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Cytoplasmic CstF-77 protein belongs to a maskingcomplex with
cytoplasmic polyadenylation
element-binding protein in Xenopus oocytes.Christel Rouget,
Catherine Papin, Elisabeth Mandart
To cite this version:Christel Rouget, Catherine Papin, Elisabeth
Mandart. Cytoplasmic CstF-77 protein belongs to amasking complex
with cytoplasmic polyadenylation element-binding protein in Xenopus
oocytes..Journal of Biological Chemistry, American Society for
Biochemistry and Molecular Biology, 2006,281 (39), pp.28687-98.
�10.1074/jbc.M601116200�. �hal-00260282�
https://hal.archives-ouvertes.fr/hal-00260282https://hal.archives-ouvertes.fr
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1
Cytoplasmic CstF-77 protein belongs to a Masking Complex
withCPEB in Xenopus Oocytes
Christel Rouget, Catherine Papin and Elisabeth MandartFrom the
Centre de Recherches de Biochimie Macromoléculaire, CNRS, 1919
route de Mende, 34293
Montpellier cedex 05, FranceRunning title: Cytoplasmic CstF-77
in masking mRNA
Address correspondence to Elisabeth Mandart: CRBM, CNRS, 1919
route de Mende, 34293 Montpelliercedex 05, France, Tel (33) 4 67 61
33 39; FAX (33) 4 67 52 15 59;
E-mail:[email protected]
Regulated mRNA translation is ahallmark of oocytes and early
embryos, ofwhich cytoplasmic polyadenylation is a majormechanism.
This process involves multipleprotein components, including the
CPSF(Cleavage and Polyadenylation SpecificityFactor), which is also
required for nuclearpolyadenylation. The CstF (Cleavagestimulatory
Factor), with CPSF, is required forthe pre-mRNA cleavage before
nuclearpolyadenylation. However, some evidencesuggests that the
CstF-77 subunit might have afunct ion independent o f nuc
learpolyadenylation, which could be related to thecell cycle. As
such, we addressed the questionwhether CstF-77 might have a role
incytoplasmic polyadenylation. We investigatedthe function of the
CstF-77 protein in Xenopusoocytes, and show that CstF-77 has indeed
arole in the cytoplasm. The Xenopus CstF-77protein (X77K) localizes
mainly to the nucleus,but also in punctuate cytoplasmic foci. We
showthat X77K resides in a cytoplasmic complexwi th e IF4E, CPEB
(Cytop lasmicPolyadenylation Element Binding protein),CPSF-100 and
XGLD2, but is not required forcytoplasmic polyadenylation per
se.Impairment of X77K function in ovo leads to anacceleration of
the G2/M transition, with apremature synthesis of Mos and
AuroraAproteins. However, the kinetic of Mos mRNApolyadenylation is
not modified. Furthermore,X77K represses mRNA translation in
vitro.These results suggest that X77K could beinvolved in masking
of mRNA prior topolyadenylation.
Regulation of gene expression plays acentral role in many
cellular functions. In
eukaryotes, formation of the mature 3’ end of amessenger RNA is
a two-step reaction thatinvolves cleavage of the nascent transcript
andsubsequent polyadenylation in the nucleus(reviewed in 1-3). In
mammals, the cleavagereaction requires two sequences, the
highlyconserved hexanucleotide AAUAAA located 30-20 nucleotides
upstream of the cleavage site, andthe G/U rich sequence that lies
downstream of the3’ cleavage site (DSE for Downstream
Element).These sequences are bound by differentmultisubunit
factors. CPSF binds to theAAUAAA sequence via the CPSF-160
subunit,and CstF recognizes the DSE element via theCstF-64 protein
(4,5). CPSF and CstF are knownto interact cooperatively at 3' end
signals (6), andallow pre-mRNA cleavage with the cleavagefactors I
and II (CFI and CFII), the carboxy-terminal domain of RNA
polymerase II and thepoly(A) polymerase (PAP). CPSF consists of
the160K, 100K, 73K, 30K and hFip proteins (7-9),and CstF is an
heterotrimeric structure composedof the 77K, 64K and 50K proteins
(10,11). CstF-77 is the subunit required for integrity of the
CstFcomplex. CstF-77 binds to CstF-64, CstF-50 (12)and CPSF-160 (5)
and therefore helps to holdtogether the much larger complete
polyadenylationcomplex.
CPSF is also involved in cytoplasmicpolyadenylation (13,14), a
mechanism ofcontrolling translation which is often used
whentranscription is quiescent, such as in oocytes orearly embryos.
In Xenopus oocytes, severaldormant mRNAs are stored with short
poly(A)tails, which are elongated when oocytes arestimulated by
progesterone, provoking theirtranslation.
Cytoplasmic polyadenylation in frogoocytes also requires two
sequences on the 3’UTR
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2
of target mRNAs, recognized by multiple proteincomponents
(reviewed in 15). CPEB directly bindsto the CPE (Cytoplasmic
PolyadenylationElement) located upstream of the AAUAAAsequence,
which is recognized by CPSF, as innuclear polyadenylation (14).
CPEB interacts withCPSF, which may help to stabilize its
associationwith the AAUAAA element and a cytoplasmicpoly(A)
polymerase. GLD2, a poly(A) polymerasewhich is structurally
distinguishable from nuclearPAP, was first identified in
Caenorhabdit iselegans (16). The Xenopus related protein(XGLD2) was
shown to bind to CPEB and CPSFand to participate in cytoplasmic
polyadenylation(17,18). Sequences other than the CPE, the
PREsequences (Polyadenylation Response Element),regulate early
cytoplasmic polyadenylation ofspecific mRNAs including Mos (19,20).
TheCPEB protein has been shown to repress mRNAtranslation in
immature oocytes and to directcytoplasmic polyadenylation and
translationalactivation in maturing oocytes. Indeed,
mRNAtranslation repression is controlled by a complexcomposed of
CPEB, Maskin and eIF4E. Maskininteracts with CPEB and eIF4E and
preventseIF4G binding to eIF4E. After stimulation, CPEBis phosphory
la ted and cy top lasmicpolyadenylation ensues, allowing disruption
ofMaskin-eIF4E binding and recruitment of eIF4G(21,22).
The yeast Saccharomyces cerevisiae andDrosophila melanogaster
homologues of CstF-77are, respectively, the Rna14 and Su(f)
proteins.Several genetic and biochemical studies haveshown a role
for Rna14 in 3’end processing (23-25), although other studies for
Rna14 havesuggested a function independent of
nuclearpolyadenylation. Indeed, rna14 mutants can beseparated into
two classes: the poly(A)-negativeclass, which contains mutants
deficient in mRNA3’ processing, and the poly(A)-positive
class,which includes mutants that are not impaired innuclear
polyadenylation (26,27). Rna14 and Su(f)localize both to the
nucleus and the cytoplasm. Inyeast, this cytoplasmic localization
is mainly inmitochondria (26,28). However, no mitochondrialfunction
for Rna14 has been described so far. InD.melanogaster , the Su(f)
protein has beenimplicated in cell cycle progression (29).
InXenopus and human cells, studies have shown thatCstF-77, CstF-64
and CPSF-100 are concentrated
in nuclear domains in Cajal or coiled Bodies (30-33) as well as
various components required fortranscription and processing of the
three classes ofnuclear transcript (reviewed in 34).
However,nothing was reported concerning a possiblecytoplasmic
localization of the CstF subunits.
The aim of our work was to study thecytoplasmic function of
CstF-77, bearing in mindpossible roles independent of
nuclearpolyadenylation that could be related to the cellcycle. We
therefore addressed the question ofwhether CstF-77 could have a
role in cytoplasmicpolyadenylation. The Xenopus oocyte is
anappropriate model to study other functions ofCstF-77, as there is
neither transcription nornuclear polyadenylation during oocyte
maturation(15). In this report, we identify the Xenopus CstF-77
protein (X77K) and show that it is partiallylocalized in the
cytoplasm. We demonstrate thatX77K belongs to a cytoplasmic complex
witheIF4E, CPEB, CPSF-100 and XGLD2. However,the protein is not
required for cytoplasmicpolyadenylation per se. Inhibition of
X77Kfunction in oocytes accelerates meiotic maturationand
precociously induces Mos and AuroraAprotein synthesis, without
modifying the kinetic ofMos mRNA polyadenylation. Moreover,
X77Kinhibits in vitro mRNA translation in a dosedependent manner.
