIF5B, and specifically implicated the region from residue
595 to 621 of Vas in eIF5B-interaction. We then tested a series of Vas
constructs that began at the N terminus and ended between residues 595 and 621
(Fig. 1C). This analysis
implicated residues 616-618 of Vas as being crucial for eIF5B interaction. We
examined whether this region of Vas was conserved in homologous proteins in
different species (Fig. 1D). Both V616 and P617 are identical among Vas homologs, while D618 does not
appear to be conserved. Some neighboring residues are also invariant among Vas
homologs. Although a proline corresponding to P617 is found in eIF4A, in
general, more distant Drosophila DEAD-box proteins such as eIF4A,
Ddx1 and Dhh1 are not highly conserved within this area
(Fig. 1D). These results
suggest that this region of Vas has been conserved within its homologous
proteins for a specific function, and is not a general feature of DEAD-box
proteins.
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616-618), as well as each residue individually (616, 617
and 618), and tested these in the yeast two-hybrid system against
eIF5B, using a liquid assay for ß-galactosidase activity to quantify the
level of interaction. All of these mutant forms of Vas expressed protein
efficiently and stably in yeast (Fig.
2A). We found that the 616-618 deletion completely
eliminated detectable interaction with eIF5B
(Fig. 2B). The single amino
acid deletions also all showed a reduction in eIF5B-interaction relative to
the full-length Vas protein, 
1.7-fold for 616, tenfold for
617 and 20-fold for 618 (Fig.
2B). In order to verify that these mutations in Vas affected
eIF5B-interaction specifically and not interaction between Vas and Osk, which
is crucial for pole plasm assembly
(Breitwieser et al., 1996), we
also tested the Vas deletions against Osk in the same assay
(Fig. 2C). We found that
relative to the full-length Vas protein, 616-618 showed a modest (less
than twofold) reduction in Osk interaction. The single amino acid deletions,
however, showed no reduction of this interaction
(Fig. 2C). We conclude that
these individual Vas deletions specifically disrupt interaction with eIF5B
without affecting the interaction between Vas and Osk. Furthermore, the
interaction site of a third known Vas-binding protein, Gustavus (Gus) maps to
a region near the N terminus of Vas
(Styhler et al., 2002), very
distant from the residues we found were crucial for eIF5B interaction. Thus,
the vas mutations we have generated that affect eIF5B interaction do
not affect its interaction with any other known binding protein, and do not
affect any conserved motif that is implicated in any function common to
DEAD-box proteins, such as ATP binding, ATP hydrolysis, RNA binding and RNA
unwinding (Pause and Sonenberg,
1992; Tanner et al.,
2003) (reviewed by Tanner and
Linder, 2001).
|
). Within the oocyte, Vas is also distributed throughout the
cytoplasm, and in later stages accumulates in the developing posterior pole
plasm, where it remains concentrated in the early embryo. The subsequent
functions of Vas all require this posterior localization, thus we wanted to
establish whether specific mutations that reduce Vas-eIF5B interaction also
affected Vas localization. To do this, we expressed GFP-fusions of various
forms of Vas in transgenic flies under the control of the endogenous
vas promoter. We tested the levels of expression of the transgenic
fusion proteins on western blots, using endogenous Vas protein as an internal
control (Fig. 3). Expression of
the wild-type transgenic protein was comparable with that of the endogenous
protein (Fig. 3C). Despite
generating multiple transgenic lines for each, the 616-618, 616
and 618 proteins were always expressed at lower levels than the
wild-type transgenic protein, when each was compared with levels of endogenous
Vas (Fig. 3F,I,O). However, the
617 transgenic protein was expressed at levels comparable with the
wild-type transgenic protein (Fig.
3L).
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616-618 transgenic protein
(Fig. 3D,E), the GFP signal was
weak because of low protein expression, but even in this case, correct
localization could be detected. From these data, we conclude that eIF5B
interaction is not required to achieve Vas localization.
To avoid complications resulting from dose effects, we chose to focus the
remainder of our analysis on the vas617 mutation
because of its high level of protein expression and the strong effect of this
mutation on eIF5B interaction. Comparing the wild-type
vas+ and the vas617 transgenes
allowed us to determine the phenotypic consequences of specifically reducing
eIF5B interaction, and to separate the functions of Vas that require that
interaction from those that depend solely on its localization and
expression.
