Developmental Biology Programme, European Molecular Biology Laboratory,
Meyerhofstrasse 1, 69117 Heidelberg, Germany
* Present address: Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX,
UK
Author for correspondence (e-mail:
ephrussi{at}embl-heidelberg.de)
Accepted 25 November 2002
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SUMMARY |
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Key words: Orb, oskar mRNA, Translation, poly(A) tail, Drosophila, Oogenesis
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INTRODUCTION |
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During embryogenesis, the establishment of the embryonic axes is a
prerequisite to proper patterning of the embryo. In Drosophila
melanogaster, both the anteroposterior (AP) and dorsoventral (DV) axes
are established during oogenesis, through the prior asymmetric localization of
cytoplasmic determinants in the oocyte. To ensure the tight and exclusive
presence of these determinants at the sites where their activity is required,
they are often synthesized in loco after prelocalization of their transcripts,
which are then subject to temporal and spatial translational regulation
(St Johnston, 1995).
oskar (osk) mRNA, which encodes the posterior
determinant, is localized at the posterior pole of the oocyte and embryo,
where Osk protein is required to assemble the germ plasm
(Beams and Kessel, 1974;
Ephrussi et al., 1991
;
Kim-Ha et al., 1991
), a
cytoplasmic sub-compartment containing the abdominal and germline determinants
of the fly (Lehmann and
Nüsslein-Volhard, 1986
). Females bearing mutations in
osk produce embryos lacking both abdomen and germline. Conversely,
mislocalization of Osk activity leads to formation of ectopic abdominal
structures at the expense of the anterior structures
(Ephrussi et al., 1991
;
Ephrussi and Lehmann, 1992
;
Smith et al., 1992
). Hence,
restriction of Osk activity to the posterior pole is also essential for normal
development to occur.
Coupling of osk mRNA translational activation to its localization
at the posterior pole of the oocyte ensures specific and exclusive
accumulation of Osk protein at the posterior pole of the oocyte
(Kim-Ha et al., 1995;
Markussen et al., 1995
;
Rongo et al., 1995
). The
oocyte develops in a 16-cell cyst, consisting of 15 transcriptionally active
nurse cells and the oocyte itself, all interconnected by cytoplasmic bridges
(Spradling, 1993
). From the
early stages of oogenesis onwards, osk mRNA is transcribed by the
nurse cells and accumulates in the oocyte. At mid-oogenesis (stage 8)
osk mRNA becomes expressed at high levels at the posterior pole,
where it is translated (Ephrussi et al.,
1991
; Kim-Ha et al.,
1991
).
Prior to its localization, osk mRNA is translationally repressed
through the binding of Bruno (Bru) repressor protein to discrete elements
(Bruno Response Elements or BRE) present in the osk 3'
untranslated region (3'UTR) (Kim-Ha
et al., 1995; Webster et al.,
1997
). Upon posterior localization, osk translation is
derepressed (Gunkel et al.,
1998
). The mechanisms that underlie repression and derepression of
osk remain elusive. In many species, however, it appears that the
translational status of a regulated transcript is determined by the length of
its poly(A) tail, and the switch from a silenced to a translationally active
state is controlled by cytoplasmic polyadenylation (reviewed by
Richter, 1999
). Accordingly,
an increase in translation is often associated with poly(A) tail elongation,
whereas translational silencing correlates with poly(A) tail shortening
(Lieberfarb et al., 1996
;
Sallés et al., 1994
;
Sheets et al., 1995
). In
Xenopus, where this phenomenon has been most extensively studied, the
cis-acting elements involved in cytoplasmic polyadenylation have been
identified and include the AAUAAA hexanucleotide (also required for nuclear
polyadenylation) and the U-rich cytoplasmic polyadenylation element (CPE)
(Fox, 1989; McGrew and Richter,
1990
). The CPE is specifically bound by the
polyadenylation-inducing protein CPEB
(Hake and Richter, 1994
;
Stebbins-Boaz et al., 1996
),
whose presumed function is to recruit and stabilize the cytoplasmic
polyadenylation machinery (Mendez et al.,
2000
).