These results suggest thatX77K could have a function in mRNA
maskingprior to cytoplasmic polyadenylation.
EXPERIMENTAL PROCEDURES
X77K cloning and protein alignment - AXenopus leavis oocyte cDNA
library in λgt11from Clontech (ZL5000b) was screened with a1.030
base pair (bp) probe obtained by RT-PCRfrom X e n o p u s RNA with
the followingdegenerated oligonucleotides (Eurogentec):
5'-ATGGCYMARGCWTAYGAYTTYGCACT-3'and
5'-ACKCKSGTRTTRTTRTCYTCRTTBAG-3' where Y=C+T, M=A+C, R=A+G,
W=A+T,K=T+G, S=C+G and B=T+C+G. Several clonescontaining the cDNA
of the Xenopus CstF-77homologue (X77K) were isolated and
sequenced.The largest cDNA (about 2.300 bp) was subclonedat the
EcoR1 site of the pBluescript II KS. Itcontains an open reading
frame encoding a proteinof 719 amino acids. This X77K amino
acidsequence was aligned with those of Homo sapiens
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3
CstF-77 (NP001317), Mus musculus CstF-77(AAH03241), Drosophila
melanogaster Su(f)(P25991), Caenorhabditis elegans
CstF-77(AAA62311) and Saccharomyces cerevisiaeRNA14 (NP013777) with
the ClustalW program.The X77K sequence was submitted to Genbankwith
the following accession number: AM071387.
Xenopus oocytes and embryos - Stage VIoocytes were selected
after surgical removal ofovaries from mature female Xenopus laevis
andtreatment with collagenase (Sigma) at 1mg/ml inOR-2 (5mM Hepes
pH 7.2, 82.5mM NaCl, 2mMKCl, 1mM MgCl2). All further manipulations
ofoocytes were performed in modified Ringer’sSolution (MMR: 5mM
Hepes pH 7.8, 100mMNaCl, 2mM KCl, 1mM MgSO4, 0.1mM EDTA,2mM CaCl2).
Progesterone was used at a finalconcentration of 1mM. For protein
oocyte extracts,oocytes were homogenized at 5 µl per oocyte inlysis
buffer (20mM Tris pH7.5, 50mM NaCl,50mM NaF, 10mM
β-glycerophosphate, 5mMNa4P2O7, 1mM Na3VO4, 1mM EDTA, 1mMEGTA,
0.1mM PMSF, plus 10 µg/ml each ofleupeptin, chymostatin, pepstatin
and aprotinin) at4°C, centrifuged at 13000 rpm for 3 minutes at4°C
and the supernatant was used for furtheranalysis. For manually
enucleated oocytes,nucleus were collected and boiled in 5X
Laëmmlibuffer and cytoplasms were homogenized at 5 µlper cytoplasm
in lysis buffer. In vitro fertilizationand embryo cultivation was
carried out asdescribed (35). The quantification of proteins
inoocyte and embryo extracts was done by Bioradprotein assay, with
quantified BSA as standard.For microinjections, the usual injected
volume forantibodies, purified protein or RNA was 20 to 40nl per
oocyte and the number of injected oocytes is35 for each
condition.
Antibodies and Western blots- The anti-pTpY-MAPK was obtained
from New EnglandBiolabs (9106S). The anti-eIF4E was provided byS.
Morley or was obtained from mouse antiserausing the same peptide
antigen. The anti-CPSF-100 and the anti-Pak5 were provided by E.
Wahleand N. Morin’s laboratories respectively. The anti-β-tubulin
and anti-HA antibodies were obtainedfrom E7 (Iowa hybridoma bank)
and from the12CA5 hybridomas respectively. The anti-RPAand
anti-AuroraA were provided by J.M.Lemaître and C. Prigent
respectively. The anti-
Mos, anti-ERK and IgG were obtained from SantaCruz (sc-086,
sc-94 and sc-2027, respectively).Other antibodies used against GST
and Xenopus77K, CPEB and GLD2 are rabbit polyclonalantisera and
were affinity purified. The 77K Cterantibody is directed against
the 17 amino-acidscarboxyl terminal of the human 77K
protein:NH2-VPPVHDIYRARQQKRIR-COOH that waskindly provided by David
Bentley, and the 573antibody is directed against the following
X77Kpeptide: NH2-LKDDVDRKPEYPKPD-COOH.The anti-CPEB antibody is
directed against GST-CPEB fusion protein as described (36). The
antiXGLD2 antibody is directed against the GST-XGLD2 2/3 carboxy
terminal fusion protein. Formicroinjections, dialyzed antibodies
were used at1 µg/µl. Western blots were probed with primaryantibody
at 50 ng/ml and the appropriatesecondary antibody horseradish
peroxidase (HRP)conjugate diluted according to
manufacturerrecommendations (Amersham) and revealed byECL
(Amersham).
RNA and recombinant proteins - The Mos3'UTR construct was
obtained by inserting theSpe1/Xba1 fragment from the pT7-G UK
XMosplasmid kindly provided by N. Sagata (37)
intoSpe1/Xba1-digested pCS2 (Dave Tumer,University of Michigan).
This 320-nucleotidefragment contains Mos PRE, CPE and
AAUAAAsequences. Mos 3'UTR RNA were prepared bylinearizing the
pCS2-Mos320 with Xba1 andcarrying out transcription reaction
according toPapin and Smith (38). The pCSH vectorcorresponds to the
pCS2 vector with two HA tagsinserted in the multiple cloning site.
CappedmRNA encoding HA-X77K, HA-XGLD2 andHA-CPEB were prepared by
cutting the pCSH-X77K ORF, pCSH-XGLD2 ORF and pCSH-CPEB ORF
respectively with Not1 and carryingout transcription reaction.
Immunoprecipitations - Protein oocyteextracts corresponding to
20 oocytes wereperformed in extract buffer (100 mM KCl, 0.1
mMCaCL2, 1mM MgCl2, 10 mM Hepes pH7.6, 50mM sucrose, plus 10 µg/ml
each of leupeptin,chymostatin, pepstatin and aprotinin). For
someexperiments, the oocyte extracts were treated 10min at room
temperature with RNAse A at 0,02mg/ml. Oocyte extracts were
incubated with 200ng of antibodies over night at 4°C on
wheel,supplemented with 20 µl of Protein A-Sepharose
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4
beads and mixed for another 45 min at 4°C onwheel. The
immunoprecipitates were then washed3 times in XRB A (0.2% Triton
X-100, 10 mMTris pH8, 150 mM NaCl and 2 mM EDTA), 3times in XRB B
(0.2% Triton X-100, 10 mM TrispH8, 500 mM NaCl and 2 mM EDTA) and
2times in 10 mM Tris pH8, eluted by boiling in 2XLaëmmli buffer,
separated by SDS-PAGE andanalyzed by Western blotting.
For immunoprecipitation of radio-labeledX77K, 1 µg of 77K
antibody was incubated or notwith 10 µg of its antigen (77K Cter
peptidecoupled with thyroglobulin) before being appliedto protein-A
Sepharose beads for 30 min at 4°C.The beads were then washed three
times withPBS, supplemented with 35S methionine X77Ktranslated in
rabbit reticulocyte lysates, and mixed1 h on ice. The
immunoprecipitates were thenwashed 3 times in XRB A, 3 times in XRB
B, and2 times in 10 mM Tris pH8, eluted by boiling in2X Laëmmli
buffer, separated by SDS-PAGE, andanalyzed by autoradiography.
