1 UMR 6061 CNRS, University of Rennes 1, Faculty of Medicine, 2 Ave. Prof.
Léon Bernard, CS 34317, 35043 Rennes Cedex, France
2 UMR 7622 CNRS, University Paris 6, Paris, France
* Author for correspondence (e-mail: jacek.kubiak{at}univ-rennes1.fr)
Accepted 13 March 2003
![]() |
Summary |
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Key words: Aurora A, Eg2, EDEN-BP, Protein phosphorylation, MPF, RNA deadenylation
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Introduction |
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Early development preceding the midblastula transition in Xenopus
laevis depends entirely on the maternal information stocked in mRNA and
proteins accumulated during oogenesis. Gene expression during early
development (as well as during oocyte maturation) depends largely on
post-transcriptional modification of maternal mRNAs (reviewed by
Richter, 1999).
Polyadenylation and deadenylation of mRNA synthesized before oocyte maturation
are the most important mechanisms involved in this process
(Osborne and Richter, 1997
).
Different classes of maternal mRNA were described in Xenopus oocytes
and embryos with respect to their pattern of polyadenylation/deadenylation.
The Eg family of mRNA containing Eg1/cdk2 (cyclin-dependent
kinase 2), Eg2/Aurora A (kinase), Eg5 (kinesin-like protein)
and c-mos are polyadenylated during oocyte maturation due to the
presence of a cytoplasmic polyadenylation element (CPE) in the
3'-untranslated region (3'-UTR)
(Paris and Richter, 1990
;
McGrew and Richter, 1990
) and
deadenylated following fertilization due to the presence of an embryo
deadenylation element (EDEN) also in the 3'-UTR
(Paillard et al., 1998
). Most
EDEN-containing mRNAs known to date are involved in cell cycle regulation
during early development. For example, c-mos must be degraded and its
synthesis/accumulation downregulated to enable the exit from M II-arrest after
fertilization, as c-mos is a key component of the CSF activity. Ectopic
expression of c-mos following fertilization induces cell cycle alterations and
eventually cell cycle arrest (Sagata et
al., 1989
; Murakami and Van de
Woude, 1998
; Murakami et al.,
1999
). Deadenylation of c-mos mRNA
(Paillard et al., 1998
) after
c-mos degradation (Lorca et al.,
1993
) could play an important role in the elimination of
c-mos gene products during early development of Xenopus
laevis (Ueno and Sagata,
2002
).
The EDEN-dependent deadenylation of maternal mRNA depends on the presence
of a trans-factor, EDEN binding protein (EDEN-BP)
(Paillard et al., 1998).
EDEN-BP is a homologue of human Nab50, or CUG-BP protein
(Timchenko et al., 1996
). This
protein was shown to be involved in maturation of mRNA in human somatic cells
(Philips et al., 1998
). It is
a phosphoprotein with a high potential to be hyper- or hypophosphorylated.
Phosphorylation and dephosphorylation of CUG-BP was thought to be one of the
key mechanisms involved in regulation of the localization (nuclear versus
cytoplasmic) and activity of this protein in mRNA maturation
(Roberts et al., 1997
).
Because EDEN-dependent deadenylation is active in early Xenopus
embryos, whereas the quantities of EDEN-BP protein are similar before and
after fertilization (Paillard et al.,
1998), we investigated whether post-translational modifications,
like phosphorylation or dephosphorylation, regulate this sequence-specific
deadenylation activity. In this paper we show that EDEN-dependent
deadenylation is sensitive to kinase and phosphatase inhibitors. EDEN-BP is
phosphorylated during oocyte maturation and dephosphorylated following egg
activation. Dephosphorylation of EDEN-BP is calcium dependent and correlates
with the increase in the activity of EDEN-dependent deadenylation. It suggests
that these post-translational modifications could regulate the efficiency of
EDEN-dependent deadenylation. MPF reactivation following egg extract
activation does not influence EDEN-dependent deadenylation, suggesting that
the regulation does not depend directly on MPF activity. These results link
calcium signals and the cell cycle regulatory machinery to the deadenylation
of certain maternal mRNAs at the very beginning of the embryonic
development.
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Materials and Methods |
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Unfertilized eggs were dejellied with 2% L-cysteine pH 8.2 in XB buffer
(Murray, 1991) (KCl 100 mM,
MgCl2 2 mM, HEPES 10 mM pH 7.65, sucrose 50 mM) and washed in XB.
