From the Division of Biological Sciences, Graduate
School of Science, Hokkaido University, Sapporo 060-0810, the
Graduate School of BioSciences, Nara Institute of Science and
Technology, Ikoma 630-0101, and the § Laboratory of
Reproductive Biology and the ¶ Center for Integrative Bioscience,
National Institute for Basic Biology, Okazaki 444-8585, Japan
Received for publication, November 21, 2000, and in revised form, March 5, 2001
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ABSTRACT |
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Translational activation of dormant cyclin B1
mRNA stored in oocytes is a prerequisite for the initiation or
promotion of oocyte maturation in many vertebrates. Using a monoclonal
antibody against the domain highly homologous to that of
Drosophila Pumilio, we have shown for the first time in any
vertebrate that a homolog of Pumilio is expressed in
Xenopus oocytes. This 137-kDa protein binds to the region
including the sequence UGUA at nucleotides 1335-1338 in the
3'-untranslated region of cyclin B1 mRNA, which is close to but
does not overlap the cytoplasmic polyadenylation elements (CPEs).
Physical in vitro association of Xenopus
Pumilio with a Xenopus homolog of Nanos (Xcat-2) was
demonstrated by a protein pull-down assay. The results of
immunoprecipitation experiments showed in vivo interaction
between Xenopus Pumilio and CPE-binding protein (CPEB), a
key regulator of translational repression and activation of mRNAs
stored in oocytes. This evidence provides a new insight into the
mechanism of translational regulation through the 3'-end of mRNA
during oocyte maturation. These results also suggest the generality of
the function of Pumilio as a translational regulator of dormant
mRNAs in both invertebrates and vertebrates.
The final inducer of oocyte maturation is the maturation-promoting
factor (MPF),1 which consists
of Cdc2 and cyclin B. MPF is stored in immature oocytes as an inactive
form (called pre-MPF), although its amount differs from species to
species (1, 2). In Xenopus (as well as fish and mammals
except for mice), the initiation of oocyte maturation requires proteins
(called "initiators") newly synthesized by translational activation
of dormant mRNAs stored in oocytes (masked mRNAs). Mos
functions as an initiator with the aid of mitogen-activated protein
kinase (MAPK) (3-9). The Cdc2 molecule of pre-MPF is phosphorylated on
threonine 14/tyrosine 15 by Myt1 and threonine 161 by
cyclin-dependent kinase-activating kinase. The Mos/MAPK
pathway probably leads to the activation of Cdc25, which
dephosphorylates threonine 14/tyrosine 15 via the polo-like kinase
(10-12), and to the inhibition of Myt1 through the activation of
p90rsk (13, 14).
Enhanced cyclin B synthesis is a ubiquitous event that occurs during
oocyte maturation in all species examined so far, and it is
indispensable for initiating oocyte maturation in fish and amphibians
except Xenopus, in which pre-MPF is absent in immature oocytes (15). The neosynthesized cyclin B is also required for leading
oocyte maturation beyond germinal vesicle breakdown (GVBD) in mammals,
in which pre-MPF in immature oocytes is only sufficient for inducing
GVBD (16, 17). In contrast, it has been shown that newly synthesized
cyclins upon progesterone stimulation are not required for initiating
oocyte maturation in Xenopus (18, 19). Like c-mos
mRNA, however, translation of cyclin B1 mRNA is activated
during Xenopus oocyte maturation (20, 21). Artificial translational activation of endogenous cyclin B1 mRNA brings about GVBD without progesterone and Mos (22, 23). Moreover, the finding that
progesterone induces cyclin B1 synthesis without any activities of Mos,
MAPK, and MPF strongly suggests that progesterone-induced cyclin B1
synthesis is a physiological trigger of pre-MPF activation (24). Taken
together, the results of past studies strongly suggest that the
translational activation of cyclin B1 mRNA universally plays a key
role in initiation of oocyte maturation in lower vertebrates, including
Xenopus (25), and an understanding of its control mechanisms
during oocyte maturation is therefore of particular importance.
Over the past decade, the mystery of how translationally repressed
mRNAs in immature oocytes are released from masking at the
appropriate time during oocyte maturation has been partially solved.
Translational control of masked mRNAs stored in oocytes is mediated
by the U-rich motif U4-6A1-2U, named
cytoplasmic polyadenylation element (CPE), present in their
3'-untranslated region (3'-UTR) (for review see Ref. 26). The CPE
sequence is the target site for CPE-binding protein (CPEB), which
facilitates polyadenylation of the target mRNA during oocyte
maturation (27). In addition to the involvement in translational
activation, CPEB has recently been shown to mediate the masking of
cyclin B1 mRNA (22, 23, 28). Thus, CPEB seems to have a dual
function: to maintain maternal mRNAs, including those of cyclin B1,
in a dormant state in immature oocytes and to activate their
translation via polyadenylation during oocyte maturation. Nevertheless,
many issues still remain to be elucidated. For example, although both c-mos and cyclin B1 mRNAs hold the CPEs in their 3'-UTR,
the following findings indicate that their translational activation is
controlled by different mechanisms: 1) translational activation of
c-mos mRNA precedes that of cyclin B1 mRNA (29); 2)
in contrast to c-mos mRNA, the protein level of cyclin
B1 can increase in the absence of polyadenylation or cap-ribose
methylation of mRNA and is independent of MAPK activity (24, 30,
31); 3) a reporter mRNA carrying the cyclin B1 3'-UTR is
translationally repressed when injected into oocytes, whereas that
carrying the c-mos 3'-UTR is not (23); and 4) the injection
of high concentrations of the CPE induces unmasking of cyclin B1
mRNA but not of c-mos mRNA (22). Therefore,
regulatory elements other than CPEs and CPEB must operate for the
coordinated activation of masked mRNAs to promote oocyte maturation.