Cap column and GST Pull-Down assay–The cap column assay was
performed as described(22). Briefly, 30 oocytes were homogenized
inhomogenization buffer (HB), and treated or not 10min at room
temperature with RNAse A (0,02mg/ml). The oocyte supernatants
weresupplemented with 0.1 mM GTP or 5 mM 7mGTPand applied in batch
to a 7mGTP-Sepharose (cap)column (Amersham) that had been
equilibratedwith HB plus bovine serum albumin (BSA, 0.1mg/ml).
Following mixing for 1h at 4°C ineppendorf, the cap column was
extensivelywashed (100 bed volumes) with 0.1 M KCl in 50mM Tris pH
7.5. The bound material was theneluted with 5 mM 7mGTP on column.
The eluatesand a fraction of oocyte lysates collected beforeand
after mixing with the cap column wereseparated by SDS-PAGE and
analyzed by Westernblotting.
The GST pull-down assay was performedas described (39). Briefly,
HA-tagged CPEB andHA-tagged XGLD2 were in vitro translated inrabbit
reticulocyte lysates (Promega) in thepresence of unlabeled amino
acids. GST-X77Kexpressed in Baculovirus and GST expressed inE.coli
were purified with glutathione sepharose(Amersham). 5 µg of
purified GST-X77K or GSTwere bound to 20 µ l of glutathione beads
in
binding buffer. The beads were subsequentlyincubated 2h at room
temperature in 400 µl ofbinding buffer plus 5 µg of the HA-CPEB or
HA-XGLD2. The beads were then extensively washedwith binding buffer
and boiled in 2X Laëmmlibuffer. The eluates and inputs were
separated bySDS-PAGE and analyzed by Western blotting.
Polyadenylation assays - Total RNA fromoocytes was extracted
using the Mini RNAIsolation IITM kit (Zymo Research) and the
PAT(PolyAdenylation Test) assay was carried outaccording to Salles
and Strickland (40) with minormodifications. Reverse transcription
was done ontotal RNA with oligo(dT)-anchor (dT-PAT) asprimer
(5'-GCGAGCTCCGCGGCCGCGT(12)-3').Subsequent PCR was carried out
using the dT-PAT primer and a specific upstream primer forM o s 3 '
U T R R N A ( 5 ' -GCACTGAAAATACAAGCAAGGATATG-3').C y c l i n B 1 O
R F R N A ( 5 ’ -GTGGAAATGGCCCGCCCACTC-3’), AuroraA3 ’ U T R R N A
( 5 ’ -GGCTGTCACCGTACAACGCTACTTG-3’) andac t in type 5 3 ’UTR RNA
(5 ’ -CAAATGTTGCAGGTACACCTG-3’). The PCRproducts were resolved in a
2.5% agarose gel andvisualized with ethidium bromide staining.
For in vitro polyadenylation assays, eggextracts were prepared
according to Murray andKirschner (41). Egg extracts were adjusted
to 1mM ATP, 1 mM MgCl2, 7.5 mM creatinephosphate and 0.1 mM EGTA
pH7.7. 30 ng ofMos 3'UTR RNA was added to the egg extract andthe
polyadenylation assay was carried out at 23°Cfor 1h, prior to RNA
extraction as described inMcGrew and Richter (42). Mos 3'
UTRpolyadenylation analysis was carried out asdescribed above (PAT
assay). For depletionexperiments, 2 µg of antibodies were incubated
30min at 4°C with 15 µl of Dynabead protein-Abeads (Dynal). Beads
were washed three timeswith PBS plus 100 µg/ml BSA and incubated
with15 µ l of egg extract for 30 min at roomtemperature. After
precipitation, depleted extractwas collected and used for
polyadenylation assayand Western blot analysis. Bead precipitates
wereeluted by boiling in 2X Laëmmli buffer andanalyzed by Western
blotting.
In vitro translation in Rabbit ReticulocyteLysate (RRL) - The
luciferase translation assay
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5
was performed according to manufacturerrecommendations
(Promega). 100 ng of LuciferasemRNA (provided in RRL kit) were
added to 10 µlof RRL in presence of [35S]methionine
(2µl,1000Ci/mmol, methionine/ cysteine mix,Amersham) with or
without the indicatedquantities of GST-77K or GST proteins, for
1.5hat 30°C. The equivalent of 20% of the reactionwas used for
autoradiography and western blotanalysis. The amount of proteins
added to thereaction was analyzed by Coomassie staining.
Theluciferase RNA was analysed by RT-PCR after thereaction. Reverse
transcription was done withrandom primers and the amplification
with thef o l l o w i n g o l i g o n u c l e a o t i d e s : 5 ’
-AAGCCACCATGGAAGA-3’ and 5 ’ -CTCTAGAATTACACGGCGATC-3’.
Cell culture and immunofluorescence -Mouse NIH 3T3 cells were
cultured in DMEMsupplemented with 10% FBS in a 37°C incubatorwith
5% CO2. Xenopus XTC cells were cultured in70% L15 Leibovitch's
Media supplemented with10% FBS in hermetic tissue culture flask at
25°C.Cultured cells were fixed using 4%paraformaldehyde (in PBS)
for 15 min at roomtemperature. The cells were then permeabilized
inPBS containing 1% Triton-X100 for 10 min atroom temperature and
blocked in PBS containing0,1% Tween and 3% BSA (blocking
reagent).Antibody incubations were done 1h at roomtemperature in
blocking reagent followed bysecondary Alexa-546-labeled goat
anti-rabbit IgG(Molecular probe) used in identical conditions.DNA
was visualized by DAPI staining.
RESULTS
Identification of Xenopus CstF-77homologue - A Xenopus laevis
oocyte library wasscreened with a probe made by RT-PCR fromX e n o
p u s oocyte RNA and degenerateoligonucleotides. A cDNA encoding
the Xenopushomologue of CstF-77 was isolated. This cDNAcontains an
open reading frame (ORF) encoding aprotein of 719 amino-acids, with
a predictedmolecular weight of 83kDa that we named X77K.We
identified a domain organization similar to theknown CstF-77
homologues, with 11 HAT (half aTPR) repeats, a bipartite nuclear
localizationsignal (NLS) and a proline-rich domain at the Cterminus
(figure 1). Apart from the NLS, these
domains are known to mediate protein-proteininteractions, and
the HAT repeats have beendescribed to be specific to a family of
proteinsinvolved in RNA processing (11,43). For thehuman CstF-77
protein, the proline-rich domainhas been shown to be necessary both
for bindingother CstF subunits and also for self association(12).
It is noteworthy that the CstF-77 proteins arehighly conserved,
since the amino acid sequenceof X77K shares 94% identity overall
with thehuman and mouse CstF-77 proteins, 57% withD.melanogaster
protein and 27% with S.cerevisiaeprotein (figure S1).
CstF-77 is localized both in the nucleusand cytoplasm - To study
the expression of X77Kprotein, we raised a polyclonal antibody
againstthe human C terminal peptide (77K antibody). ByWestern
blotting, this antibody recognizes the HA-tagged X77K expressed in
oocytes, and thepurified Baculovirus GST-X77K protein (figure2A
lanes 2 and 5). It also detects endogenousXenopus CstF-77 protein
from oocytes or celllysates (figure 2A lanes 1 and 3 respectively)
andendogenous mouse (lane 4) and human protein(not shown).
Moreover, the in vitro translatedX77K protein is immunoprecipitated
by the 77Kantibody but not by the 77K antibody pre-incubated with
the peptide antigen (figure 2B).These results show the specificity
of the 77Kantibody for the 77K protein.A Western blot analysis on
oocyte and embryolysates shows that X77K is expressed at a
constantlevel dur ing X e n o p u s oogenesis andembryogenesis up
to stage 40 (figure 3A and 3B).In S. cerevisiae and D.
melanogaster, the CstF-77homologues Rna14 and Su(f) respectively
localizeboth to the nucleus and cytoplasm (28,44). InXenopus
oocytes, the nuclear localization ofX77K has been well determined,
where it ispresent in Cajal bodies and lampbrushchromosomes (32).
However, nothing has beenreported concerning a possible
cytoplasmiclocalization. To investigate whether X77K couldbe
cytoplasmic in Xenopus laevis, lysates fromnucleus, cytoplasm or
total oocytes were analyzedby Western blotting with 77K antibody.