In Drosophila, no discrete cis-acting elements involved in
cytoplasmic polyadenylation have been identified so far. This has made it
difficult to assess directly the involvement of cytoplasmic polyadenylation in
translational control of regulated transcripts. A putative CPEB is encoded by
the oo18 RNA binding (orb) locus. Strong orb
alleles affect osk mRNA localization
(Christerson and McKearin,
1994; Lantz et al.,
1994
), preventing an analysis of their effect on osk
translation. Weaker orb alleles, for which the pattern of
osk mRNA localization has not yet been analyzed, are available and
might provide a useful tool with which to dissect the role of Orb in
modulating osk translation. Among those, the hypomorphic orb
allele, orbmel, completes oogenesis and has been
previously shown to affect the establishment of the AP axis, by interfering
with Osk protein accumulation (Christerson
and McKearin, 1994
). The observation by Chang et al.
(Chang et al., 1999
) that in
this mutant osk poly(A) tail length is shortened supports the idea
that cytoplasmic polyadenylation might be involved at least in some aspects of
osk translational regulation. However, as posterior localization is
required for osk mRNA translation, characterization of the effect of
the orbmel mutation on osk mRNA localization is
an essential prerequisite to any assessment of a possible role of Orb in
regulation of osk polyadenylation and Osk protein accumulation.
At least two non-mutually exclusive scenarios may be hypothesized.
Polyadenylation of osk transcript might be required to overcome
translational repression and to activate translation upon posterior
localization, a mechanism widely used to repress/derepress translation of
several developmentally regulated transcripts. This would predict that, even
upon posterior localization, no Osk activity is synthesized in the absence of
polyadenylation. Alternatively, poly(A) tail elongation might not be a
prerequisite for translation per se, but rather be required to enhance
translation efficiency, allowing for Osk protein accumulation. In fact, it is
already established that the poly(A) tail of an mRNA can synergize with its
5' cap structure to enhance translation initiation. This synergistic
effect of the cap and the poly(A) tail arises from the simultaneous binding of
the poly(A)-binding protein PABP and the cap-binding protein eIF4E, to the
translation initiation factor eIF4G, promoting reinitiation of translation by
terminating ribosomes on the same transcript
(Gallie, 1991;
Tarun and Sachs, 1996
;
Wells et al., 1998
). This
second hypothesis predicts that upon posterior localization of osk
mRNA, translation would initiate, but with limited efficiency, unless enhanced
by polyadenylation. In this light it is interesting that posterior patterning
and germline differentiation require different thresholds of Osk activity
(Markussen et al., 1995
;
Rongo et al., 1995
). Posterior
patterning is robust and requires only low levels of Osk, whereas pole cell
formation appears to be very sensitive to any reduction in Osk protein levels.
To guarantee production of sufficient amounts of Osk for fulfilment of both
functions, translational derepression and enhancement might be involved.
We address the role of cytoplasmic polyadenylation in control of osk translation. We have measured osk poly(A) tail length in vivo and correlated its polyadenylation status to its translation status both in vivo and in vitro. We show that within RNA corresponding to all stages of oogenesis, a discrete population of osk mRNA bears a long poly(A) tail. A poly(A) tail of the maximum length observed for osk mRNA in vivo is necessary and sufficient to enhance translation of a chimeric osk transcript in vitro, in the absence of Bruno repressor protein. However, addition of a poly(A) tail of any length does not suffice to overcome BRE-mediated repression, at least in vitro. We also show that maintenance of a long poly(A) tail on osk transcript requires Orb and is essential for Osk protein accumulation. In orbmel mutant egg chambers, the osk poly(A) tail is shorter than in wild type. Shortening of the osk poly(A) tail correlates with a reduction in Osk protein accumulation despite posterior localization of the transcript. This leads to complete sterility and, in extreme cases, to loss of posterior embryonic patterning. However, osk translation in orbmel appears to be only attenuated but not abolished. We therefore suggest a role for Orb in posterior patterning by enhancing osk translation through the addition or maintenance of a long poly(A) tail.