Some of the eggs were collected and the others were activated using 0.5
µg/ml calcium ionophore A23187 for 2 minutes and then extensively washed in
XB. Eggs collected at different times after activation were frozen in liquid
nitrogen and kept at -70°C.
Extracts were prepared by the addition of homogenization buffer (ß-glycerophosphate 60 mM, p-nitrophenyl phosphate 15 mM, MOPS 25 mM pH 7.2, EGTA 15 mM, MgCl2 15 mM, dithiothreitol 2 mM, Na-orthovanadate 1 mM, NaF 1 mM, di-Na-phenylphosphate 1 mM, leupeptin 10 µg/ml, aprotinin 10 µg/ml, chymostatin 10 µg/ml and pepstatin 10 µg/ml; 200 µl for 40 oocytes) to the thawing oocytes and pipetting. After centrifugation at 12,000 g at 4°C the supernatant was used for the experiment.
CSF extracts were prepared using joint protocols previously described
(Lohka and Masui, 1984) and
Blow (Blow, 1993
) with few
modifications: additional EGTA up to 7 mM final concentration and sucrose 50
mM in the CSF extraction buffer (Blow et al., 1993) (KCl 50 mM, HEPES 50 mM pH
7.6, MgCl2 5 mM, EGTA 5 mM and DTT 2 mM) and two centrifugations at
15,000 g. Calcium-activated extracts were prepared from CSF
extracts supplemented with CaCl2 (0.8 mM final concentration). To
check the integrity of our extracts, 10-20 µl of extract were incubated at
21°C for 1 to 1.5 hours with sperm nuclei. The chromatin condensation (CSF
extract) and chromatin decondensation (activated extract) was visualized by
fluorescence and phase contrast microscopy using Hoechst dye in fixing media.
In some cases we also measured histone H1 kinase activity or determined the
state of phosphorylation of Cdc25 phosphatase by western blotting.
Calcium ionophore-activated embryo extract was prepared as described by
Legagneux et al. (Legagneux et al.,
1995).
Drugs and recombinant proteins
Extracts were supplemented with recombinant bacterial 90 sea urchin
cyclin B to 0.2 ng/µl (Murray and
Kirschner, 1989
). Okadaic acid (OA)
(Goris et al., 1989
) was used
at a concentration of 1 µM, and roscovitine
(Meijer et al., 1997
) at 100
µM.
Transcripts
Two plasmid constructs pEg2-410
(Legagneux et al., 1992) and
pEg2-410a (Bouvet et al., 1994
)
were used. Both were derived from the 3'-UTR of Eg2/Aurora A mRNA.
Eg-410 is a substrate for EDEN-dependent deadenylation. Eg2-410a RNA is a
substrate for default deadenylation as it contains neither a CPE nor an EDEN.
These plasmids were used to produce capped radiolabelled transcripts as
previously described (Legagneux et al.,
1992
). The radiolabelled transcripts were purified by
electrophoresis on a 4% polyacrylamide-urea gel.
In vitro deadenylation and RNA analysis
The deadenylation experiments were realized in vitro as previously
described (Legagneux et al.,
1995). In brief, 6 µl extracts were incubated with 1 µl
capped radiolabelled transcripts Eg2-410 and Eg2-410a at 21°C for 1 to 3
hours. The transcripts were extracted as described by Harland and Misher
(Harland and Misher, 1988
),
then precipitated and analysed by electrophoresis on 4% polyacrylamide-urea
gel and phosphorimaging. 0.5 µl of radiolabelled transcripts were loaded
directly on the gel without precipitation (lanes T) as a size marker for the
input RNA, the starting point of deadenylation.
Immunoprecipitation
Anti-EDEN-BP sepharose beads were prepared with anti-EDEN-BP rabbit serum
(Paillard et al., 1998) loaded
on protein A-sepharose beads. The IgG were then covalently coupled to the
beads using dimethyl pimelimidate coupling agent according to standard
procedure.
Oocyte or unfertilized egg extracts were added to anti-EDEN-BP sepharose beads (200 µl of extract for 20 µl of beads) and incubated at 4°C for 40 minutes followed by extensive washing in bead buffer (Tris 50 mM pH 7.4, NaF 5 mM, NaCl 250 mM, EDTA 5 mM, EGTA 5 mM, Nonidet P-40 0.1%, leupeptin 10 µg/ml, aprotinin 10 µg/ml, chymostatin 10 µg/ml and pepstatin 10 µg/ml). The bound material was either used for further experiment or eluted at 90°C in elution buffer (Tris 62.5 mM pH 6.8, 2% sodium dodecyl sulfate) and collected after centrifugation. Concentrated Laemmli denaturating buffer was added to the eluted material. Samples were boiled before analysis.