Figure 3Cshows that X77K is mainly nuclear, but is alsopresent in
the cytoplasm. We verified thatcytoplasmic extracts were not
contaminated withnuclear extracts by probing the membrane for
thenuclear protein RPA (Replication Protein A). This
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6
double localization was confirmed byimmunofluorescence
experiments on XenopusXTC and mouse NIH3T3 cell lines (figure
3D):CstF-77 protein is mainly in the nucleus but is alsolocalized
in punctuated bodies distributed in thewhole cytoplasm. These
results show that theCstF-77 protein is localized both in the
nucleusand cytoplasm in vertebrates, as in S. cerevisiaeand
Drosophila.As X77K has a functional NLS but partiallylocalizes to
the cytoplasm, we investigatedwhether the protein shuttles between
the GV(Germinal Vesicle) and the cytoplasm, even if itdoes not
present a NES (Nuclear Export Signal).To answer this question,
oocytes were treated ornot overnight with leptomycin B, a nuclear
exportinhibitor. They were then enucleated, to analyzewhether the
protein accumulated in the nucleus inpresence of leptomycin B. In
this condition, nochange in the relative levels of X77K between
thenucleus and the cytoplasm was observed, showingthat X77K does
not shuttle between the twocompartments (figure 3E). Surprisingly,
the CPEBprotein was sensitive to leptomycin B andaccumulated in the
nucleus (figure 3E). This resultvalidates the leptomycin B
treatment and impliesthat CPEB shuttles between the cytoplasm and
thenucleus. This observation was not expected sinceCPEB was not
detected in the nucleus undernormal conditions (35 and figure 3E)
and suggestsnew functions for CPEB in the nucleus, or
innucleo-cytoplasmic transport.
X77K is part of a cytoplasmic complexwith CPEB, eIF4E, CPSF-100
and XGLD2 - Aswe asked whether X77K could have a function
incytoplasmic polyadenylation, we checked forX77K partners in the
cytoplasm. In Xenopusoocytes, CPEB plays a key role in
translationalrepression in immature oocytes, and in
directingcytoplasmic polyadenynation during oocytematuration. We
investigated whether X77K couldbelong to a complex which includes
CPEB. Weperformed co-immunoprecipitations from totalstage VI oocyte
extracts, treated or not with RNaseA, with antibodies directed
against differentproteins known to be in a cytoplasmic complexwith
CPEB. In this experiment, X77K, CPEB,eIF4E and CPSF-100 proteins
were co-immunoprecipitated with 77K, eIF4E and CPEBantibodies but
not with nonspecific IgG (figure4A). As a negative control we used
an antibody
directed against the Pak5 (P21 activated kinase)protein. This
antibody did not immunoprecipitateany of the proteins tested.
Similarly, Pak5 proteinwas not immunoprecipitated by the 77K, eIF4E
orCPEB antibodies. These results show that X77Kspecifically
interacts with eIF4E, CPEB andCPSF-100 independently of the mRNA.To
show that cytoplasmic X77K is involved in thiscomplex, we
enucleated oocytes and performedco-immunoprecipitation experiments
from oocytecytoplasm extracts treated with RNase A. TheX77K, CPEB
and CPSF-100 proteins were co-immunoprecipitated by X77K, eIF4E and
CPEBantibodies (figure 4B). These results show thatcytoplasmic X77K
belongs to a complex withCPEB, eIF4E and CPSF.It has been reported
that XGLD2, a divergentpoly(A) polymerase, directly binds to CPEB,
evenin stage VI oocytes (18). We asked whetherXGLD2 was also in the
complex with X77K instage VI oocytes. As XGLD2 is expressed at a
lowlevel in oocytes (18), immunoprecipitations wereperformed from
oocytes overexpressing XGLD2.HA-tagged XGLD2 mRNA was injected
intooocytes and, after 14 hours of expression, theoocyte extracts
were subjected to differentantibody immunoprecipitations in
presence orabsence of RNase A. In these experiments, HA-XGLD2,
X77K, eIF4E and CPEB proteins wereco-immunoprecipitated with XGLD2,
X77K,eIF4E and CPEB antibodies (figure 4C), showingthat XGLD2 is
also part of the complex. It isinteresting to note that two forms
of eIF4E wereimmunoprecipitated by the eIF4E antibody butthat only
the lower form is mainly co-immunoprecipitated (figure 4A and C).To
better characterize the complex and investigatewhether the complex
exists in metaphase IIoocytes, we used another approach. Cap
columns(7mGTP chromatography), which allow eIF4Erecruitment, were
used to identify proteins presentin the complex. Stage VI and
metaphase IIextracts, treated (figure 4D lanes 6 and 7) or
not(lanes 2 and 3) with RNase, were supplementedwith GTP to reduce
non-specific adsorption. As anegative control, some extracts were
supplementedwith free cap (7m GTP) (figure 4D lanes 4 and 5).The
extracts were then applied on a cap columnand the bound material
was eluted with free capand detected by Western blot. A fraction of
bothX77K and CPEB from stage VI and metaphase II
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7
oocytes with or without RNAse was retained onthe column in
presence of GTP but not when7mGTP was added in the extracts (figure
4D). Thisexperiment was done using GTP at a lowerconcentration than
7mGTP in the extracts. Toconfirm the specificity of the binding, a
controlexperiment was done either, using the sameconcentration of
GTP and 7mGTP in the extracts,or, by eluting the bound proteins
with GTP or7mGTP (figure S2). We observed that GTP did notcompete
for eIF4E, 77K and CPEB binding while7mGTP did (figure S2 compare
lane 6 with lane 8)and that GTP only weakly eluted bound
proteinscompare to 7mGTP (compare lane 4 with lane 2).Asmall amount
of total X77K was retained on theresin (figure 4D and S2), which is
coherent withthe fact that only a small fraction of X77K
iscytoplasmic and can bind to eIF4E. It isnoteworthy that CPEB was
also poorly retained onthe resin (figure 4D and S2), even though it
isabundant in the cytoplasm. However, this resultcould be explained
by the fact that CPEB, knownto be part of the masking complex,
interacts onlytransiently with eIF4E (20). In metaphase IIoocytes,
the electrophoretic mobility of CPEB isshifted and the protein is
partially degraded (45).However, the same proportion of total CPEB
wasretained on the column in stage VI and metaphaseII extracts. As
negative controls, we looked forthe binding of ERK and β−tubulin
proteins, andobserved, as expected, that they were not retainedon
the column (figure 4D). We failed to detectendogenous XGLD2 on the
resin, probablybecause of its low expression level.To assess
whether the interactions were direct, weperformed GST-pull-down
experiments withpurified GST-X77K from Baculovirus and
eitherHA-CPEB or HA-XGLD2 proteins translated invitro. Figure 4E
shows that GST-X77K bound toHA-CPEB and HA-XGLD2, whereas GST
alonedid not. In the same kind of experiment, a directinteraction
between GST-X77K and HA-eIF4Ewas not conclusive, due to a high
background inthe GST control (data not shown).Altogether, these
results show that X77K belongsto a RNA-independent cytoplasmic
complex witheIF4E, CPEB, CPSF-100 and XGLD2, and thatX77K is
capable of directly interacting with CPEBand XGLD2.
X77K is not required for cytoplasmicpolyadenylation - We showed
that X77K binds to
proteins required for cytoplasmic polyadenylationin oocytes. To
address whether X77K is requiredfor cytoplasmic polyadenylation, we
performed invitro polyadenylation tests in egg extracts thatwere
subjected to 77K, or control IgGimmunodepletions. As a control, we
also did theexperiment on egg extracts depleted for CPEB, aprotein
that is known to be required forcytoplasmic polyadenylation. Figure
5A showsthat immunodepletions of X77K and CPEBcompletely removed
the endogenous proteins (leftpanel), which were retained on the
antibody beads(right panel). Moreover, even if CPEB wasapparently
not depleted by 77K antibody, and viceversa, we observed a
co-immunoprecipitationbetween X77K and CPEB in egg extracts
(figure5A right panel) that confirmed our results obtainedwith
oocyte lysates (figure 4).To do the polyadenylation test, Mos 3'UTR
RNA,bearing wild-type CPE and PRE elements, wasincubated for one
hour with egg extracts or X77K,CPEB or mock depleted extracts. The
total RNAwas isolated at the beginning of the reaction andone hour
later. Then, Mos 3’UTR polyadenylationwas checked by PAT
(PolyAdenylation Test)assay, to measure the poly(A) tail length.