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MATERIALS AND METHODS |
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osk poly(A) tail was measured mainly using an RNaseH-based method,
as described by Zangar et al. (Zangar et
al., 1995), including some modification as described by Lie and
Macdonald (Lie and Macdonald,
1999
). After denaturation at 85°C for 5 minutes, 20 µg of
total ovary RNA were hybridized to 5 µg of a complementary DNA oligo
specific for osk mRNA (5'-CGC CAG AAT TCT ACA CTG TG-3')
with and without 5 µg of dT16 oligo, at 42°C for 10 minutes.
The RNA-DNA hybrid was than specifically digested with RNase H (1 U from
Gibco) at 30°C for 30 minutes in 20 µl of RNase H buffer (40 mM
Tris-HCl pH 8, 4 mM MgCl2, 1 mM DTT, 30 ng/ml BSA). Reactions were
stopped by phenol-chloroform extraction and ethanol precipitation. The RNA
fragments were resuspended in Ambion RNA loading buffer, separated on 5%
polyacrylamide denaturing gel and transferred by electro-blotting for 3-4
hours at 20 V to NEN Genescreen hybridization membrane. osk mRNA was
detected by northern hybridization. Quantification of the RNA populations
bearing different poly(A) tail length was performed on a Macintosh computer
using the public domain NIH Image program (developed at the US National
Institutes of Health and available on the internet at
http://rsb.info.nih.gov/nih-image/).
Alternatively, poly(A) tail length was measured using the poly(A) test
(PAT). cDNAs were synthesized from 500 ng of total ovary RNA, using the BRL
Superscript retrotranscriptase. The protocol used was modified to maintain the
poly(A) tract in the cDNA. Prior to first strand synthesis, the poly(A) tail
was hybridized to saturation with dT16 oligo and the oligo
dT16 subunits ligated before adding the oligodT-anchor, as
described by Sallés et al.
(Sallés et al., 1994).
Specific osk cDNAs were amplified by PCR in the presence of
[
32P]dCTP using the oligodT-anchor and a specific
osk oligo (5'-AAG CGC TTG TTT GTA GCA CA-3'). PCR
products were separated on 5% denaturing polyacrylamide gels.
DNA constructs
m1m2lacWT was previously described
(Gunkel et al., 1998).
m1m2lac
ABC was derived from m1m2lacWT by deletion of an
EcoRI/DraI fragment containing the AB repressor region, and
additional deletion of the C region by PCR-directed mutagenesis, using the PCR
primers
CbclIIup (5'-ACT GTC TAG AAC GTT TTT TTT GTC C-3')
and T3XL (5'-CGA AAT TAA CCC TCA CTA AAG GGA-3'). This construct
lacks nucleotides 3660-3778 and 4416-4487 of the m1m2lacWT chimeric
osk-lacZ transcript.
Cassettes of 36, 73, 98 and 150 adenosines (As) were cloned downstream of the various 3'UTRs using the unique NotI and KpnI sites in the plasmids. The derived constructs were named after the original plasmids and differ only in the length of the poly(A) cassette. Cassettes encoding a poly(A) tract longer than 150 nucleotides could not be cloned because of instability in bacteria. Poly(A) tails longer than 150A were added by enzymatic polyadenylation, using recombinant yeast Poly(A) Polymerase (yPAP) from Amersham. Transcript (1 µg) was polyadenylated at 30°C for 20 minutes in the presence of 10 mM ATP, 1x PAP buffer and 600 U of yPAP.
In vitro transcription
Capped chimeric osk-lacZ mRNAs were synthesized using the SP6
mMessage mMachine kit from Ambion. After a 2 hour reaction, the template DNA
was eliminated by digestion with DNAseI, and the RNA purified using RNeasy
columns from QIAGEN. Prior to transcription, the template was linearized with
an appropriate restriction enzyme, cutting at a unique site downstream of the
3'UTR. C36luca mRNA was synthesized as described
(Gray and Hentze, 1994). The
RNAs were trace labeled with [
32P]UTP to facilitate
assessment of their concentration and integrity. All RNAs used in the same
experiment were synthesized in parallel.