Vas-eIF5B interaction is required for female fertility and for grk regulation
In order to investigate the requirement of the Vas-eIF5B interaction during
oogenesis, we examined the vas617 transgene in the
background of a vas null allele, vasPH165
(Styhler et al., 1998). The
wild-type vas+ transgene, and the
vas617 transgene express comparable levels of
protein in the vas null background
(Fig. 4A). Most
vasPH165 egg chambers arrest early in oogenesis, producing
few mature eggs, none of which hatches into embryos
(Styhler et al., 1998)
(Fig. 4B). Strikingly,
vasPH165;P{vas617} females
exhibit a very similar arrest in oogenesis to that of
vasPH165 itself, producing few mature eggs that do not
hatch (Fig. 4D). In both
vasPH165 and
vasPH165;P{vas617} females of
the same age (3-4 days), the number of stage 14 eggs per ovary ranges widely
from 0-32 with an average of nine, roughly a third the average of wild-type.
Examples of severely atrophied vasPH165 and
vasPH165;P{vas617} ovaries are
shown in Fig. 4B,D. Similar to
vasPH165 (Styhler et
al., 1998), eggs produced by
vasPH165;P{vas617} females
sometimes exhibit a duplicated micropyle at both the anterior and posterior
ends (data not shown). By contrast, ovaries from
vasPH165;P{vas+} females produce
abundant mature eggs that can hatch into viable embryos
(Fig. 4C). Thus, the
vas617 transgene does not rescue the oogenesis
arrest caused by the vasPH165 mutation, indicating that
interaction between Vas and eIF5B is crucial for the progression of
oogenesis.
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;
Tinker et al., 1998;
Tomancak et al., 1998). In
agreement with previous reports (Styhler
et al., 1998), we observed that 63% of eggs produced by
vasPH165 females exhibited one fused or semi-fused dorsal
appendage, 12% had no dorsal appendages and 25% had two dorsal appendages. We
examined dorsal appendages in our transgenic lines and found that in
vasPH165;P{vas617} females, 67%
of eggs exhibited one semi-fused or fused dorsal appendage
(Fig. 5C-E), 11% formed no
dorsal appendages (Fig. 5F) and
22% had two dorsal appendages (Fig.
5B). Conversely, 82% of control
vasPH165;P{vas+} eggs had two dorsal
appendages (Fig. 5A). Thus, the
vas617 transgene does not rescue the ventralization
phenotype of the vasPH165 mutation.
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617} ovaries, and
found that it also was indistinguishable in early stages of development from
that observed for vasPH165
(Fig. 5H,I,K,L) (Styhler et al., 1998).
grk RNA is strongly enriched in the oocyte
(Fig. 5H,I); however, the
protein level is severely reduced (Fig.
5K,L). In control
vasPH165;P{vas+} ovaries,
concentration of both grk RNA and protein in the oocyte resembles
wild type (Fig. 5G,J). Thus,
the vas617 transgene does not support efficient
expression of Grk in a vas-null background. We conclude that the
requirements for Vas for the progression of oogenesis, for dorsoventral
patterning of the egg chamber, and for grk regulation, all rely on
its interaction with eIF5B.
Pole plasm components assemble in vasPD;P{vas617} ovaries
Pole plasm assembly requires the sequential posterior localization of
multiple proteins and RNAs (reviewed by
Mahowald, 2001). Osk, which is
at the top of a complex hierarchy of factors involved in pole plasm assembly,
is required for recruitment of Vas to the posterior
(Lasko and Ashburner, 1990).
Downstream of Vas localization, Tud protein is recruited
(Bardsley et al., 1993), and
Osk, Vas and Tud are required for pole cell formation and posterior
segmentation. In order to investigate the requirement for the Vas-eIF5B
interaction in pole plasm assembly, we examined the
vas617 transgene in the background of a hypomorphic
vas allele, vasPD, as
vasPH165; P{vas617} ovaries
produce few late-stage egg chambers. The vasPD allele, in
which Vas is detectable only in the germarial stages of oogenesis
(Lasko and Ashburner, 1990),
completes oogenesis normally, but the embryos produced lack pole cells and
posterior segmentation. Sequencing of the vasPD allele did
not reveal any alteration in the coding sequence
(Liang et al., 1994), thus
this mutation is believed to affect only the level of vas expression
and not the nature of Vas protein.