In early Drosophila embryos, Pumilio represses translation
of maternal hunchback (hb) mRNA, in
conjunction with Nanos (32-34). The translational repression is due to
the direct binding of Pumilio to two copies of a bipartite sequence,
the nanos response elements (NREs), located in the 3'-UTR of
hb mRNA (35-38). Loss-of-function mutations of
pumilio and nanos induce the premature expression of cyclin B protein in pole cells (39). Deletion from cyclin B mRNA
of cis-elements (named TCEs) resembling the NREs in
hb mRNA results in a phenotype similar to that caused by
pumilio and nanos mutations (40). These results
strongly suggest that Pumilio, in cooperation with Nanos, is involved
in translational repression of cyclin B mRNA through the NRE-like
sequence TCE, although it remains to be elucidated biochemically
whether or not the direct binding of Pumilio to cyclin B mRNA
brings about its translational repression.
Our goal is to elucidate the molecular mechanism of translational
activation of dormant cyclin B1 mRNA upon hormonal stimulation, which is prerequisite for initiating oocyte maturation in fish and
amphibians, probably inclusive of Xenopus (25). NRE-like motifs are found in the 3'-UTR of cyclin B1 mRNA in fish and
amphibians, including goldfish (41), medaka (42),
zebrafish,2
Xenopus (43), and Rana (44). It is therefore
expected that a homolog of Pumilio or its related protein(s) binds
directly to cyclin B1 mRNA to control its translation. To date,
however, there has been no report on protein expression of Pumilio
homologs in vertebrates, although cDNAs probably encoding human
homologs have been isolated in a cDNA project (45, 46). In the
present study, we biochemically characterized a Xenopus
Pumilio protein, and we investigated its involvement in the
translational control of cyclin B1 mRNA. We isolated a cDNA
clone encoding a protein homologous (78% identity) to
Drosophila Pumilio in the domain that defines the Pumilio
family. A monoclonal antibody raised against this domain has revealed
for the first time in any vertebrate that Xenopus oocytes
have a protein homologous to Pumilio (named XPum for Xenopus
Pumilio), which has an apparent molecular mass of 137 kDa and binds to
the NRE of Drosophila hb mRNA, as in the case of the
Drosophila counterpart. Using UV cross-linking assays with
various RNA probes, we have also provided the first biochemical evidence for the sequence-specific direct binding of Pumilio to the
3'-UTR of cyclin B1 mRNA. Moreover, the results of protein pull-down and co-immunoprecipitation assays indicated that XPum associates with a Xenopus homolog of Nanos, Xcat-2 (47), and CPEB.
Animals and Oocytes--
Xenopus laevis
was purchased from a dealer (Hamamatsu Seibutsu Kyozai, Shizuoka,
Japan) and maintained in laboratory aquaria at 20 °C. Full-grown
immature oocytes at stage VI (48) were isolated with forceps or 0.2%
collagenase (Wako, Osaka, Japan) in modified Barth's saline buffered
with Hepes (MBS-H) (49) and induced to mature in vitro by
cultivating in MBS-H supplemented with 10 µg/ml progesterone.
cDNA Cloning and Recombinant Proteins--
A cDNA clone
encoding the conserved region of Pumilio was isolated from ovary RNA by
reverse transcription-polymerase chain reaction (RT-PCR) using two
primers, Pum1 (GAYCARCAYGGITCICGITTYATICA) and Pum4R
(TARTTIGCRTAYTGRTCYTTCATCAT). Pum1 and Pum4R correspond to the highly
conserved amino acid sequences in Pumilio, DQHGSRFIQ and MMKDQYANY,
respectively. The sequence isolated by RT-PCR (named Pum1-4R) was
extended in both 5' and 3' directions by 5' and 3' RACE systems (Life
Technologies, Inc., Rockville, MD) according to the manufacturer's
instructions using the following primers: XlPumGSP1 (GCTGCCAAACTCAAA),
XlPumGSP2 (CATAAGCTGGTAAGCTGCTTG), and XlPumGSP3
(GCTGCTTGAGGATCTCGTTGAACACCA) for 5' RACE, and XlPum3'F1 (CACATGCATCTCGTACGGAAAGGGCCATGC) and XlPum3'F2
(GGGCCATGCTGATAGACGAGGTGTGCACAA) for 3' RACE.
To construct a glutathione S-transferase (GST) fusion
protein, the cDNA Pum1-4R was amplified by PCR with a 5'-primer
(TGGAATTCTAGACCARCAYGGGTCGCGG) and a 3'-primer
(TCGAATTCGCRTAYTGGTCTTTCATCAT). The PCR product was digested with
EcoRI and ligated into EcoRI-cut pGEX-KG (50). GST fusion protein (GST-XPum1-4R) was expressed in Escherichia coli TB1 and purified by SDS-polyacrylamide gel electrophoresis followed by electroelution in Tris-glycine buffer without SDS, as
described previously (41).
A cDNA clone encoding Xcat-2 (47) was isolated from a
To construct a histidine-tagged CPEB, the ORF of Xenopus
CPEB (27) was amplified by PCR using a 5'-primer
(GGAATTCCCATGGCCTTCCCACTGAAAGAT) and a 3'-primer
(ATGCTCGAGGCTGGAGTCACGACTTTTCTG). The PCR product was digested with
EcoRI and XhoI and ligated into the same cloning site of pET21c (Novagen, Madison, WI). Histidine-tagged
Xenopus CPEB (T7-XlCPEB-His) was expressed in E. coli BL21(DE3)pLysS and purified by SDS-polyacrylamide gel
electrophoresis. A histidine-tagged version (T7-Xdazl-His) of
Xenopus Daz-like protein (51) was similarly produced by
inserting the ORF into the EcoRI-site of pET21c (52).
Production of Antibodies--
The recombinant protein
GST-XPum1-4R was injected into mice to produce monoclonal antibodies
according to the procedures described previously (53). To obtain
antibodies specific to XPum1-4R, hybridoma clones were screened with
XPum1-4R cleaved from GST with thrombin (50). One clone (Pum2A5, IgG1
with a Immunoprecipitation and Immunoblotting--
Oocytes were
homogenized with a pestle (Pellet Pestle, Kontes, Vineland, NJ) in 1 µl/oocyte of ice-cold extraction buffer (EB; 20 mM Hepes,
pH 7.5, 100 mM Affinity Chromatography and Northwestern Blotting--
The ovary
(100 mg) was homogenized in 250 µl of buffer A (10 mM
Tris-HCl, pH 7.5, 83 mM KCl, 17 mM NaCl).