In vitro translation assay
Translation assays were performed as described previously
(Castagnetti et al., 2000).
Briefly, 50 ng of template osk mRNA were translated in a 12.5 µl
reaction containing 60 µM amino acids, 16.8 mM creatine phosphate, 80
ng/µl creatine kinase, 24 mM HEPES (pH 7.4), 0.6 mM Mg(OAc)2, 60
mM KOAc, 0.1 mM Spermidine, 1.2 mM DTT, 100 ng/µl calf liver tRNA and 40%
ovary or embryo extract. Luciferase mRNA (20 ng) was co-translated as an
internal control. The reactions were incubated for 90 minutes at 25°C. The
translation efficiency of the osk chimeric mRNAs was quantified using
the chemiluminescent ß-Gal Reporter Gene Assay (Roche), following the
protocol provided by the manufacturer. Luciferase activity was measured
according to Brasier et al. (Brasier et
al., 1989
).
In situ hybridization and immunostaining
Two- to three-day-old females were dissected in PBS and ovaries were fixed
for 20 minutes in 4% paraformaldehyde in PBS. After washing twice in PBT (0.1%
Triton X-100), ovaries were extracted for 1.5 hours in PBS 1% Triton X-100,
blocked for 1 hour in PBS 0.3% Triton X-100, 0.5% BSA and then incubated
overnight with primary antibodies (-Stau at 1/250,
-Osk at
1/3000). After washing, samples were incubated with FITC/rhodhamine-coupled
secondary antibodies (1/500 in PBT) for detection. Microscopy was carried out
using a Leica TCS SP confocal microscope.
For in situ hybridization, ovaries were fixed in 4% paraformaldehyde and
processed according to Glotzer and Ephrussi
(Glotzer and Ephrussi, 1999)
using osk digoxigenin-labeled probes.
Immunoprecipitation
Total ovarian extract, prepared as for the in vitro translation assay, was
incubated with 3 µl of specific antibody in 100 µl of hybridization
buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 2.5 mM MgCl2, 250 mM
sucrose, 0.05% NP-40, 0.5% Triton X-100, 1X EDTA-free protease inhibitors
cocktail from Roche]. After an overnight incubation, sepharose-protein A beads
were added for 50 minutes to allow binding. Beads were washed three times with
hybridization buffer without MgCl2. Protein were denatured in
Laemmli buffer, run on a 10% SDS polyacrylamide gel and detected using
monoclonal Orb antibodies (1:500). The monoclonal antibodies orb6H4 and orb4H8
developed by P. Schedl were obtained from the Developmental Studies Hybridoma
Bank developed under the auspices of the NICHD and maintained by The
University of Iowa, Department of Biological Sciences, Iowa City, IA
52242.
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RESULTS |
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Previous evaluations of the length of the osk poly(A) tail
involved different methods of measurement, yielding differing results
(Chang et al., 1999;
Lie and Macdonald, 1999
;
Sallés et al., 1994
).
We therefore decided to perform the two assays typically used for this purpose
on the same samples: the PAT assay, involving PCR amplification; and the more
direct, RNaseH/northern blot-based assay. For the PAT assay, ovaries were
divided into early (stage 1-5) and late stages (stage 7-14). During the early
stages of oogenesis, osk mRNA is unlocalized and translationally
silent. During the late stages, at least a portion of osk mRNA is
localized and translated. In both pools, osk poly(A) tails ranging
from 100 to 230 A were present (Fig.
1A). Using the RNaseH-based assay, poly(A) tails 200 in length
were also detected in both samples (Fig.
1B and data not shown). Hence, a significant fraction of
osk mRNA molecules bears remarkably long poly(A) tails, consistent
with some of the previous reports (Chang et
al., 1999
; Sallés et
al., 1994
).
|
As a first step towards investigating a potential role of cytoplasmic
polyadenylation and the poly(A) tail in osk translational control, we
measured the length of the osk poly(A) tail in ovaries of the
hypomorphic orb mutant, orbmel. orbmel
mutant females complete oogenesis and produce embryos out of which 25% show
posterior patterning defects, reflecting the reported reduction in Osk protein
accumulation (Christerson and McKearin,
1994; Markussen et al.,
1995
). As measured by the RNaseH-based assay, the osk
poly(A) tail is
50 A shorter in orbmel mutant females
than in the wild type (Fig.