Figure5 B shows that Mos 3'UTR RNA waspolyadenylated in control and
X77K depletedextracts, while it was not polyadenylated in
CPEBdepleted extracts (compare lanes 2, 4, 6 with lane8). These
results show that depletion of X77Kdoes not impair cytoplasmic
polyadenylation invitro.To verify that this is also the case in
vivo, weperformed a polyadenylation test in oocytes.Oocytes were
first injected with Mos 3’UTRRNA. Some of them were subsequently
injectedwith 77K, CPEB or nonspecific IgG. One hourlater, the
oocytes were induced, or not, to maturewith progesterone, and
harvested when 30% of thecontrol oocytes have undergone GVBD
(GerminalVesicle Break Down). The total RNA was isolatedand scored
for Mos mRNA polyadenylation. Incontrol oocytes, Mos 3’UTR was
polyadenylatedonly when the oocytes were submitted toprogesterone
(Figure 5C compare lane 2 with lane3) . The change in
electrophoretic mobilityobserved was due to the lengthening of
thepoly(A) tail, since the addition of oligo (dT) andRNaseH before
the PAT assay suppressed themobility shift (figure 5C right panel).
As in egg
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8
extracts, the impairment of X77K function did notmodify the Mos
3’UTR polyadenylation in theoocytes (figure 5C compare lane 6 with
lanes 3and 8). In CPEB antibody injected oocytes, Mos3’UTR was
polyadenylated to a lesser extent thanin control oocytes (compare
lane 10 with lanes 3and 8). This short poly(A) tail of Mos
3’UTRobserved was probably due to a PRE-dependentpolyadenylation.
Indeed it has been shown thatMos 3’UTR could be precociously
polyadenylatedindependently of CPE/CPEB, but the length of
thepoly(A) tail was shorter than in the presence ofCPEB (20). The
above experiment shows thatX77K is not required for polyadenylation
ofexogenous Mos mRNA.To confirm this result, we performed
apolyadenylation test on endogenous mRNAs fromoocytes injected with
IgG, 77K or CPEBantibodies, or non-injected (figure 5D).
CyclinB1and AuroraA mRNAs were polyadenylated incontrol and 77K
antibody injected oocytes, whichis in agreement with the result
obtained withexogenous Mos mRNA. In CPEB antibodyinjected oocytes,
there is no polyadenylation ofcyclinB1 mRNA that has no PRE
sequence, whilewe observed a short poly(A) tail on AuroraAmRNA
which bears a PRE element (19). Thepolyadenylation observed with
our PAT assay is aspecific reaction, as endogenous actin mRNA,which
is not subjected to polyadenylation duringoocyte maturation, is not
polyadenylated in ourtest (figure 5D). Furthermore, we observed a
weakdeadenylation of actin mRNA from 6 hours ofprogesterone
addition, showing that the injectedantibodies did not impair the
deadenylationreaction during meiotic maturation. Altogetherthese
results obtained in vitro and in ovo show thatX77K is not required
for cytoplasmicpolyadenylation.
77K antibody injected into oocytesaccelerates the G2/M
transition - During theexperiments of polyadenylation test in
oocytes, weobserved that X77K antibody injection acceleratesoocyte
maturation. The antibodies were injectedinto and maintained in the
cytoplasm (figure 6Ainsert), arguing that 77K antibody affected
thefunction of the cytoplasmic X77K protein.Moreover, as maturation
is accompanied with acessation of transcription (15), an effect on
thenuclear polyadenylation could be ruled out. Werepeated the
experiment several times and the 77K
antibody injected oocytes always underwent 50%GVBD one hour or
more before the controloocytes (figure 6A), depending on the batch
ofoocytes. This was also the case when we injectedanother antibody
directed against X77K (the 573antibody) (data not shown). In
agreement withthese results, this acceleration was accompaniedby a
premature synthesis of Mos protein andactivation of MAP kinase
(figure 6B). In 77Kantibody injected oocytes, Mos protein is
welldetected 2 hours after progesterone induction,while in control
oocytes (uninjected or IgGinjected) it is clearly detected only 5
hours afterprogesterone addition. The MAPK activation,revealed by
the bi-phosphorylation of ERK, is alsoearlier in 77K antibody
injected oocytes (2 hours)than in control oocytes (4 hours). To
investigatewhether downstream events of maturation wereaccelerated,
we checked for proteins where thesynthesis is induced at GVBD. In
77K antibodyinjected oocytes, AuroraA synthesis increased 2hours
after progesterone induction while, incontrol oocytes (IgG injected
and uninjectedoocytes), this increase is well observed at 5
hours(figure 6B). This acceleration of protein synthesisalso
occurred for cyclinB1 (data not shown).One supposition for this
premature proteinsynthesis was that the impairment of X77Kfunction
in oocytes accelerates mRNAcytoplasmic polyadenylation. To test
thishypothesis, we performed a time course of Mos3’UTR
polyadenylation in 77K antibody injectedoocytes. The oocytes were
first injected with Mos3’UTR RNA and then with 77K antibody or
IgG.Oocytes were harvested at different times afterprogesterone
induction, total RNA was extractedand checked for Mos mRNA
polyadenylation. Incontrol oocytes and 77K antibody
injectedoocytes, Mos mRNA polyadenylation occurred atthe same time,
showing that injection of 77Kantibody does not modify the kinetics
ofcytoplasmic polyadenylation (figure 6C). CPEB isknown to belong
to a masking complex witheIF4E and Maskin. It has been reported
thatmRNA cytoplasmic polyadenylation allows thedisruption of
Maskin/eIF4E interaction by eIF4Grecruitment, and translation
ensues (22). Thissuggests that the role of
cytoplasmicpolyadenylation is to allow the binding of eIF4Gto
eIF4E. In 77K antibody injected oocytes, Mos,AuroraA and CyclinB1
proteins were precociously
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9
synthesized, while Mos mRNA polyadenylationwas not accelerated.
This phenotype, with the factthat X77K belongs to a complex with
CPEB andeIF4E, suggests that X77K could have a role inmRNA masking
before cytoplasmicpolyadenylation.
X77K represses mRNA translation in vitro- The above results
document that X77K functionsin vivo as part of a complex involved
in mRNAmasking, thereby preventing a prematuretranslation of
protein that must be synthesized at aprecise time. To test a
potential function of theX77K protein in mRNA translation
inhibition, weperformed an in vitro translation experiment. Inthis
assay, we added increasing quantities ofpurified GST-77K or GST
proteins in rabbitreticulocyte lysates programmed with a
luciferasereporter, and checked for mRNA translation bymeasuring
and quantifying 35S methionineincorporation. Figure 7 shows that
luciferasemRNA translation was inhibited by addition ofrecombinant
X77K in a dose dependent manner,while no translation inhibition was
observed withthe GST protein. Importantly, this effect is not dueto
destabilization of Luciferase mRNA by theaddition of the
recombinant proteins. This result isin agreement with a role for
X77K in mRNAmasking.
DISCUSSION
In this study, we identified the XenopusCstF-77 protein (X77K)
and showed that the CstF-77 proteins are very well conserved in
vertebrates(figure 1 and S1). X77K is expressed in oocytesand
embryos at a constant level, and is localizedmainly to the nucleus,
but also in discretecytoplasmic foci in vertebrate cells (figure
3).However, X77K does not shuttle between the twocompartments
(figure 3E ). The cytoplasmiclocalization, together with other
arguments (seeintroduction), suggested a cytoplasmic role
forCstF-77 protein. We established that X77K is partof a
cytoplasmic complex with eIF4E, CPEB,CPSF and XGLD2 (figure 4) but
is not requiredfor cytoplasmic polyadenylation per se (figure
5).Nevertheless, impairment of X77K function inoocytes, accelerates
oocyte maturation and proteinsynthesis without modifying the
kinetic of MosmRNA polyadenylation (figure 6). Moreover,X77K
represses mRNA translation in vitro (figure
7). Altogether, these results support a role forX77K in mRNA
masking.