Phosphatase assays
For these experiments the extracts were made in homogenization buffer
without di-Na phenyl phosphate and p-nitrophenyl phosphate. The
phosphatase (Lambda Protein phosphatase P0753S Biolabs) was incubated
according to the manufacturer's instructions with either nonactivated egg
extract or ionophore-activated egg extract (40 units/µl of extract) or with
EDEN-BP immunoprecipitated from unfertilized egg extract on sepharose beads
(160 Unit/µl of beads). The incubation was realized at 30°C for 30
minutes to 2 hours. The reaction was stopped by addition of Laemmli
denaturating buffer. The samples were boiled before analysis.
Electrophoresis and western blotting
Protein was analysed on a sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS/PAGE) gel (Laemmli,
1970); when required the separating gel consisted of 15%
acrylamide and 0.13% bisacrylamide (ratio 29.8:0.2) to allow better separation
of different forms of EDEN-BP (Anderson et
al., 1973
). Otherwise, 10% SDS/PAGE gels were used
(Laemmli, 1970
). Separated
proteins were transferred to nitrocellulose membranes (Hybond C; Amersham)
according to standard procedures and probed with either purified rabbit
polyclonal anti-EDEN-BP antibodies 1/1000 (nonimmunoprecipitated extracts),
polyclonal anti-EDEN-BP guinea pig serum 1/3000 (immunoprecipitation
experiments) or anti-Cdc25 (gift from Marcel Dorée, CRBM, Montpellier,
France) and followed by alkaline phosphatase-coupled secondary antibody
(Sigma) 1/30000.
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Results |
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|
Calcium triggers changes in the electrophoretic mobility of EDEN-BP
in CSF extract and M II oocytes
EDEN-BP migrates as a doublet of 55/53 kDa in SDS/PAGE gels
(Legagneux et al., 1992).
Therefore, we evaluated the effect of increasing the concentration of calcium
ions in the CSF extracts on the electrophoretic mobility of EDEN-BP. Using
Anderson's modified SDS/PAGE gels (Anderson
et al., 1973
), we detected a more complex pattern of EDEN-BP
electrophoretic mobility. Three bands (Fig.
2A,B; named a, b, c) were observed. The uppermost weak band (a) is
only visible in CSF extracts collected before calcium addition. The second
band (b) corresponds to the major band in the CSF extracts and its intensity
decreases after calcium activation. This decrease appears sensitive to the
amount of calcium added (compare extracts 3 hours after activation with 0.4,
0.8 and 1.6 mM CaCl2, Fig.
2A). The lower major band (c) is clearly observed in the activated
extracts; however, it is also present as a minor band in CSF extracts. The
intensity of this band increases in parallel with the increase in calcium
concentration (compare extracts 3 hours after activation with 0.4, 0.8 and 1.6
mM CaCl2, Fig.
2A).
|
To determine whether the calcium-induced changes in the electrophoretic mobility of EDEN-BP occur in vivo, we studied the protein in parthenogenetically activated M II oocytes. The protein was detected by western blotting either directly (data not shown) or after immunoprecipitation (Fig. 2B). In nonactivated eggs (arrested in M II of meiosis), similarly to CSF extracts, three EDEN-BP bands (a, b, c) with a strong upper major band (b) and a very weak lower band (c), were detected (Fig. 2B, NA; compare with untreated CSF extracts, time 0, in Fig. 2A). In activated eggs the uppermost band (a) was absent, the intensity of major band (b) significantly diminished and the lower major band (c) dramatically increased (Fig. 2B, A). Immunoprecipitation of EDEN-BP enabled us to more clearly visualize the intermediate bands (b'), sometimes visible as a doublet in between the major bands (b) and (c) (Fig. 2B, A). Therefore, on egg activation the electrophoretic mobility of EDEN-BP increases as observed in the calcium-treated CSF extracts.
The electrophoretic mobility changes in EDEN-BP occur during oocyte
maturation and following egg activation
To characterize the dynamics of the changes in the electrophoretic mobility
of EDEN-BP, we studied the protein in detail during oocyte maturation and
following egg activation. Samples of maturing oocytes were collected every
hour following progesterone addition (Fig.
3A) and at short intervals following parthogenetic activation of M
II oocytes with calcium ionophore (Fig.