In wild-type stage 10 egg chambers, strong posterior accumulation of Osk,
Vas and Tud protein is evident (Fig.
6B,F,J), while in vasPD ovaries, which have
severely reduced levels of Vas protein
(Fig. 6A), posterior Osk is
abundant, but neither Vas nor Tud is detected at the posterior
(Fig. 6C,G,K). We next compared
the accumulation of these three proteins in vasPD;
P{vas+} and vasPD;
P{vas617} ovaries, which contain comparable levels
of Vas protein as assayed by western blotting
(Fig. 6A). We found that Osk,
Vas and Tud protein can all be readily detected at the posterior of stage 10
oocytes (Fig. 6D-E,H-I,L-M), at
apparently equivalent levels for both transgenic genotypes. Thus,
GFP-Vas617 not only localizes correctly to the posterior, but it is
also able to recruit the downstream pole plasm component Tud. We conclude that
interaction with eIF5B is not required for the role of Vas in the initial
assembly of the pole plasm.
|
617 transgene in the
vasPD background allowed us to address this question, as
Vas protein is expressed abundantly from this transgene, localizes correctly
and is able to recruit Tud, an essential factor for germ cell specification
(Ephrussi and Lehmann, 1992).
We examined pole cell formation in the progeny of flies bearing either the
vas+ transgene or the vas617
transgene in the vasPD background. For simplicity,
transgenic embryos will be referred to by the genotype of the mother. Using
detection of GFP-Vas as a marker in live embryos at the cellular blastoderm
stage, we found that the majority of
vasPD;P{vas+} embryos had formed pole
cells at this stage, indicating that the wild-type transgene rescues this
vas phenotype (Fig.
7A). By contrast, only one
vasPD;P{vas617} embryo out of
216 examined exhibited pole cells at the same stage, indicating that
expression of the vas617 transgene could not rescue
this vas phenotype (Fig.
7B). We verified this result using Nos as an independent marker
for pole cells. Although 58% of
vasPD;P{vas+ }embryos exhibited
Nos-positive cells at the posterior of the embryo at the cellular blastoderm
stage (Fig. 7C), 81% of
vasPD;P{vas617} embryos
examined did not have any Nos-positive cells
(Fig. 7D). Five percent of
these embryos formed one to three Nos-positive cells at the posterior
(Fig. 7E), while in 14% of
embryos, Nos-positive cells were visible, but at inappropriate positions
(Fig. 7F). These results
demonstrate that, downstream of initial pole plasm assembly, the Vas-eIF5B
interaction is vital for embryonic germ cell formation.
|
), and
mutations in posterior group genes such as vas inhibit the expression
of stripes 4-6. In contrast to vasPD, in which all embryos
exhibit severe posterior segmentation defects
(Schüpbach and Wieschaus,
1986), we observed that half of
vasPD;P{vas617} embryos have a
wild-type segmentation pattern, as inferred from a wild-type ftz
distribution (Fig. 8A). In
fact, when allowed to complete development, many
vasPD;P{vas617} embryos hatched
into viable larvae. A further 20% of the
vasPD;P{vas617} embryos
exhibited a weak posterior group phenotype in which stripes 4-6 of
ftz were present but were reduced in width and intensity relative to
the other stripes (Fig. 8B),
although the remaining 30% exhibited a stronger phenotype
(Fig. 8C), more closely
resembling that of vasPD
(Schüpbach and Wieschaus,
1986). Over 90% of
vasPD;P{vas+} embryos expressed
ftz in the normal pattern of seven stripes (data not shown). From
this analysis, we conclude that the vas617
transgene can partially rescue the abdominal segmentation defect of the
vasPD allele, suggesting that the Vas-eIF5B interaction is
less crucial for posterior patterning than it is for pole cell specification.
nos translation is tightly regulated such that it is repressed
outside of the pole plasm and active in the pole plasm
(Gavis and Lehmann, 1994). In
existing vas mutants, nos is unlocalized and untranslated,
and the absence of a Nos protein gradient prevents abdominal formation
(Gavis and Lehmann, 1994;
Wang et al., 1994). We
examined Nos protein distribution in
vasPD;P{vas617} embryos
directly. Consistent with the ftz expression data, 55% of
vasPD;P{vas617} embryos
exhibited a detectable Nos gradient (Fig.