The known function of nuclear CstF-77protein is to enhance
cleavage by stabilizing theinteraction between CPSF and AAUAAA in
thepre-cleavage complexes (46). We envisage thatcytoplasmic X77K
could have the same functionof complex stabilization on cytoplasmic
mRNA.This could explain why cytoplasmic X77K is notrequired for
cytoplasmic polyadenylation per se,since its nuclear counterpart is
required for pre-mRNA cleavage but not for the subsequentpoly(A)
addition step of the reaction. In thenucleus, CstF-77 interacts
with CPSF via itsCPSF-160 subunit (5). In the cytoplasm, X77Kalso
binds to CPSF and CPEB (figure 4B ).Moreover, X77K directly
interacts with XGLD2and CPEB in vitro, showing that X77K, CPSF,CPEB
and XGLD2 are part of the samecytoplasmic complex. CstF-77 has no
RNAbinding domain, thus, one or several bindingpartners must
recruit X77K to increase the affinityof the polyadenylation
complexes (nuclear orcytoplasmic) on the target RNA. In the
nucleus,CstF-64 is the RNA binding protein that targets,with
CPSF-160, CstF-77 on the pre-mRNA. In thecytoplasm, CPEB could
replace CstF-64 for thisfunction. Moreover, CstF-77 has a
functionalNLS, leading to its intranuclear targeting (31 andour
unpublished data), and does not shuttlebetween the nucleus and the
cytoplasm (figure3E ), which could explain the low level of
itscytoplasmic counterpart. This suggests that CstF-77 can stay
into the cytoplasm only in interactionwith other proteins, and that
a fraction of X77Knewly synthesized can be recruited by CPEB,XGLD2
or CPSF-160 and probably other proteins(see below).
CstF-77 is not the only protein thatbelongs to both nuclear and
cytoplasmicpolyadenylation complexes: besides CPSF, shownto be
involved in cytoplasmic polyadenylation(14), Symplekin was recently
identified as part ofthe cytoplasmic polyadenylation machinery
(18).Symplekin is a dual location protein that has beenlocalized to
the cytoplasmic plaques of tightjunctions, and in the nucleus where
it interactswith CPSF, CstF-77 and CstF-64 (12,47).However,
Symplekin does not interact with thefully assembled CstF,
suggesting that Symplekinand CstF-77 can compete for the same site
in
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10
CstF-64 (12). CPEB binds both X77K (this study)and Symplekin
(18), strengthening the idea thatCPEB could have the same function
in thecytoplasm than CstF-64 in the nucleus.
A cytoplasmic localization for the otherCstF subunits has not
been described invertebrates, and the S.cerevisiae homologueRNA15
is only nuclear (28). However, we cannotrule out a possible
cytoplasmic function for theCstF-64 or CsF-50 subunits, as gametes
expressdifferent protein isoforms from those present insomatic
cells. Indeed, a second form of CstF-64protein (tau CstF-64),
expressed in meiotic andpost meiotic male germ cells, has been
described(48). Searches for specific CstF-64 or CstF-50isoforms in
oocytes, and their possible functions inthe cytoplasm, have not
been tested, due to a lackof purified antibodies recognizing
thecorresponding Xenopus proteins.
We observed an acceleration of the G2/Mtransition in 77K
antibody injected oocytessubmitted to progesterone. This phenotype
isprobably due to premature Mos protein synthesis,that activates
the MAPK pathway and MPF(Maturation Promoting Factor). However, the
MosmRNA polyadenylation kinetic is not accelerated,suggesting that
impairment of X77K functionallows Mos mRNA recruitment on the
ribosomeindependently of its polyadenylation state. It hasbeen
suggested that the function of cytoplasmicpolyadenylation was to
displace eIF4E from amasking complex (22). One possibility is that
77Kantibody, by interacting with the X77K protein,disrupts the
masking complex, allowing thebinding of eIF4E to eIF4G, and the
recruitment ofthe small ribosomal subunit by the mRNA. Thus,X77K
could play a role in mRNA masking prior tocytoplasmic
polyadenylation.
However, as X77K represses CPE-independent mRNA translation
(figure 7), itprobably has a more general function in
mRNAtranslation inhibition. In that case, X77K would betargeted on
the mRNA by one or several proteinsother than CPEB. CPSF can
recruit X77K on themRNA by itself. Nevertheless, other RNA
bindingproteins, in cooperation with CPSF, could alsoaddress X77K
on the messenger RNA. Xp54, aubiquitous translational repressor
that alsointeracts with CPEB (49), could be one of them.XPumilio
(50,51) and XDAZL (52), othertranslational regulators independent
of CPE
dependent cytoplasmic polyadenylation, are alsopotential
candidates. Thus, CPSF and an X77K-containing complex could
interact cooperatively tostabilize a RNA masking complex, until it
isdisrupted or modified by a signal allowing mRNAtranslation.
CstF-77 protein is localized in discretecytoplasmic foci in
NIH3T3 and XTC cells(Figure 3D). In oocytes, Xp54 and p47,
theXenopus and Spisula homologues of the yeastdecapping activator
Dhh1, are localized in storedmRNP and bind to CPEB (49,53,54).
Moreover, ithas been reported that human CPEB1 expressioninduces
the assembly of stress granules, that inturn, recruit the
processing bodies (PB) (55), andthat eIF4E is localized in PB
(56,57). Thus,proteins initially involved in mRNA translationcan be
localized in PB and, inversely, decappingactivator can be stored in
mRNP in oocytes.Recently, Brengues et al (58) have shown thatmRNA
molecules move from PB to polysomesand vice versa. These data show
that stressgranules, PB and stored mRNP are dynamicallylinked sites
of mRNP remodeling.
The fact that: 1) X77K belongs to acomplex with eIF4E, CPEB and
XGLD2; 2) Theimpairment of 77K function leads to a prematureprotein
synthesis in oocytes; 3) RecombinantX77K represses mRNA translation
in vitro and 4)we observed CstF-77 in NIH3T3 and XTCcytoplasmic
foci, suggest that cytoplasmic CstF-77could have a ubiquitous
function in mRNPremodeling events, for the transition of mRNAfrom
inactive to active translation.
Acknowledgments- We thank the Thierry Lorcalab for reagents,
advice and assistance, Cyril Bernis foregg extracts and Jamal Tazy
for luciferaseoligonucleotides. We are grateful to Simon
Morley,Jean-Marc Lemaître, Nathalie Morin and ClaudePrigent for the
antibodies, Dr N.Sagata for the Mosvector, Dr D. Bentley for the
CstF 77 carboxy terminalpeptide and Elmar Wahle and Uwe Kuehn for
the giftof unpublished anti CPSF-100 antibody. We also thankA.
Bernet, J.M. Donnay, Y. Boublik and G. Herrada fortechnical
assistance. We are grateful to D. Fisher forlooking over the
English. This work was supported bythe Centre National de la
Recherche Scientifique andthe Association pour la Recherche sur le
Cancer(contrat numbers 4469 and 3147 to E.M.). CR issupported by
the Ministère de l'éducation nationale, de
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11
la Recherche et de l'enseignement supérieur and theAssociation
pour la Recherche sur le Cancer.
REFERENCES
1. Zhao, J., Hyman, L., and Moore, C. (1999) Microbiol Mol Biol
Rev 63, 405-4452. Edmonds, M. (2002) Prog Nucleic Acid Res Mol Biol
71, 285-3893. Proudfoot, N. (2004) Curr Opin Cell Biol 16,
272-2784. Wilusz, J., Shenk, T., Takagaki, Y., and Manley, J. L.