3B), and analysed as above. Stage VI prophase I-arrested oocytes
contain two forms of EDEN-BP: the major lower band (c) and a smear of minor
ones corresponding to the bands (b'). Four hours after the addition of
progesterone, when 50% of oocytes had undergone germinal vesicle breakdown
(GVBD), as judged by the appearance of the maturation spot, the mobility of
EDEN-BP changed and the upper band (b) appeared. The uppermost band (a) was
detected after 6 hours of progesterone treatment. After 7 hours (100% GVBD),
the intensity of the upper bands (a) and (b) clearly increased, whereas the
intensity of the lower (c) band decreased
(Fig. 3A). Following activation
of M II oocytes we observed a rapid decrease in the intensity of the upper
bands (a) and (b) (Fig. 3B). The uppermost band (a) disappeared within 15 minutes, whereas the band (b)
persisted until 2 hours postactivation. The intensity of lower bands
(b') and (c) increased concomitantly. Thus, EDEN-BP undergoes rapid
post-translational modifications at the time of the GVBD during oocyte
maturation and following egg activation. The dynamics of these changes in
EDEN-BP electrophoretic mobility resemble those observed for other proteins
that undergo cell-cycle-dependent phosphorylation during GVBD and
dephosphorylation following entry into development (e.g. ERK2 MAP kinase)
(Ferrell et al., 1991).
|
EDEN-BP is post-translationally modified by
phosphorylation/dephosphorylation
To verify whether the electrophoretic changes of EDEN-BP were due to
changes in the phosphorylation of the protein, extracts were treated with the
phage phosphatase at 30°C for 0, 0.5 or 1 hour. The samples were
then analysed by SDS/PAGE and anti-EDEN-BP western blotting. The phage
phosphatase treatment caused an increase in the electrophoretic
mobility of EDEN-BP, whereas without phage
phosphatase treatment, the
major upper band (b) of EDEN-BP was still detectable after 1 hour of
incubation at 30°C (Fig.
4A). To show the direct effect of the phage
phosphatase
on EDEN-BP, we immunoprecipitated EDEN-BP followed by l phosphatase treatment.
In these conditions a similar modification of EDEN-BP was observed
(Fig. 4B). However, the
conversion of the immunoprecipitated protein appeared to be less complete and
residual amounts of bands (a) and (b) were sometimes observed. This reduced
conversion to the dephosphorylated form could be due to a masking effect
because EDEN-BP is complexed with the antibodies during the phosphatase
treatment. The data presented show that the phage
phosphatase
treatment mimics to a large part the down shift of EDEN-BP observed after
calcium addition to CSF extract or calcium ionophore treatment of M II
oocytes. These experiments show that the post-translational modifications of
EDEN-BP are, at least in part, due to phosphorylation and dephosphorylation.
EDEN-BP is phosphorylated during maturation and dephosphorylated following egg
activation.
|
Kinase and phosphatase inhibitors downregulate EDEN-dependent
deadenylation
A large number of kinases are inactivated on egg activation
(Karsenti et al., 1987). The
temporal correlation between the beginning of EDEN-BP dephosphorylation and
the acceleration of EDEN-dependent deadenylation following egg activation
suggests that the two processes, which are triggered by free calcium increase,
could be due to a change in the equilibrium between kinases and phosphatases
activities. Therefore, we determined whether kinase and phosphatase inhibitors
could influence the process of EDEN-dependent mRNA deadenylation.
To inhibit a broad range of serine/threonine kinases we used roscovitine at
high concentration (100 µM) added to CSF extract activated by the addition
of CaCl2 (Fig. 5).
At this concentration roscovitine inhibits a large spectrum of kinases
(Meijer et al., 1997). We
observed a complete inhibition of the EDEN-dependent deadenylation; no
poly(A)- form of Eg2-410 chimeric RNA was detected even after 3
hours of incubation (Fig. 5).
Only slight changes (higher accumulation of intermediate forms of Eg2-410a
chimeric RNA) in the dynamics of the default deadenylation were observed.
|
Similar experiments were performed by inhibiting a broad range of phosphatases. A mixture of concentrated phosphatase inhibitors was added to the CSF extract containing chimeric RNAs before CaCl2 addition. EDEN-dependent deadenylation was very efficiently arrested by these inhibitors both in CSF and calcium-activated extracts (data not shown).
Okadaic acid is a potent inhibitor of phosphatases 1 and 2A
(Goris et al., 1989).