1B), extending to only 150 A. Thus, the presence of a long poly(A)
tail on osk mRNA requires orb function. This confirms the
observation that osk mRNA is subject to cytoplasmic polyadenylation
in vivo (Chang et al.,
1999
).
The fact that the osk poly(A) tail is somewhat shorter in
orbmel than in wild type suggested that this difference
might be the basis of the observed defect in Osk protein accumulation in the
mutant. However, despite their shorter poly(A) tails in
orbmel ovaries, the osk transcripts still bear
relatively long tails that should in principle be able to stimulate
translation. To determine whether the shorter poly(A) tail of osk
mRNA in orbmel could affect Osk accumulation, we tested
whether the length of the poly(A) tail is critical for translational
stimulation of osk mRNA expression. We compared the in vitro
translation efficiency of chimeric osk-lacZ transcripts bearing
poly(A) tails of different lengths. As investigation of the role of the
poly(A) tail in osk translational stimulation requires an environment
in which osk translation is not otherwise silenced, we used the
cell-free translation system obtained from Drosophila embryos, which
contains no Bru repressor protein. As shown in
Fig. 1C, a poly(A) tail as long
as 150 A does not stimulate translation above that of transcripts bearing no
poly(A) tail. This result is consistent with previous reports from Lie and
Macdonald (Lie and Macdonald,
1999) and Castagnetti et al.
(Castagnetti et al., 2000
) in
which poly(A) tails of 36 A and 73 A failed to stimulate translation of
osk reporter transcripts in vitro. Remarkably, however, addition of a
tail in excess of 200 A enhances translation 20- to 100-fold above that of a
transcript bearing no poly(A) tail (Fig.
1C). This increase in translation following polyadenylation is due
to a substantial increase in the rate of translation initiation, rather than
to stabilization of the transcripts, whose half-lives are comparable
(Fig. 1C; data not shown).
These results suggest that a tail longer than 150 A may also be necessary for
efficient Osk accumulation in vivo, consistent with a role of cytoplasmic
polyadenylation and Orb protein in osk mRNA translational
regulation.
A poly(A) tail longer than 150A is necessary for Osk accumulation in
vivo
Previous reports have shown the involvement of Orb in the establishment of
anteroposterior polarity (Christerson and
McKearin, 1994). To study the potential dependence of osk
translation on the poly(A) status of the mRNA in vivo, we first had to
determine whether the mRNA is correctly localized in the weak
orbmel mutants, as analysis of strong orb alleles
has highlighted a role of Orb in osk mRNA posterior localization. In
situ hybridization revealed that osk mRNA is correctly localized in
67% and partially localized in 10% of orbmel oocytes
(Fig. 2, upper panel).
osk mRNA is not detected at the posterior pole of the remaining 23%
of orbmel oocytes. Staufen (Stau) protein, whose
distribution has been shown to mirror that of osk mRNA during
oogenesis (St Johnston et al.,
1991
), shows a similar degree of localization in
orbmel egg chambers
(Fig. 2 lower panel). As shown
in Fig. 2, 100% of wild-type
and 81% of orbmel oocytes at stage 9/10 accumulate Stau at
the posterior pole. In 80% of orbmel oocytes that show
posterior Stau accumulation, the protein is fully localized at the posterior
pole, and in 20% Stau is also detected in the cytoplasm. Thus, in contrast to
the strong orb alleles
(Christerson and McKearin,
1994
; Lantz et al.,
1994
), orbmel only mildly affects osk
mRNA localization, rendering it suitable for an analysis of the role of
cytoplasmic polyadenylation in osk translation.
|
An indication that Orb is required for osk translation is that, as
shown in Fig. 3A and previously
reported (Markussen et al.,
1995), the amount of both Osk isoforms is dramatically reduced in
orbmel when compared with wild type. This reduction could
in principle be a consequence of the osk mRNA localization defect.