(1990) Mol Cell Biol 10, 1244-12485. Murthy, K. G. K., and Manley,
J. L. (1995) Genes Dev. 9, 2672-26836. Gilmartin, G. M., and
Nevins, J. R. (1991) Mol. Cell. Biol. 11, 2432-24387. Bienroth, S.,
Wahle, E., Suter-Crazzolara, C., and Keller, W. (1991) J Biol Chem
266, 19768-
197768. Murthy, K. G., and Manley, J. L. (1992) J Biol Chem 267,
14804-148119. Kaufmann, I., Martin, G., Friedlein, A., Langen, H.,
and Keller, W. (2004) Embo J 23, 616-62610. Takagaki, Y., Manley,
J. L., MacDonald, C. C., Wilusz, J., and Shenk, T. (1990) Genes Dev
4,
2112-212011. Takagaki, Y., and Manley, J. L. (1994) Nature 372,
471-47412. Takagaki, Y., and Manley, J. L. (2000) Mol Cell Biol 20,
1515-152513. Bilger, A., Fox, C. A., Whale, E., and Wickens, M.
(1994) Genes Dev. 8, 1106-111614. Dickson, K. S., Bilger, A.,
Ballantyne, S., and Wickens, M. P. (1999) Mol Cell Biol 19,
5707-
571715. Mendez, R., and Richter, J. D. (2001) Nat Rev Mol Cell
Biol 2, 521-52916. Wang, L., Eckmann, C. R., Kadyk, L. C., Wickens,
M., and Kimble, J. (2002) Nature 419, 312-
316.17. Rouhana, L., Wang, L., Buter, N., Kwak, J. E., Schiltz,
C. A., Gonzalez, T., Kelley, A. E.,
Landry, C. F., and Wickens, M. (2005) Rna 11, 1117-113018.
Barnard, D. C., Ryan, K., Manley, J. L., and Richter, J. D. (2004)
Cell 119, 641-65119. Charlesworth, A., Cox, L. L., and MacNicol, A.
M. (2004) J Biol Chem 279, 17650-1765920. Charlesworth, A., Ridge,
J. A., King, L. A., MacNicol, M. C., and MacNicol, A. M. (2002)
Embo
J 21, 2798-280621. Stebbins-Boaz, B., Cao, Q., de Moor, C. H.,
Mendez, R., and Richter, J. D. (1999) Mol Cell 4,
1017-102722. Cao, Q., and Richter, J. D. (2002) Embo J 21,
3852-3862.23. Minvielle-Sebastia, L., Preker, P. J., and Keller, W.
(1994) Science 266, 1702-170524. Mandart, E., and Parker, R. (1995)
Mol Cell Biol 15, 6979-698625. Mandart, E. (1998) Mol Gen Genet
258, 16-2526. Rouillard, J. M., Brendolise, C., and Lacroute, F.
(2000) Mol Gen Genet 262, 1103-111227. Brendolise, C., Rouillard,
J. M., Dufour, M. E., and Lacroute, F. (2002) Mol Genet
Genomics
267, 515-52528. Bonneaud, N., Minvielle-Sebastia, L., Cullin,
C., and Lacroute, F. (1994) J. Cell. Science. 107,
913-92129. Audibert, A., and Simonelig, M. (1999) Mech Dev 82,
41-5030. Schul, W., Groenhout, B., Koberna, K., Takagaki, Y.,
Jenny, A., Manders, E. M., Raska, I., van
Driel, R., and de Jong, L. (1996) Embo J 15, 2883-289231. Schul,
W., van Der Kraan, I., Matera, A. G., van Driel, R., and de Jong,
L. (1999) Mol Biol Cell
10, 3815-382432. Gall, J. G., Bellini, M., Wu, Z., and Murphy,
C. (1999) Mol Biol Cell 10, 4385-4402
-
12
33. Bleoo, S., Sun, X., Hendzel, M. J., Rowe, J. M., Packer, M.,
and Godbout, R. (2001) Mol BiolCell 12, 3046-3059
34. Gall, J. G. (2003) Nat Rev Mol Cell Biol 4, 975-98035.
Newport, J., and Kirschner, M. (1982) Cell 30, 675-686.36. Hake, L.
E., and Richter, J. D. (1994) Cell 79, 617-62737. Sagata, N.,
Oskarsson, M., Copeland, T., Brumbaugh, J., and Vande Woude, G. F.
(1988) Nature
335, 519-52538. Papin, C., and Smith, J. C. (2000) Dev Biol 217,
166-172.39. Nakamura, A., Sato, K., and Hanyu-Nakamura, K. (2004)
Dev Cell 6, 69-7840. Salles, F. J., and Strickland, S. (1995) PCR
Methods Appl 4, 317-32141. Murray, A. W., and Kirschner, M. W.
(1989) Nature 339, 275-28042. McGrew, L. L., and Richter, J. D.
(1990) EMBO J. 9, 3743-375143. Preker, P. J., and Keller, W. (1998)
Trends Biochem Sci 23, 15-1644. Audibert, A., Juge, F., and
Simonelig, M. (1998) Mech Dev 72, 53-6345. Reverte, C. G., Ahearn,
M. D., and Hake, L. E. (2001) Dev Biol 231, 447-45846. Weiss, E.
A., Gilmartin, G. M., and Nevins, J. R. (1991) EMBO J. 10,
215-21947. Hofmann, I., Schnolzer, M., Kaufmann, I., and Franke, W.
W. (2002) Mol Biol Cell 13, 1665-
167648. Wallace, A. M., Dass, B., Ravnik, S. E., Tonk, V.,
Jenkins, N. A., Gilbert, D. J., Copeland, N. G.,
and MacDonald, C. C. (1999) Proc Natl Acad Sci U S A 96,
6763-676849. Minshall, N., and Standart, N. (2004) Nucleic Acids
Res 32, 1325-133450. Nakahata, S., Katsu, Y., Mita, K., Inoue, K.,
Nagahama, Y., and Yamashita, M. (2001) J Biol
Chem 276, 20945-2095351. Padmanabhan, K., and Richter, J. D.
(2006) Genes Dev 20, 199-20952. Collier, B., Gorgoni, B.,
Loveridge, C., Cooke, H. J., and Gray, N. K. (2005) Embo J 24,
2656-
266653. Ladomery, M., Wade, E., and Sommerville, J. (1997)
Nucleic Acids Res 25, 965-97354. Minshall, N., Thom, G., and
Standart, N. (2001) Rna 7, 1728-174255. Wilczynska, A., Aigueperse,
C., Kress, M., Dautry, F., and Weil, D. (2005) J Cell Sci 118,
981-
99256. Andrei, M. A., Ingelfinger, D., Heintzmann, R., Achsel,
T., Rivera-Pomar, R., and Luhrmann, R.
(2005) Rna 11, 717-72757. Kedersha, N., Stoecklin, G., Ayodele,
M., Yacono, P., Lykke-Andersen, J., Fitzler, M. J.,
Scheuner, D., Kaufman, R. J., Golan, D. E., and Anderson, P.
(2005) J Cell Biol 169, 871-88458. Brengues, M., Teixeira, D., and
Parker, R. (2005) Science 310, 486-489
FIGURE LEGENDS
Fig. 1. A, Amino acid sequence of X77K. The HAT repeats and the
Prolin-rich domain are underlinedwith solid and broken lines
respectively. The bipartite nuclear localization signal (NLS) is in
bold. Aminoacids are numbered on the right. B, Schematic
representation of X77K protein.
Fig. 2. 77K antibody characterization. A, Western blot analysis
of stage VI oocyte extract (lane 1), HA-tagged X77K expressed in
oocytes (lane 2), XTC and NIH (3T3) cell lines (lanes 3 and 4) and
purifiedBaculovirus GST-tagged X77K (lane 5) probed with 77K
antibody. B, Immunoprecipates from 35Smethionine labeled X77K
protein (1ng) with control IgG, or 77K antibody or 77K antibody
pre-incubatedwith 77K antigen were analyzed by autoradiography.