Extracts were loaded onto a poly(U)-Sepharose column (Amersham
Pharmacia Biotech, Tokyo, Japan). After washing with buffer A, bound
proteins were step-eluted with buffer A containing 100-400
mM KCl, precipitated with 15% trichloroacetic acid, washed
with acetone, and analyzed by immunoblotting with anti-XPum antibody.
Oligo(dT)-cellulose chromatography and Northwestern blotting were
performed as described previously (55).
For RNA-affinity chromatography, RNA (500 µg) was coupled with 1 ml
of cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech).
The beads suspended in binding buffer (10 mM Hepes, pH 7.2, 100 mM KCl, 3 mM MgCl2, 5%
glycerol, 1 mM dithiothreitol) were incubated with 500 µl
of oocyte extracts for 1 h at 4 °C. After washing the beads
with binding buffer, bound proteins were step-eluted with 200-1000
mM KCl and analyzed by anti-XPum immunoblotting.
Protein Pull-down Assay--
For GST pull-down, ~5 µg of
gel-purified GST-Xcat-2 or GST as a control was incubated with 20 µl
of glutathione-Sepharose (Amersham Pharmacia Biotech) for 30 min on
ice. After removal of unbound proteins, 20 µl of oocyte extracts was
mixed with the beads in a binding buffer (50 mM Tris-HCl,
pH 7.5, 100 mM KCl, 1 mM MgCl2, 80 mM Plasmid Construction and RNA Synthesis--
A cDNA encoding
the full-length Xenopus cyclin B1 (43) in pBluescript II
SK(+) was utilized as the original template for producing various
probes used in this study (this cDNA being named B
For a UV cross-linking assay and an injection experiment, a cDNA
fragment including the entire 3'-UTR of cyclin B1 (nucleotides 1245-1395, named B3') was created by PCR using B
For deletion mutants of B3' (see Figs. 4B and 5A
for structural details and nomenclature of probes), PCR products
obtained with the following primer sets were ligated into pGEM-T Easy
Vector (Promega, Madison, WI): probe 1-118, primer 1 and primer 3 (CCGGATCCACATTAAAAACACAATACACTAT); 19-118, primer 4 (GGAAATGGCCCGCCCACTC) and primer 3; 53-118, primer 5 (TGGCATTCCAATTGTGTA) and primer 3; 76-118, primer 6 (GGCACCATGTGCTTCTGT) and primer 3; 95-151, primer 7 (AATAGTGTATTGTGTTTT) and T3 primer (ATTAACCCTCACTAAAG); 105-151,
primer 8 (TGTGTTTTTAATGTTTTACTGG) and T3 primer; 119-151, primer 9 (TTTACTGGTTTTAATAAAGCTC) and T3 primer.
Substitution mutants were produced by PCR with the following primers
(mutation sites are underlined; see Figs. 4B and
5A for structural details): probe sub13-18
(CAGATGTGCACCAGGAAA), sub63-68 (TGGCATTCCATCATCTTATTGTTGGC), sub70-74
(CCAATTGTGTTCATCTGGCACCATG), sub82-86
(GGCACCCCCCCCTTCTGTAAATAGTGT), sub91-94
(GGCACCATGTGCTTCGTGCAATAGTGT), sub103-108
(ATAGTGTTCATCTTTTTTAATGTGGATCCGG), mut105-108
(ACGCTTTTTAATGTTTTACTGG), mut109-113
(TGTGGGGGGAATGTTTTACTGGTT), and mut114-119
(TGTGTTTTTGAACGCTTACTGGTTT). Using a cDNA of the NRE
sequence in Drosophila hb mRNA
(ATTATTTTGTTGTCGAAAATTGTACATAAGCC), we also
produced a deletion mutant (named Mut1) bearing the sequence ATTATTTTGTTGTCGA and a substitution mutant (named Mut2)
bearing the sequence
ATTATTTTGTTGTCGAAAATacgcCATctGCC (The NRE
consensus bases are underlined, and the mutated bases are
written in lowercase letters; cf., Fig.
3A).
For in vitro transcription, the DNA templates were
linearized with BamHI. Radiolabeled RNA probes were prepared
using an RNA transcription kit (Stratagene, La Jolla, CA), with 25 µCi of [32P]UTP (400 Ci/mmol; Amersham Pharmacia
Biotech). Unlabeled transcripts were synthesized in the presence of 400 µM UTP. Large-scale transcription reactions were
performed using a MEGAscript T7 kit (Ambion, Austin, TX). All in
vitro synthesized RNAs were phenol/chloroform-extracted, ethanol-precipitated, and dissolved in distilled water.
UV Cross-linking--
UV cross-linking assays were carried out
essentially as described by Walker et al. (56). Briefly,
reactions were performed in 96-well microtiter plates (Falcon 3911, Becton Dickinson, Franklin Lakes, NJ). In each 10-µl reaction, probe
RNA (60-100,000 cpm) was incubated with 2 µl of extract for 15 min
at room temperature and irradiated in a UV cross-linker (XL-1000,
Funakoshi, Tokyo, Japan) at an energy setting of 860 mJ/cm2. A competition assay was performed by preincubation
of the reactions with a 25-, 50-, or 100-fold molar excess of unlabeled
RNA for 5 min prior to the addition of radiolabeled RNA.
In Vitro Selection--
RNA sequences required for interaction
with Xenopus Pumilio were determined by an in
vitro selection assay using RNAs having a random sequence of 25 nucleotides and a baculovirus-expressed Xenopus Pumilio
protein fused with GST (GST-XPum-Bac). GST-XPum-Bac was produced as
follows. A DNA fragment including the ORF of GST was created by PCR
using pGEX-KG (50) as a template with a 5'-primer (GAAGATCTGTCATGTCCCCTATACTAGGTTA) and a 3'-primer
(TCCGGGAGCTGCATGTGTCAG). The PCR product was digested with
BglII and HindIII and ligated into
BamHI/HindIII-cut pFastBac1 (Life Technologies,
Inc.) to generate pFastBac-GST. A 2.0-kb EcoRI fragment of
the XPum cDNA (see "Results") was inserted into the same
cloning site of pFastBac-GST. The plasmid DNA was transformed into
DH10Bac cells (Life Technologies, Inc.). After transposition,
high-molecular-weight DNA was isolated and transfected into
Spodoptera frugiperda (Sf21) cells using Cellfectin
(Life Technologies, Inc.) according to the manufacturer's instructions. The cells were harvested 4 days after transfection, and
GST fusion protein was purified on glutathione-Sepharose (Amersham Pharmacia Biotech) as described above.