8E,F). In control
vasPD;P{vas+} embryos, a Nos protein
gradient could be observed in 95% of the embryos
(Fig. 8D). Thus, the
vas617 transgene partially rescues the Nos
accumulation defect of the vasPD allele, suggesting that
either the Vas-eIF5B interaction is dispensable for Nos translation, or that
the low level of interaction between Vas617 and eIF5B is sufficient to
activate Nos.
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|
Discussion |
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|
TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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617, which has greatly
reduced ability to interact with eIF5B. As residue 617 is not involved in
binding to any known Vas-interacting protein other than eIF5B, and it is
outside the region of Vas that contains the well-characterized catalytic
domains that are present in all DEAD-box proteins, we are confident that
Vas617 specifically disrupts the Vas-eIF5B interaction, and that this
mutation can be used to identify developmental processes that are sensitive to
an association between Vas and the general translational machinery. We found
that the VaseIF5B interaction is crucial for the progression of oogenesis, for
correct dorsoventral patterning of the egg, and for expression of high levels
of Grk in the developing oocyte. These results are most easily explained if
grk is a target for Vas-mediated translational activation acting
through its association with eIF5B.
A role for Vas in positively regulating grk translation is
consistent with previous work (Styhler et
al., 1998; Tomancak et al.,
1998; Ghabrial and
Schüpbach, 1999). Vas-mediated regulation of grk is
in turn regulated in response to a meiotic checkpoint, activated when DNA
double-strand break (DSB) repair is prevented during meiotic recombination
(Ghabrial et al., 1998;
Ghabrial and Schüpbach,
1999). In response to this checkpoint, Vas is post-translationally
modified, and Grk accumulation is reduced. It will be important to understand
the nature of the DSB-dependent modification of Vas, and to determine whether
it affects the Vas-eIF5B interaction, in order to gain insight into the
mechanism connecting cell cycle regulation with oocyte patterning.
The RNA-binding and unwinding activities of wild-type Vas and several
mutant forms of Vas have previously been assessed through in vitro assays
(Liang et al., 1994). Two
mutant forms of Vas, encoded by the vasO14 and
vasO11 alleles, were found to be severely reduced for
binding to an artificial RNA substrate, and a third form, encoded by
vasD5, was defective for RNA unwinding but not for
binding. Although vasD5 leads to defects in oogenesis,
vasO11 phenotypically resembles vasPD,
and vasO14 is a weak temperature-sensitive allele
(Lasko and Ashburner, 1990).
In the light of our present results, it is surprising that a mutant form of
Vas that cannot interact with RNA would nevertheless support oogenesis.
Perhaps in vivo, the RNA-binding and helicase activities of Vas are stimulated
or enhanced through a co-factor or through posttranslational modifications,
and the in vitro assay used in our earlier study may not accurately reflect
Vas activity in vivo.
Are there target RNAs for Vas-eIF5B regulation in the pole plasm?
Reduction of the Vas-eIF5B interaction by expressing Vas617 severely
reduces pole cell formation. This happens despite the ability of
vasPD;P{vas617} oocytes to
accumulate Osk, Vas and Tud at the posterior pole, demonstrating an essential
role for Vas in pole cell specification that is dependent upon its association
with eIF5B, and that cannot be substituted by Osk and Tud. The simplest
interpretation of these results is that Vas derepresses translation of a
localized RNA required for pole cell specification, in a manner analogous to
what appears to be the case for grk.
We considered the possibility that the Vas-eIF5B interaction could target
osk mRNA. It has previously been shown that whereas Osk protein
accumulates normally in vas mutant ovaries, Osk levels are severely
reduced at the posterior of vas mutant embryos
(Harris and Macdonald, 2001),
suggesting a role for Vas in posterior accumulation of Osk after its initial
recruitment, and/or in stabilizing Osk at the posterior. We observed
comparable and substantial levels of Osk at the posterior in
vasPD;P{vas617} and
vasPD;P{vas+} embryos (data not
shown), arguing against a direct role for the VaseIF5B interaction in
activating translation of osk mRNA. A requirement for Vas in
Par1-mediated phosphorylation and stabilization of Osk has been suggested
(Breitwieser et al., 1996;
Markussen et al., 1997;
Riechmann et al., 2002). As
Vas617 localizes normally and is able to interact with Osk, we would
not expect this mutation to have any effect on this Osk modification pathway.