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13
Fig. 3. Endogenous expression of CstF-77 protein. A and B,
Immunoblot analysis of Xenopus oocyteextracts (25 µg by lane) A and
embryo extracts (12,5 µg by lane) B with 77K and β-tubulin
antibodies.The β-tubulin, consistently expressed throughout oocyte
maturation and embryogenesis, serves as aloading control. C,
Oocytes were manually enucleated and pools of nucleus and
cytoplasms werecollected separately. Proteins extracts were
analyzed by Western blotting with 77K and RPA antibodies.The
equivalent of one oocyte (StVI), one nucleus (N) or one cytoplasm
(Cyto) was loaded. TheReplication Protein A (RPA) is exclusively
expressed in nucleus and serves as a control of enucleation.
D,Immunofluorescence of NIH3T3 and XTC cells. Cells were fixed,
stained with 77K antibody and withDAPI and observed by fluorescence
microscopy. A nuclear and cytoplasmic expression of endogenous77K
is observed in both cell lines. E, X77K localization is not
sensitive to leptomycin B. Oocytes weretreated or not over night
with leptomycin B (100 nM final) and were manually enucleated.
Pools ofnucleus and cytoplasms were collected separately. Proteins
extracts were analyzed by Western blottingwith 77K, CPEB, RPA and
β-tubulin antibodies. The equivalent of one oocyte (StVI), one
cytoplasm(Cyt) or 0.5 nucleus (N) was loaded. The RPA and β-tubulin
proteins are respectively expressed innucleus and cytoplasm and
serves as enucleation and loading controls.
Fig. 4. X77K is part of a complex with eIF4E, CPEB, CPSF and
XGLD2. A, StVI oocyte extracts, treatedor not with RNase A as
indicated, were immunoprecipated with 77K, eIF4E, CPEB, Pak5
antibodies orwith control IgG. The immunoprecipitates were analyzed
by Western blotting with 77K, CPEB, eIF4E,CPSF-100 and Pak5
antibodies. B, Oocytes were manually enucleated and cytoplasmic
extracts weretreated with RNase A and immunoprecipated with 77K,
eIF4E, CPEB or control IgG. Theimmunoprecipitates were analyzed by
Western blotting with 77K, CPEB and CPSF-100 antibodies. C,Oocytes
were injected with mRNA encoding HA-tagged XGLD2 and, after 14
hours of HA-XGLD2expression, protein extracts were treated or not
with RNase A and immunoprecipated with differentantibodies as
indicated. The immunoprecipitates were analyzed by Western blotting
with HA, 77K, eIF4Eand CPEB antibodies. The equivalent of 1 oocyte
(A and C) and one cytoplasm (B) was loaded as input.D, Cap column
assay. Protein extracts from StVI oocytes or mature oocytes (MII)
treated or not withRNase A were supplemented with GTP or 7mGTP and
applied to 7mGTP-Sepharose beads (cap column).After beads washes,
the bound proteins were eluted with 7mGTP. All of the eluted
proteins (B: Bound),the equivalent 1/10 of StVI oocytes or mature
oocytes extracts (input StVI or input MII), 1/8 of thesupernatant
of protein extracts after cap column binding (U: Unbound) were
analyzed by Western blottingwith eIF4E, 77K, CPEB, ERK and
β-tubulin antibodies. E, GST pull-down assay. 5 µg of purified
GST-X77K or GST were bound to glutathione beads that were
subsequently incubated with in vitro translatedHA-tagged CPEB (left
panel) or HA-tagged XGLD2 (right panel). The bound proteins were
boiled inSDS sample buffer. The eluates and the equivalent of 1/10
of the in vitro translated proteins (input) wereanalyzed by Western
blotting with HA and GST antibodies.
Fig. 5. X77K is not required for cytoplasmic polyadenylation. A
and B, Polyadenylation assay in eggextracts. A, Egg extracts were
immunodepleted with 77K or CPEB antibodies or with control IgG.
1/10 ofthe egg extracts, immunodepleted or not (left panel) and
1/10 of the immunoprecipitates (right panel)were analyzed by
Western blotting with 77K or CPEB antibodies. B, Egg extracts,
immunodepleted ornot as indicated, were incubated with exogenous
Mos 3'UTR RNA. Total RNA extracted at 0 and 1 hourafter Mos 3'UTR
RNA addition was submitted to Mos polyadenylation analysis (PAT
assay) usingspecific primers. C and D, Polyadenylation assay in
oocytes. C, Exogenous Mos mRNA polyadenylation.Left panel, Oocytes
were first injected with Mos RNA and 30 min later were injected
with 77K, CPEBantibodies or with nonspecific IgG. After 1 hour
incubation, maturation was induced or not withprogesterone. Total
RNA was extracted from pools of 5 oocytes collected at the time of
progesteroneaddition (time point 0h) or when 30% of control oocytes
have undergone GVBD (time point 6h). TotalRNA was submitted to Mos
polyadenylation analysis. Right panel, total RNA, corresponding to
lane 1and 3 of the left panel, were submitted or not to RNaseH
treatment before Mos polyadenylation analysis
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(PAT assay). D, Endogenous mRNA polyadenylation. Oocytes were
injected with 77K, CPEB antibodiesor with nonspecific IgG. After 1
hour incubation , maturation was induced or not with progesterone.
TotalRNA was extracted from pools of 5 oocytes collected at the
time of progesterone addition (time point 0h),when 30% of control
oocytes have undergone GVBD (time point 6h), and two hours later
(time point 8h).Total RNA was submitted to endogenous CyclinB1,
AuroraA and actin type5 polyadenylation analysis(PAT assay) using
specific primers. C and D, Lane 4 corresponds to RNA from a mix of
oocytes injectedwith the different antibodies at time point 0.
Fragment size (M) is indicated on the left in base pair (bp).
Fig. 6. Injection of 77K antibody accelerates oocyte maturation.
A, Oocytes were injected with 77Kantibody or nonspecific IgG or
left uninjected and maturation was induced by progesterone one hour
later.The percentage of GVBD was scored at the indicated times and
graphed. For each treatment, 35 oocyteswere injected. This graph is
representative of six experiments. Insert, 5 hours after 77K
antibodyinjection, oocytes were manually enucleated and pools of
cytoplasms and nucleus were collectedseparately. Proteins extracts
were analyzed by Western blotting with 77K and RPA antibodies.
Theequivalent of one uninjected oocyte (StVI), one cytoplasm (cyto)
and one nucleus (N) from injectedoocytes was loaded. The
Replication Protein A (RPA) serves as a control of enucleation. An
exclusivecytoplasmic localization of 77K IgG is observed. B,
Western blot analysis of Mos and AuroraA synthesis,activated MAPK
(ppERK) and β-tubulin in oocytes collected during an experiment
similar to the onedepicted in A. The β-tubulin serves as a loading
control. C, Oocytes were first injected with Mos 3’UTRRNA and 30
min later were injected with 77K antibody or nonspecific IgG. 1
hour later, maturation wasinduced with progesterone. Total RNA was
extracted from pools of 5 oocytes collected at the indicatedtimes
and was submitted to Mos polyadenylation analysis. Fragment size is
indicated on the left in basepair (bp).
Fig. 7. X77K protein inhibits mRNA in vitro translation in a
dose dependent manner. A, LuciferasemRNA was translated in rabbit
reticulocyte lysates (RRL) in presence of 35S methionine alone
(Control)or with increasing amount of purified GST-77K or GST as
indicated (0.5 µg to 3 µg). 20% of each treatedRRL were analyzed by
SDS-PAGE. Translated luciferase was revealed by autoradiography and
ß-tubulin,as a loading control, was analyzed by Western blotting.
The integrity of luciferase mRNA in presence ofthe recombinant
proteins was verified by RT PCR. The increasing amount of GST-77K
and GST wereanalyzed by Coomassie staining. Two bands are detected
for GST-77K, the lower one corresponding toGST-77K lacking the
carboxy terminal end. B, Quantification of luciferase translation
by Phosphoimagerwhere control is referred as 100%.
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