In vitro selection assays were carried out according to
Sakashita et al. (57). Briefly, the starting RNA source was
synthesized from a mixture of DNA templates containing a T7 promoter
and a random sequence of 25 nucleotides, using a MEGAscript T7 kit
(Ambion). A total of five rounds of selection and amplification were
performed. The final cDNA products were subcloned into pBluescript
II SK(+) and sequenced.
Oocyte Injection--
Manually isolated stage VI oocytes were
injected with 70 ng of competitor RNAs (in 40 nl of distilled water).
After incubation of the injected oocytes in MBS-H for 24 h at
18 °C, 10 oocytes were homogenized, subjected to Suc1-precipitation
to partially purify the Cdc2-cyclin B complex (58), and analyzed by
immunoblotting with a monoclonal antibody that recognizes amphibian
cyclin B1 (Bufo 2F5) (59). The remaining oocytes were boiled, cut with a razor, and scored for the percentage of GVBD.
Presence of a Pumilio Homolog in Xenopus
Oocytes--
Drosophila Pumilio and a Caenorhabditis
elegans Pumilio homolog, FBF, are members of the Pumilio-homology
domain (Pum-HD) family, also known as Puf (for Pumilio and
FBF) family (60, 61). The sequence in the C-terminal region
of the Pum-HD family is highly conserved in many species, including
human homologs (DDBJ/EMBL/GenBankTM accession numbers KIAA0099
and KIAA0235) deduced from their cDNAs (45, 46). We
therefore designed oligonucleotide primers corresponding to this region
to isolate a cDNA clone encoding Xenopus Pumilio by
RT-PCR. We initially obtained an 800-bp cDNA fragment (Pum1-4R)
that encodes a nucleotide sequence highly homologous to
Drosophila Pumilio. We then extended this fragment by 5' and 3' RACE and finally obtained a 2.0-kb sequence (DDBJ/EMBL/GenBankTM accession number AB045628) containing a domain equivalent to those of
Drosophila Pumilio (78% identity) and the human homolog KIAA0099 (95% identity) (Fig. 1). This
domain is known as the diagnostic hallmark of the Pum-HD family and is
defined by the presence of eight copies of an imperfect repeat
sequence, comprising a specific RNA-binding domain (60-62) (Fig. 1).
Based on the existence of this feature in the obtained clone (Fig. 1),
we identify it as a Xenopus homolog of Pumilio. Although the
known Pum-HD proteins share few sequences outside the Pum-HD (60),
considerable sequence conservation is seen between the
Xenopus and the human homologs (69% and 81% identity in
the N-terminal and the C-terminal regions, respectively). This finding
and biochemical characterization of the protein encoded by this
cDNA (see later sections) also confirmed that the obtained cDNA
encodes a Xenopus Pumilio homolog. From the lengths of those
found in other species, the obtained cDNA apparently lacked the 5'
sequence. However, we did not persist with isolation of the full-length
cDNA, because the obtained clone was long enough to judge it as a
Xenopus Pumilio homolog and to produce recombinant proteins
necessary for raising specific antibodies and characterizing the
encoded protein.
Using a monoclonal antibody raised against the recombinant protein
encoded by the cDNA Pum1-4R, we investigated the existence of
Pumilio homolog proteins in Xenopus oocytes. A single
137-kDa protein, the size of which is comparable to those of
Drosophila Pumilio (157 kDa) and the human homolog KIAA0099
(>128 kDa), was recognized specifically by the antibody in both
immature and mature oocyte extracts (Fig.
2A). The 137-kDa protein was
also detected by immunoprecipitation (Fig. 2B). Because this
protein exhibits biochemical characteristics similar to those of
Drosophila Pumilio, as described below, we refer to this as
Xenopus Pumilio (XPum). No apparent differences in protein
content or electrophoretic mobility of XPum were detected between
immature and mature oocytes (Fig. 2B). XPum was present
throughout oogenesis, although its apparent concentration decreased
during the accumulation of yolk (Fig. 2C).
XPum as a Cyclin B1 mRNA-binding Protein--
To test the
capability of endogenous XPum to bind to mRNAs in oocytes, we first
employed oligo(dT)-cellulose and poly(U)-Sepharose affinity
chromatography. The anti-XPum immunoblots clearly showed that XPum was
enriched in 10-25% of formamide eluates from an oligo(dT)-cellulose
column (Fig. 2D) and in 400 mM KCl eluate from a
poly(U)-Sepharose column (Fig. 2E), suggesting that this protein is physically associated with mRNAs in oocytes. We then analyzed the interaction between XPum and cyclin B1 mRNA by cyclin B1 mRNA-affinity chromatography. XPum was bound to the resin
coupled with cyclin B1 mRNA but not to the control resin without
mRNA (Fig. 2F). Similar experiments using resins coupled
with either a 5'- or a 3'-fragment of cyclin B1 mRNA showed
that XPum had an affinity for the 3'-fragment but not for the
5'-fragment (Fig. 2G).
The interaction between XPum and cyclin B1 mRNA was finally
confirmed by Northwestern blotting. Oocyte extracts eluted from an
oligo(dT)-cellulose column were probed with the 3'-fragment of cyclin
B1 mRNA. The blot showed that, in addition to abundant 56-kDa
Xenopus Y-box protein (FRGY2) that can bind to mRNAs in a sequence-nonspecific manner (63, 64), a 137-kDa protein recognized
cyclin B1 mRNA (Fig. 2H, lane 1). Anti-XPum
immunostaining of the identical blot demonstrated that the 137-kDa
cyclin B1 mRNA-recognizing protein is XPum itself (Fig.
2H, lane 2).