Therefore, using this drug enabled us to identify the spectrum of phosphatases
inhibiting EDEN-dependent deadenylation. CSF extracts supplemented with OA
were incubated at 21°C for 15 minutes and then activated with
CaCl2, and radiolabelled transcripts were added. We observed that
OA inhibited the deadenylation process in calcium-activated extracts
(Fig. 6A). The inhibition of
EDEN-dependent deadenylation was correlated with the inhibition of the
dephosphorylation of EDEN-BP (Fig.
6B). The dynamics of default deadenylation was only slightly
modified by OA, although the accumulation of several partially deadenylated
products became visible.
|
From these experiments, we can conclude that EDEN-dependent deadenylation is regulated by a balance between phosphorylation and dephosphorylation activities and that this process is sensitive to OA.
MPF pathway does not inhibit EDEN-dependent deadenylation
MPF is finely regulated via phosphorylation/dephosphorylation. Cdc25
phosphatase is a major activator of MPF and this phosphatase is itself
activated by a series of phosphorylations on the entry into the M-phase
(Kumagai and Dunphy, 1991;
Gautier et al., 1991
). MPF
activity depends on the stability of cyclin B
(Murray and Kirschner, 1989
;
Murray et al., 1989
).
Moreover, OA is known to indirectly activate MPF in Xenopus oocytes
and extracts (Goris et al.,
1989
; Rime et al.,
1990
; Jessus et al.,
1991
; Lorca et al.,
1991
). Therefore, the sensitivity of EDEN-dependent deadenylation
to OA could be due, in part, to activation of MPF. To determine whether this
activity interferes with the process of EDEN-dependent deadenylation we
specifically activated MPF via the addition of indestructible
90 cyclin
B of sea urchin into CSF extracts followed by calcium treatment
(Fig. 7A). Addition of
exogenous D90 cyclin B into CSF extract treated with Ca2+
maintained condensed chromosomes (Fig.
7A, lower panel, compare left and middle images), indicating that
the extract remained in M-phase despite calcium addition, whereas interphase
nuclei were present in calcium-activated extract
(Fig. 7A, lower panel, right).
However, the high MPF activity in the extract had no effect on EDEN-dependent
deadenylation (Fig. 7A, upper
panel).
|
To check whether MPF activity could affect the deadenylation activity,
which had already been accelerated on egg activation, we added 90
cyclin B to ionophore-activated embryo extract and followed EDEN-dependent
deadenylation (Fig. 7B). In
parallel, we checked the inhibitory effect of OA in the same extract, as well
as the combined effect of both
90 cyclin B and OA
(Fig. 7B). As expected,
90 cyclin B restored an M-phase state in the extract, which was
confirmed in western blot with anti-Cdc25; the disappearance of the fast
migrating (dephosphorylated) band is concomitant with the appearance of a new,
upshifted band (Fig. 7B, lane 2
in the lower panel). The addition of
90 cyclin B did not affect
EDEN-dependent deadenylation in this extract when compared with the control,
interphase extract (Fig. 7B,
upper panel, compare experiments 1 and 2). As already shown for the calcium
activated CSF extract, 1 µM OA slowed this process and this reduced rate of
deadenylation was not affected when the OA treatment extract was supplemented
with
90 cyclin B (Fig.
7B, compare experiments 3 and 4). As expected, the combined effect
of OA and
90 cyclin B treatment induced a more pronounced
phosphorylation of Cdc25 (Fig.
7B, lower panel, lane 4). Such a combined treatment seems to have
a synergistic effect on the M-phase state of the extract, but does not
influence significantly EDEN-dependent deadenylation compared with the OA
treatment alone (Fig. 7B, upper
panel, compare experiments 3 and 4). From these experiments we conclude that
EDEN-dependent deadenylation of RNA is not affected by MPF activity.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphorylation and dephosphorylation modulate EDEN-dependent
deadenylation of mRNA
Calcium waves that occur after fertilization provoke a rapid inactivation
of CSF and MPF activity, resulting in metaphase to anaphase transition
followed by the first embryonic interphase
(Lorca et al., 1993) (reviewed
by Maller, 1998
). Inactivation
of MPF via cyclin B degradation (Murray et
al., 1989
) triggers changes in the
phosphorylation/dephosphorylation equilibrium in favour of dephosphorylation
(Karsenti et al., 1987
).