However, the amount of Osk protein in orbmel is at best
25% of the wild type, in spite of the fact that 65% of
orbmel oocytes show normal posterior osk mRNA
localization. This suggests an involvement of Orb in osk translation.
Consistent with this, antibody staining of ovaries reveals that only 67% of
orbmel oocytes that localize Stau also accumulate Osk at
the posterior (Fig. 3B). In the
remaining 33%, no Osk protein is detected. However, trace amounts of Osk are
presumably also produced in these oocytes, as maintenance of Stau and
osk mRNA at the posterior pole requires Osk protein itself
(Rongo et al., 1995
;
Vanzo and Ephrussi, 2002
). It
therefore appears that, in orbmel ovaries, osk
translation is impaired but not abolished. In these egg chambers, osk
mRNA bears a poly(A) tail that is insufficient to support efficient
translation in vitro. Taken together these observations suggest that
Orb-mediated cytoplasmic polyadenylation is required to enhance translation,
but not for translational derepression of osk mRNA.
|
To confirm this hypothesis, we checked if the addition of a long poly(A) tail could overcome BRE-mediated repression in vitro. For this purpose, we used the ovarian extract, which contains Bru and recapitulates BRE-mediated repression. As shown in Fig. 4, addition of a >250 A tail does not overcome Bru-mediated repression, as BRE-containing transcripts are less efficiently translated than their BRE-deleted counterparts, whether or not they bear a poly(A) tail. As in the embryo extract, the polyadenylated BRE+ and BRE- transcripts are translated more efficiently than the corresponding transcripts lacking a poly(A) tail. Taken together, these results suggest that addition of a long poly(A) tail is not sufficient to overcome repression, but has a stimulatory effect on translation of both BRE+ and BRE- transcripts. The physiological relevance of cytoplasmic polyadenylation in osk regulation is strengthen by the observation that, in orbmel egg-chambers, even when levels of Osk sufficient to support abdomen formation are produced, those embryos that develop into adult females are sterile (Table 1).
|
|
Orb interacts with Bic-C and Bru
Cytoplasmic polyadenylation of mRNA requires CPEB to recruit the enzyme
poly(A) polymerase (PAP) on the regulated mRNA. Until recently, only one
family of PAP, containing both a catalytic domain and an RRM-like domain, was
known. Wang et al. have now identified a novel family of PAP that differs from
the canonical PAP for the absence of the RRM-like domain. The prototype of
this family is represented by C. elegans GLD-2 whose binding to the
RNA is mediated by GLD-3, a KH domain containing protein of the BicC family
(Wang et al., 2002).
Interestingly, we found that Drosophila BicC interacts physically
with Orb in co-immunoprecipitation experiments
(Fig. 5A). As the phenotype of
BicC mutants implicates BicC protein as a negative regulator of
osk translation (Saffman et al.,
1998), we tested whether Orb interacts with the translational
repressor Bru. Indeed, we could detect a physical interaction between Bru and
Orb (Fig. 5B), as revealed by
the co-immunoprecipitation of Bru with Orb. By contrast, we detected no direct
interaction between Bru and BicC (data not shown) in our assay.
|
The relevance of these interactions in vivo is further confirmed by the
genetic interactions between the BicC locus and the orb and
aret loci the latter encoding Bru protein. Females
heterozygous for BicC show a number of AP patterning defects, ranging
from head defects to bicaudal embryos
(Mahone et al., 1995). The
BicC phenotype is suppressed when the mutation is combined with an
orb allele or the strong aret allele,
aretQB72. Table
2 shows that 85% of embryos produced by
Bic-CYC33/+ females fail to hatch and of those 60% are
bicaudal. The null allele orbF343 efficiently suppresses
the Bic-C phenotype and only 13% of the embryos produced by
BicCYC33/+; orbF343/+ fail to hatch,
none of which shows a bicaudal phenotype. As shown in
Table 2, the strength of the
phenotype and the extent of the suppression depends on the orb
allele. Embryonic viability is also improved, up to 72%, in embryos produced
by BicCYC33/ aretQB72 females.