Identification of the XPum-binding Site in Cyclin B1
mRNA--
To determine the cyclin B1 mRNA sequence required
for the binding of XPum, we carried out UV cross-linking assays with
various RNA probes. First, we confirmed that XPum can bind to the
wild-type NRE, but not to two mutant NREs, of hb mRNA
(Fig. 3, A and B), similar to Drosophila Pumilio (38). The results of UV
cross-linking assays with the 3' and 5' sequences of cyclin B1 mRNA
also confirmed the results of mRNA-affinity chromatography showing
that XPum binds to the 3' region of cyclin B1 mRNA but not to the
5' region (Fig. 3B). The binding of XPum to the labeled B3'
probe was inhibited by unlabeled B3' or wild-type NRE but not by mutant
NRE (Fig. 3C), indicating that the XPum recognition sequence
in the cyclin B1 3'-UTR resembles that of NRE.
To map the XPum-binding site in cyclin B1 mRNA more precisely,
various deletion and substitution mutants of B3' RNA were used for UV
cross-linking (Fig. 4). Among five
NRE-like elements present in B3' RNA (Fig. 4A), neither
deletion nor substitution of the three elements in the 5'-half affected
the binding of XPum to the probes (Fig. 4, B and
C). These results indicated that the XPum-binding site
resides in a 43-nucleotide stretch from positions 76 to 118, harboring
two NRE-like elements; the first element consists of the
tetranucleotide UGUA located at positions 91-94 and the second one is
the sequence AUUGU at positions 103-108 (Fig. 4A). Mutation
of the second element, AUUGU, to UCAUC (sub103-108) had no
effect on the binding, as in the case of mutation in an NRE-unrelated
sequence in the same area (sub82-86). However, mutation of
the first element, UGUA, to GUGC (sub91-94) completely
abolished the binding of XPum (Fig. 4C). It is therefore
likely that the UGUA sequence at positions 91-94 (nucleotides
1335-1338) is essential for the XPum binding. The finding that
deletion or mutation of the UGUA sequence of hb mRNA
prevented the binding of XPum (Fig. 3, A and B)
also supports this notion.
As a further test of the sequence requirement for XPum-RNA interaction,
we carried out in vitro selection experiments using an RNA
population with 25 nucleotides of a randomized sequence and GST-fused
XPum produced in baculovirus-infected insect cells. After a total of
five cycles of selection, the sequences recognized by XPum were
determined (Table I). The highly
conserved sequence is represented by the tetranucleotide UGUA.
Moreover, in 11 of 15 UGUA sites, the consensus sequence is followed by
an A residue at the 3'-end, which closely matches the sequence at
positions 1335-1339 of cyclin B1 mRNA (Fig. 4A). Thus,
it is most likely that the UGUA(A) sequence at positions 1335-1338
(1339) is a component important for the XPum binding.
Sequence Elements Responsible for Masking Cyclin B1
mRNA--
Endogenous cyclin B1 mRNA can be translationally
activated by injection into oocytes of a high concentration of the
cyclin B1 3'-UTR that includes the CPEs, probably due to displacement of putative masking factors bound to the CPEs (22, 23). Because the
cyclin B1 3'-UTR comprises the XPum binding site, as well as the CPEs,
we investigated whether injection of excess amounts of the XPum-binding
sequence can activate endogenous cyclin B1 mRNA (Fig.
5). Consistent with the previous finding
(22), when B3' RNA was injected into Xenopus oocytes, cyclin
B1 protein accumulated in the absence of progesterone and more than
75% of the oocytes underwent GVBD (Fig. 5B). In contrast,
76-118 RNA, which can bind to XPum (Fig. 5C), stimulated
neither cyclin B1 synthesis nor GVBD (Fig. 5, B and
D), indicating that the XPum-binding sequence itself is
unable to titrate masking proteins required for translational repression of cyclin B1 mRNA.
To identify the cis-element responsible for masking cyclin
B1 mRNA, we continued similar injection experiments using several deletion and substitution mutants (Fig. 5A). The shortest
RNA that retained the GVBD-inducing ability equivalent to B3' comprised the 3'-terminal one-third of B3' (105-151 RNA, Fig. 5D).
This RNA harbors two CPEs, named CPE1 at nucleotides 1353-1360 and CPE2 at nucleotides 1371-1377 (Fig. 5A) (21). Injection of
119-151 RNA, which lacks CPE1 but retains CPE2, did not induce GVBD
(Fig. 5, A and D), suggesting that CPE1, but not
CPE2, is involved in masking. Experiments with substituted mutant RNAs
(mut105-108 carrying a mutation in four nucleotides
upstream of CPE1, mut109-113 with a mutation in the 5'-half
of CPE1, and mut114-119 with a mutation in the 3'-half of
CPE1) showed that any mutations near or in CPE1 hampered the
GVBD-inducing activity of 105-151 RNA (Fig. 5, A and
D), indicating the necessity of CPE1 and its neighboring sequences for masking cyclin B1 mRNA. The necessity of the
sequences near CPE1 was also indicated by the finding that 76-118 RNA,
which contains complete CPE1 but only an additional two nucleotides on
its 3'-side, had little activity for unmasking cyclin B1 mRNA and
inducing GVBD (Fig. 5, A, B, and
D).
XPum-interacting Proteins--
Given that Pumilio regulates
translation of mRNAs with the aid of Nanos in Drosophila
(37, 39), we analyzed the interaction between XPum and Xcat-2, a
Xenopus homolog of Nanos (47), by protein pull-down assays
using recombinant Xcat-2 and endogenous XPum. XPum bound to GST-Xcat-2
but not to GST as a control (Fig. 6A), indicating that XPum
interacts with Xcat-2 at least in vitro.
The injection experiments reported above (Fig. 5) strongly suggested
that CPEB plays an essential role in masking and unmasking cyclin B1
mRNA. We therefore investigated the interaction between XPum and
CPEB by protein pull-down assays. It was found that XPum bound to the
beads prebound to T7-XlCPEB-His but not to those prebound to
T7-Xdazl-His as a control (Fig. 6B), indicating the ability
of the recombinant CPEB to associate with XPum. To know their
interaction in vivo, we performed co-immunoprecipitation assays with a monoclonal antibody raised against Xenopus
CPEB (XlCPEB12), which recognizes a 62-kDa CPEB (and an additional 64-kDa phosphorylated CPEB in mature oocytes) with high specificity both by immunoprecipitation and immunoblotting (Fig. 6C).