Thus, our findings are consistent with a model whereby Vas influences Osk
activity through effects on phosphorylation, anchoring and/or stability,
perhaps through Par1, rather than directly regulating osk
translation.
Another candidate target for the Vas-eIF5B interaction is germ
cell-less (gcl), the activity of which is important for pole
cell specification but not for posterior patterning
(Jongens et al., 1992).
Unfortunately, with current reagents, and with new antisera we have generated,
we cannot reliably detect Gcl protein even in wild-type embryos prior to pole
bud formation, thus we cannot presently address the effects on gcl
translation of any mutation that abrogates pole cell formation. In addition,
effects on gcl cannot fully explain the severe consequences of the
Vas617 mutation on pole cell formation, because the number of pole
cells formed in maternal gcl-null embryos is somewhat higher than in
vasPD;P{vas617} embryos
(Robertson et al., 1999). This
suggests that even if gcl is a target, the Vas-eIF5B interaction may
regulate translation of more than one target RNA involved in pole cell
formation.
Is the Vas-eIF5B interaction required for posterior patterning?
Although the Vas-eIF5B interaction is vital for pole cell specification, it
is perhaps less so for posterior patterning and establishment of the Nos
gradient. Previous analysis of hypomorphic mutations in posterior-group genes,
including vas has indicated that a higher level of activity is
required for pole cell specification than for posterior patterning. For
example, all embryos produced by females homozygous for
vasO14 (Lasko and
Ashburner, 1990), osk301
(Lehmann and Nüsslein-Volhard,
1986) and tudWC
(Schüpbach and Wieschaus,
1986), lack pole cells, but some have normal posterior patterning
and are able to hatch. Our present results suggest two alternative
explanations for these observations. One possibility is that the Vas-eIF5B
interaction is required for posterior patterning, but that the residual
activity present in Vas617 is sufficient to achieve the low activity
level that is necessary. Alternatively, the Vas-eIF5B interaction may be
dispensable for posterior patterning, and the fact that we do not observe
complete rescue of this phenotype with the vas617
transgene may be due to an indirect effect of this mutation, resulting from a
general destabilization of the pole plasm that occurs in embryos that do not
form pole cells (Iida and Kobayashi,
2000). In such embryos, pole plasm components localize initially
but become fully delocalized by the blastoderm stage
(Lasko and Ashburner, 1990).
Consistent with this idea, all of the pole plasm components examined that are
downstream of Vas, including nos RNA, could be detected at the
posterior of vasPD;P{vas617}
embryos, although to variable degrees (data not shown).
Previous work has suggested that nos may be a target for
Vas-mediated translational regulation
(Gavis et al., 1996). Outside
of the pole plasm, nos translation is repressed through the binding
of Smg, and possibly other repressors, to its 3' UTR
(Smibert et al., 1996;
Dahanukar et al., 1999;
Crucs et al., 2000;
Nelson et al., 2004). Smg
achieves this regulation at least in part through interaction with the
eIF4E-binding protein Cup, thus influencing the cap-binding stage of
translation (Nelson et al.,
2004). Within the pole plasm, in complexes with Osk and Vas,
nos translational repression is overcome, potentially through a
direct interaction between Osk and Smg
(Dahanukar et al., 1999), which
may displace Smg-Cup interaction (Nelson
et al., 2004). Our analysis of Vas617 does not support an
important role for the VaseIF5B interaction in activating nos
translation in the pole plasm, as clearly translation of nos is far
less sensitive to the level of this interaction than is translation of
grk in early oocytes. The primary function of Vas in nos
accumulation may therefore be in anchoring nos mRNA in complexes
within the pole plasm, consistent with recent observations that nos
mRNA is trapped at the posterior by complexes containing Vas
(Forrest and Gavis, 2003). It
of course remains possible that the low level of residual eIF5B binding
provided by Vas617 is sufficient to fulfill a role of Vas in activating
translation of this transcript.