Inactivation of multiple kinases participates in this process and correlates
with dephosphorylation of EDEN-BP. Using nondegradable sea urchin
90
cyclin B, which enables the MPF kinase activity to be artificially maintained
in calcium-treated CSF extracts, or to activate MPF in interphase extracts, we
show that the EDEN-dependent deadenylation is independent of MPF activity. In
contrast, OA reduces the activity of EDEN-dependent deadenylation. OA is known
to activate MPF and ERK2 MAP kinase besides inactivating phosphatases PP1 and
PP2 (Jessus et al., 1991
).
Whether the effect of OA on EDEN-dependent deadenylation is via ERK2
activation or phosphatase inactivation is not clear at present. However, in
preliminary experiments we observed that inactivation of ERK2 by the addition
of recombinant phosphatase CL 100 to OA-treated extract restored the
EDEN-dependent deadenylation activity (our unpublished results). In addition,
the timing of MAP kinase ERK2 activation and inactivation during oocyte
maturation and after fertilization correlates with phosphorylation and
dephosphorylation of EDEN-BP (Ferrell et
al., 1991
; Abrieu et al.,
1996
; Chau and Shibuya,
1999
). A positive feedback between MPF and ERK2 activation in CSF
extracts through the phosphorylation of c-mos, an upstream regulator of the
ERK2, was reported recently by Castro et al.
(Castro et al., 2001
). In some
of our experiments in which
90 cyclin B was added to CSF extracts
followed by calcium addition, we observed an intermediate rate of
EDEN-dependent deadenylation compared with CSF extracts and calcium-activated
extracts (data not shown). In these extracts we detected the phosphorylated
form of ERK2 MAP kinase. This suggests that the feedback loop described by
Castro et al. (Castro et al.,
2001
) is provoking ERK2 MAP kinase activation in these extracts,
supposedly when c-mos was not totally degraded. Further studies are necessary
to examine the potential role of ERK2 MAP kinase, and/or a combined role of
MPF and MAP kinase pathways, in the regulation of EDEN-dependent deadenylation
and, in particular, of EDEN-BP phosphorylation.
The insensitivity of EDEN-dependent deadenylation to MPF contrasts with the
recent data by Groisman et al. (Groisman
et al., 2002) showing that cyclin B1 mRNA polyadenylation is
stimulated via MPF and that its deadenylation is induced by MPF inactivation.
There is no proof that cyclin B1 3'-UTR contains an EDEN, suggesting
that the deadenylation of this mRNA is independent of EDEN-BP. Therefore, two
groups of maternal Xenopus mRNAs can be distinguished on the basis of
their behavior regarding deadenylation during early development: namely, MPF-
or cell-cycle-dependent deadenylation, like cyclin B1 mRNA
(Groisman et al., 2002
), and
MPF-independent, but activation-dependent deadenylation, like Eg2 (this
paper). Because calcium is as an important regulator of the cell cycle, we
cannot exclude that mRNA deadenylation is controlled by cytoplasmic calcium in
both cases.
EDEN-BP phosphorylation and dephosphorylation seems a very complex process. We could distinguish four different forms of this protein on western blots. The complex pattern of EDEN-BP phosphorylation suggests that multiple phosphorylation sites are modified during oocyte maturation and following egg activation. The inhibitory effects of both kinase and phosphatase inhibitors on EDEN-dependent deadenylation supports the hypothesis of a network of kinases and phosphatases controlling this process.
Could EDEN-BP phosphorylation regulate EDEN-dependent
deadenylation?
EDEN-BP is a good candidate for the regulatory molecule involved in the
modulation of EDEN-dependent deadenylation on M II oocyte fertilization or
activation. After calcium ionophore-induced activation this protein undergoes
a series of dephosphorylations. The hyperphosphorylated band (a) disappears
rapidly, whereas the intensity of the dephosphorylated band (c) increases to
reach a maximum 2.5 to 3 hours after egg activation. The intensity of the
hyperphosphorylated band (b), which is the major band in CSF extracts,
decreases gradually and disappears 2 hours after activation. This correlation
suggests that phosphorylation of EDEN-BP could be involved in the inhibition
of EDEN-dependent deadenylation. However, EDEN-BP is part of a multimeric
complex of high molecular weight
(Legagneux et al., 1995) that
probably contains several other factors required for deadenylation. Therefore,
at present we can not exclude that changes in the phosphorylation of one or
several of these factors are not involved in the regulation of EDEN-dependent
deadenylation. Identification of the amino acids of EDEN-BP that are
phosphorylated on oocyte maturation and studies of mutant proteins will permit
us to evaluate the exact role of EDEN-BP phosphorylation and dephosphorylation
during oocyte maturation and early embryonic development.
![]() |
Acknowledgments |
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![]() |
References |
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