XPum, as well as CPEB, was detected in anti-CPEB immunoprecipitates from immature and mature oocyte extracts but not in anti-goldfish cyclin B1 (B63) (41) immunoprecipitates as a control (Fig.
6C). Treatment of oocyte extracts with SDS (0.25% at final
concentration) prior to immunoprecipitation caused the loss of XPum,
but not CPEB, in anti-CPEB immunoprecipitates (Fig. 6C),
confirming that XPum was not precipitated directly with the anti-CPEB
antibody but was precipitated through an interaction with CPEB.
Treatment of extracts with RNase A (0.2 mg/ml, 5 min, at room
temperature) did not prevent the precipitation of XPum by the anti-CPEB
antibody (Fig. 6D), ruling out the possibility that
co-precipitation of XPum and CPEB was mediated by cyclin B1 mRNA to
which they simultaneously bind. Anti-CPEB immunoblotting of anti-XPum
immunoprecipitates gave essentially the same results (data not shown).
These results provide evidence of physical interaction between XPum and
CPEB in vivo for the first time in any species.
We have focused on Pumilio as one of the possible translational
regulators of cyclin B1, because the 3'-UTR of cyclin B1 mRNA has
sequences resembling those required for translational repression by
Pumilio in Drosophila. We report here the following main
findings: 1) A 137-kDa Xenopus homolog of Pumilio (XPum)
exists in oocytes and eggs, the first demonstration of Pumilio protein
expression in vertebrates; 2) XPum binds to the cyclin B1 3'-UTR
containing the UGUA at nucleotides 1335-1338; 3) The sequence
responsible for masking cyclin B1 mRNA, as determined by
translational activation of endogenous cyclin B1 mRNA by injection
of various cis-elements, is located around CPE1 (nucleotides
1353-1360), close to but different from the XPum-binding site; 4) XPum
physically associates with a Xenopus Nanos homolog, Xcat-2,
at least in vitro and with CPEB in vivo.
The amino acid sequence alignment of Pumilio homologs exhibits a high
degree of conservation in the Pum-HD (Fig. 1), which is known to have a
dual function, not only recognizing the NREs but also interacting with
some component of the translational machinery (36, 37, 65). The
sequence similarity is suggestive of a common function of Pumilio.
Indeed, our results showed that XPum can bind to the NRE of
hb mRNA, the sequence recognized by the
Drosophila counterpart. Moreover, the present UV
cross-linking assays using endogenous Pumilio protein and various
mutant probes indicated that the Pumilio-binding site contains the
sequence UGUA, which is identical to the sequence determined by
gel-shift experiments with recombinant Pumilio proteins from human and
Drosophila (60). Recent studies have also demonstrated that
yeast Pumilio-related proteins play a role in negative control of
translation by recognizing a cis-element similar to that for
Drosophila, Xenopus, and human Pumilio homologs
(66, 67). Taken together, the results strongly suggest that the
function of Pumilio as a translational regulator and its
cis-element of the target RNAs are widely conserved in evolution.
Drosophila Pumilio represses translation of maternal
hb mRNA by direct binding to the NRE in its 3'-UTR (36,
38). The results of genetic analyses also suggest that Pumilio, in
cooperation with Nanos, controls translation of cyclin B mRNA in
the migrating pole cells (39), although there has been no direct
biochemical evidence for the binding of Pumilio to cyclin B mRNA in
any species to date. The results of the present study have provided the
first biochemical evidence of a Pum-binding site in cyclin B1 mRNA
as well as evidence of the binding of endogenous Pumilio to cyclin B1
mRNA in a sequence-specific manner. These findings led us to verify
the possibility that, as in Drosophila, XPum plays an
essential role in translational control of cyclin B1 mRNA during
oocyte maturation. To this end, we injected various
cis-elements into oocytes in the expectation that, if the
injected sequence is responsible for masking cyclin B1 mRNA, it
will titrate the masking proteins and thereby induce translational
activation of endogenous cyclin B1 as already demonstrated (22, 23). We
confirmed that the injection of large amounts of cyclin B1 3'-UTR into
Xenopus oocytes induced cyclin B1 synthesis and GVBD. We
also demonstrated that the cis-element necessary for
translational repression of cyclin B1 mRNA exists in the
neighborhood of CPE1 (nucleotides 1353-1360) but not in CPE2
(nucleotides 1371-1377). Injection of the XPum-binding sequence,
however, induced neither translational activation of endogenous cyclin
B1 mRNA nor GVBD (Fig. 5). These results seem to imply that the
binding of Pumilio to cyclin B1 mRNA is not responsible for masking
cyclin B1 mRNA. Nevertheless, this suggestion needs to be proved by
experimental approaches other than the injection of
cis-elements, because it has been reported that injection of large amounts of CPE-containing RNA into mouse oocytes does not induce
translational activation of cyclin B1 mRNA despite the involvement
of CPEB in both the repression and the stimulation of cyclin B1
mRNA in this species, as in the case of Xenopus (28). In
cooperation with CPEB as a major control element, XPum might contribute
as a fine tuner of cyclin B1 mRNA translation. This notion is also
supported by the present finding that XPum physically interacts with
CPEB both in vivo and in vitro (Fig. 6,
B-D).
The function of Drosophila Pumilio in control of the
translation of mRNAs is dependent on Nanos (68, 69). FBF, a
C. elegans Pumilio-related protein, also controls the
sperm/oocyte switch in a hermaphrodite germ line by regulating the
translation of fem-3 mRNA with interaction with one of
the C. elegans Nanos homologs (61, 65). XPum may likewise
collaborate with one or more partners to regulate the translation of
specific maternal mRNAs (for review see Ref. 70). The most likely
candidate for the XPum partner is a Xenopus homolog of
Nanos, Xcat-2 protein (71, 72). In fact, we have provided evidence of
association between XPum and Xcat-2, at least in vitro (Fig.
6A). However, Xcat-2 protein is reported to be absent in
oocytes and eggs despite the presence of its mRNA. The protein
appears in accordance with the movement of germ plasm during early
embryogenesis (73). Consistent with this, Xcat-2 is undetectable in
anti-XPum immunoprecipitates from oocyte
extracts.3 Although we cannot
exclude the possibility that one or more Nanos homologs other than
Xcat-2 collaborate with XPum in oocytes, it remains a mystery whether
XPum acts together with a Nanos homolog to govern the translation of
mRNAs in oocytes.