How might Vas-eIF5B interaction regulate translation of grk and potentially other target mRNAs?
Cap-dependent translation initiation in eukaryotes requires many
translation initiation factors, and involves several main steps (reviewed by
Pestova et al., 2001). Most
known mechanisms of translational regulation impinge on the recruitment of the
cap-binding complex eIF4F to the mRNA, which represents the rate-limiting
first step of initiation. mRNA circularization through proteins such as Cup
serves an important role in translational control by allowing 3'
UTR-bound regulatory factors to influence translation initiation at the
5' end of the transcript. (Nakamura
et al., 2004; Nelson et al.,
2004; Wilhelm et al.,
2003).
60S ribosomal subunit joining represents the interface between translation
initiation and elongation, and the VaseIF5B interaction suggests a distinct
mechanism of translational control occurring at this last stage of initiation.
Although this step has not historically been considered a target for
regulation, several examples have emerged to suggest that subunit joining may
in fact be subject to regulation. Translational repression of mammalian
15-lipoxygenase (LOX) mRNA is mediated by hnRNP proteins that bind to a
specific 3' UTR regulatory element, and which are thought to act by
blocking the activity of either eIF5 or eIF5B
(Ostareck et al., 2001). An
additional link between mRNA 3' regulatory regions, and eIF5B activity,
comes from analysis of two DEAD-box proteins in yeast, Ski2p and Slh1p
(Searfoss et al., 2001). These
proteins are required to achieve the selective translation of
poly(A)+ mRNAs, relative to poly(A)- mRNAs, and genetic
experiments suggest that they specifically repress the translation of
poly(A)- mRNAs by acting through eIF5 and eIF5B.
Together with these studies, our work suggests that in the
Drosophila germline, specific translational repression events may
target eIF5B and the ribosomal subunit joining step of initiation. Vas, which
potentially functions at the 3' UTR through interaction with specific
repressor proteins, may act to alleviate a translation block occurring at this
step. Such a model is consistent with what is known about translational
regulation of grk. For example, grk translation is repressed
by Bru, which binds to a Bruno-response element within its 3' UTR
(Filardo and Ephrussi, 2003).
Vas interacts with Bru (Webster et al.,
1997), suggesting that Vas could function as a derepressor by
overcoming Bru-mediated repression of grk translation. However, the
inability of a vas transgene to ameliorate the phenotype of
nosGAL4VP16-driven overexpression of Bru, might argue against this
model (Filardo and Ephrussi,
2003). The mechanism by which Bru regulates grk remains
unclear. Translational repression of osk by Bru relies on direct
interaction with Cup, linking Bru with eIF4E
(Nakamura et al., 2004).
However, mutations in cup that prevent interaction with Bru do not
appear to affect Grk expression, suggesting that Bru may operate through a
distinct mechanism to regulate grk translation
(Nakamura et al., 2004). In
addition, in vitro translation assays have suggested that Bru can mediate
translational repression through a cap-independent mechanism
(Lie and Macdonald, 1999).
Thus, Bru may be capable of regulating translation at more than one stage.
Based on the observations for the mammalian hnRNP proteins on the LOX mRNA,
and the Ski2p and Slh1p helicases in yeast, specific translational repressors
such as Bru could target the subunit joining step of initiation.
eIF5B is thought to form a molecular bridge between the two ribosomal
subunits, and to play a fundamental role in stabilizing the initiator
Met-tRNAiMet in the ribosomal P site (reviewed by
Pestova et al., 2000a).
Inhibition of eIF5B activity could occur while the factor is bound to the
initiation complex, at the start codon, and block its ability to link or
stabilize the ribosomal subunits. Through circularization of the mRNA, this
block could be achieved by trans-acting factors at the 3' UTR, and the
Vas-eIF5B interaction may be involved in alleviating these specific repression
events, potentially through displacement of a repressor protein.
Alternatively, Vas could play a role in recruitment of eIF5B to specific
transcripts. As eIF5B is required for all cellular translation, a general
mechanism must exist to recruit this factor to all transcripts. However, in a
scenario where repressor proteins may be blocking the subunit joining step,
either through a direct effect on eIF5B, or another mechanism, it is
conceivable that eIF5B could become limiting for translation. In this
situation, Vas could play a role in recruiting this factor to specific
transcripts.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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