|
![]() |
DISCUSSION |
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The prevailing model, which is based on studies of translational control in
the Xenopus oocyte, suggests that the polyadenylation status of a
transcript correlates with its translational status: a short poly(A) tail
corresponding to a silenced mRNA and poly(A) tail elongation triggering
translational activation. In Xenopus, upon progesterone treatment a
wave of cytoplasmic polyadenylation activates translation of deadenylated and
silenced maternally derived mRNAs
(Richter, 1999). In
Drosophila, translation of bicoid mRNA, which encodes the
anterior determinant of the embryo, is repressed until egg activation when
poly(A) tail elongation triggers translation initiation
(Sallés et al., 1994
).
Although the correlation between adenylation and translation still holds for
several transcripts, a growing body of evidence suggests that the two events
may be coincidental but not directly connected. Interestingly, deadenylation
and translational repression of Drosophila hunchback (hb)
(Chagnovich and Lehmann, 2001
)
and mouse tPA (Stutz et al.,
1998
) mRNAs can occur independently of each other. The transcripts
are deadenylated concomitant with translational repression, yet repression can
occur in the absence of ongoing deadenylation. In arrested primary mouse
oocytes, polyadenylation of the tPA mRNA is necessary to counteract the
default deadenylation that affects most other oocyte mRNAs, thus preventing
its degradation (Stutz et al.,
1998
).
Our observations suggest that silencing and awakening of osk mRNA
translation can occur in the absence of changes in poly(A) tail length and, in
fact, osk mRNA bears a long poly(A) tail at all stages of oogenesis,
including when it is unlocalized and translationally silent. However, it is
still formally possible that at intermediate stages of oogenesis osk
mRNA undergoes a deadenylation that goes undetected in our measurements on
bulk RNA, and that elongation of the poly(A) tail causes displacement of the
repressor complex, leading to translational derepression. This hypothesis is
supported by the fact that the repressor protein Bru shares a 50% sequence
identity with the Xenopus deadenylation promoting factor EDEN-BP
(Kim-Ha et al., 1995).
However, we did not detect any obvious pattern of deadenylation in vitro when
Bruno was added to the embryonic extract, nor could we observe a shortening of
the poly(A) tail of translationally silenced osk transcript recovered
form ovarian extract (data not shown). Nevertheless, our results show that
BRE-mediated repression is effective independently of the length of the
poly(A) tail on osk transcripts, and that a silenced mRNP can be
assembled on a naked osk transcript, whether or not it bears a
poly(A) tail. These results suggest that polyadenylation is not the sole
determining event leading to translational derepression of osk mRNA
at the posterior pole, but that the maintenance of a long poly(A) tail, by
cytoplasmic polyadenylation, accounts for the enhancement of osk
translation and is required for efficient osk translation, to ensure
sufficient accumulation of Osk at the posterior pole of the
Drosophila oocyte to promote abdominal patterning and germline
differentiation.
Furthermore, the physical interaction detected between Orb and Bru, and Orb
and BicC suggests the existence of a multi-protein complex containing both
positive and negative regulators of osk translation. In this
scenario, translational silencing and polyadenylation are linked through Bru
protein, offering a possible explanation as to how CPEB might be recruited to
mRNAs in Drosophila, where no canonical CPE has so far been
identified. Transcripts properly repressed by Bru, upon localization, could be
adenylated by the recruitment of Orb by Bru itself. Loss of Bru repression
would, therefore, result in loss of Orb binding with consequent deadenylation
and translational silencing. In this model, modulation of the poly(A) tail
would be part of the mechanism that regulates translation, ensuring a second
level of control over ectopic expression while localizing all the components
necessary for efficient translation. Remarkably, mutations in the BRE sites do
not result in ectopic osk translation
(Kim-Ha et al., 1995),
suggesting the existence of a second layer of translational control. Moreover,
during embryonic development when osk translation is no longer
required, both Orb and Bru proteins are depleted in the embryo and
osk mRNA undergoes complete deadenylation
(Sallés et al.,
1994
).
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ACKNOWLEDGMENTS |
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