The actual biological roles of XPum are completely unknown at present,
but we speculate that XPum plays an important role in translational
control of cyclin B1 mRNA like in Drosophila (39, 40).
CPEB directly binds to maskin, a protein that can also bind directly to
the cap-binding translation initiation factor elF-4E, which leads to
translational repression. The dissociation of maskin from elF-4E allows
elF-4G to bind to elF-4E, which brings elF-3 and the 40 S ribosomal
subunit to the mRNA to initiate translation via cap-ribose
methylation (30, 74, 75). Recent studies have also shown that a
progesterone-induced early phosphorylation of CPEB at serine 174 is
catalyzed by Eg2 (76) and that this phosphorylation recruits cleavage
and polyadenylation specificity factor into an active cytoplasmic
polyadenylation complex (77). Thus, CPEB plays a key role in both
translational repression and activation of mRNAs stored in oocytes
(for review see Ref. 78). We demonstrated in this study that XPum is
physically associated with CPEB in oocytes. In cooperation with CPEB,
XPum may control the CPEB/maskin-mediated translational masking and
unmasking to assure the highly coordinated successive translational
activation of masked mRNAs during oocyte maturation. Further
studies are required to understand the biological significance of the
interactions among XPum, CPEB, and cyclin B1 mRNA, as well as to
elucidate the functions of XPum in oocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP II
library constructed from Xenopus eggs. The open reading
frame (ORF) was amplified by a XNos2-F primer
(CGGAATTCTAATGGATGGCGGTCTCTGC) and a T7 primer (TAATACGACTCACTATAGGG).
The PCR product was digested with EcoRI and XhoI,
and ligated into EcoRI/XhoI-cut pGEX-KG to produce a GST fusion protein (GST-Xcat-2). E. coli-produced
GST-Xcat-2 or GST was gel-purified as described above.
light chain) was selected to detect XPum by
immunoprecipitation and immunoblotting. Monoclonal antibodies against
Xenopus CPEB were raised against T7-XlCPEB-His (54). One
clone (XlCPEB12, IgG1 with a
light chain) was used in this study.
-glycerophosphate, 15 mM
MgCl2, 5 mM EGTA, 1 mM
dithiothreitol, 100 µM
(p-amidinophenyl)methanesulfonyl fluoride, 3 µg/ml
leupeptin) and centrifuged at 15,000 × g for 10 min at
4 °C. The extracts were subjected to immunoprecipitation followed by
immunoblotting as described previously (53).
-glycerophosphate, 0.2% sodium azide, 0.1% Nonidet
P-40) and incubated for 12 h at 4 °C. Following washing six
times with binding buffer, the beads were boiled in SDS sample buffer,
and solubilized proteins were analyzed by anti-XPum immunoblotting. For
histidine pull-down, ~1 µg of Xenopus CPEB
(T7-XlCPEB-His) or Daz-like protein (T7-Xdazl-His) as a control was
bound to 10 µl of nickel-nitrilotriacetic acid agarose (Qiagen,
Tokyo, Japan). After washing with EB, the beads were incubated with 100 µl of oocyte extracts in EB for 12 h at 4 °C and analyzed
with anti-XPum immunoblotting, as in the case of GST pull-down assay.
0). For a
Northwestern assay and RNA-affinity chromatography, a 3' part of cyclin
B1 (nucleotides 1137-1395, named 3'-cyclin B) was constructed as
follows: B
0 was digested with EcoRI and NdeI,
and the larger fragment was isolated, filled in with a Klenow fragment
of DNA polymerase I, and self-ligated. An RNA probe for a 5' part of
Xenopus cyclin B1 (nucleotides 1-206, named 5'-cyclin B)
was produced by linearizing B
0 in pGEM1 (a gift from Dr. Hideki Kobayashi, Kyushu University) with StyI and transcribing
with T7 RNA polymerase as described below.
0 as a template with primer 1 (CGGAATTCGAGGTCAGATGTTGTTGTGG) and primer 2 (CGGAATTCCATGTTAAAATGAGCTTTAT). The PCR product was digested with
EcoRI and ligated into EcoRI-cut pBluescript II
SK(+). A plasmid with the T7-sense oriented insert was further used as
a PCR template to produce various deletion and substitution mutants.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence alignment of Pumilio homologs.
Eight repetitive motifs (1-8) with flanking residues at
both the N- and C-terminal ends of Pumilio homologs from
Drosophila (GenBankTM accession X62589) (79),
Xenopus (accession AB045628) (this study), and human
(accession D43951) (46) are shown. Identical residues are marked
(#).
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Fig. 2.
Biochemical characterization of XPum.
A and B, presence of XPum in immature and mature
oocytes. Extracts from ~1/8 immature stage VI oocytes (lanes
1 and 3) and progesterone-matured oocytes (lanes
2 and 4) were immunoblotted with (lanes 1 and 2) or without (lanes 3 and 4)
anti-XPum antibody (A). Bands found in lanes 3 and 4 are nonspecific signals due to the secondary antibody.
Extracts from 25 immature stage VI oocytes (lanes 1 and
3) and progesterone-matured oocytes (lanes 2 and
4) were immunoprecipitated with anti-XPum antibody and then
immunoblotted with (lanes 1 and 2) or without
(lanes 3 and 4) the same antibody (B).
Anti-XPum immunoprecipitate without oocyte extract (lane 5)
shows the positions of immunoglobulins (Ig). C,
XPum expression during oogenesis. Extracts from oocytes at stages I-VI
and progesterone-matured oocytes (M) were applied to each
lane (3.7 µg of protein/lane, equivalent to 1.7 oocytes at stage VI)
and immunoblotted with anti-XPum antibody. Nonspecific bands that were
also detected in the control blots without the primary antibody are
indicated. D and E, association of XPum with
mRNAs. Proteins retained on an oligo(dT)-cellulose column
(D) or poly(U)-Sepharose column (E) were
step-eluted with 10%, 25%, and 60% formamide or 100, 200, and 400 mM KCl, respectively. The eluted proteins were
immunoblotted with anti-XPum antibody. F and G,
binding of XPum to cyclin B1 mRNA. RNA-affinity chromatography with
full-length cyclin B1 mRNA (F). Oocyte extracts were
incubated with Sepharose coupled with (+) or without ( ) cyclin B1
mRNA. Bound proteins were eluted stepwise with 250, 500, and 1000 mM KCl, and immunoblotted with anti-XPum antibody. Note the
reduction in XPum protein content in the supernatant of
mRNA-coupled beads (Sup, +). RNA-affinity chromatography
with a 5'-terminal fragment (nucleotides 1-206, 5' cyclin
B) or a 3'-terminal fragment (nucleotides 1137-1395, 3'
cyclin B) of cyclin B1 mRNA (G). Eluates were
assayed as in F. H, Northwestern blotting of
oocyte extracts fractionated by oligo(dT)-cellulose chromatography
(25% formamide-eluate) using a 3'-terminal fragment of cyclin B1
mRNA (lane 1). The same membrane was probed with
anti-XPum antibody (lane 2). The cyclin B1 mRNA-bound
137-kDa protein (arrow in lane 1) is identical to
the anti-XPum reactive 137-kDa protein (XPum). Abundant 56-kDa Y-box
proteins (FRGY2) were also detected on the Northwestern blot.
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Fig. 3.
Binding of XPum to the NRE of
Drosophila hb mRNA. A, sequence of
the wild-type hb NRE (WT) and two mutant NREs
(Mut1 and Mut2). Lowercase letters
designate nucleotides that were changed in Mut2. Sequences conserved
among the NREs of hb mRNAs are underlined
(35). B, UV cross-linking assay with the wild-type
hb NRE (WT), two mutant hb NREs
(Mut1 and Mut2), cyclin B1 5' sequence
(B5'), and cyclin B1 3' sequence (B3').
Arrows indicate XPum. C, RNA competition assay.
Oocyte extracts were incubated with radiolabeled B3' RNA in the
presence of a 25-, 50-, or 100-fold molar excess of unlabeled
competitor RNAs as indicated.
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Fig. 4.
Identification of the XPum-binding
sequence. A, the sequence of B3' RNA. Nucleotides are
numbered from the base 1245 of cyclin B1 cDNA sequence
to the last nucleotide before the poly(A) tract. The termination codon
UGA is marked by double underlining and the NRE-like
elements (cf., Fig. 3A) are underlined.
B, schematic diagrams of RNA probes and summary of the ability of
RNA probes to bind XPum. Potential XPum-binding sites (NRE-like
sequences) in B3' RNA are shown by arrowheads. RNA probes
are indicated by the nucleotide numbers (The first
nucleotide of B3' is designated as number 1 and thus 1-151
RNA is identical to B3'), and their relative positions are shown as
open bars. Substituted mutant regions are indicated by
solid bars. The XPum-binding ability is shown as similar (+)
or significantly reduced ( ) relative to B3'. C, UV
cross-linking assays (summarized in B) with the RNA probes
indicated. The arrow shows XPum.
In vitro selected RNA sequences by XPum
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Fig. 5.
Identification of the masking element in the
3'-UTR of cyclin B1 mRNA. Oocytes were injected with various
RNAs containing the XPum-binding site and/or the CPEs to determine the
ability of each RNA to induce translational activation of cyclin B1
mRNA and GVBD. A, partial sequence of B3', showing the
XPum-binding site in a black box, two CPEs (CPE1 and CPE2)
in white boxes, and polyadenylation signal (AAUAAA) in
boldface. Schematic diagrams of RNA probes and summary of
the ability of RNA probes to bind to XPum and to induce GVBD are also
shown. The XPum-binding site (XPB, shaded box),
CPE1 and CPE2 (open boxes), and substituted mutant regions
(solid bars) in the probes are indicated. The XPum-binding
ability is shown as similar (+) or significantly reduced ( ) relative
to B3'. Percentage of GVBD induced by the injection of each RNA is
shown as
(less than 10%),
* (10-20%), and + (more than
70%). ND, not determined. B, protein expression
of cyclin B1 in oocytes injected with either the RNAs indicated or
distilled water (Water), uninjected immature oocytes
(Ni), and progesterone-induced mature oocytes
(Nm). Suc1 precipitates were analyzed by anti-cyclin B1
immunoblotting. C, UV cross-linking with the RNA probes
indicated. D, GVBD in oocytes injected with the RNAs
indicated. Data are shown as the average of three independent
experiments using 35-45 oocytes in each experiment.
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Fig. 6.
Association of XPum with Xcat-2
(A) and CPEB (B-D).
A, GST-Xcat-2 pull-down assay. Beads prebound to E. coli-expressed GST (lane 1), GST-Xcat-2 (lane
2), or no protein (lane 3) were incubated with immature
oocyte extracts, and bound proteins were analyzed by anti-XPum
immunoblotting. B, T7-XlCPEB pull-down assay. Immature
oocyte extracts were mixed with beads prebound to T7-Xdazl-His
(lane 1) or T7-XlCPEB-His (lane 2), and examined
by anti-XPum immunoblotting. C, co-immunoprecipitation
assay. Untreated (lanes 2 and 4) or SDS-treated
(lane 6) immature oocyte extracts, mature oocyte extracts
(lane 5), and extraction buffer alone (lanes 1 and 3) were subjected to immunoprecipitation with control
antibody (lanes 1 and 2) or anti-XlCPEB antibody
(lanes 3-6). The precipitates were analyzed by
immunoblotting with anti-XPum and anti-CPEB antibodies. D,
immature oocyte extracts treated without (lane 1) or with
(lane 2) RNase and extraction buffer alone (lane
3) were immunoprecipitated with anti-CPEB antibody, then
immunoblotted with anti-XPum and anti-CPEB antibodies.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Hideki Kobayashi (Kyushu University) for providing the cDNA for Xenopus cyclin B1 in pGEM1 and Dr. Satoru Kobayashi (Tsukuba University) for his helpful discussions. Thanks are also due to Drs. Nancy Standart and Ann Kaminski (University of Cambridge, UK) for technical advice on UV-crosslink and RNA affinity chromatography, respectively.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (10160201 to M. Y.) and JSPS-RFTF 96L00401 and BioDesign Program from the Ministry of Agriculture, Forestry and Fisheries, Japan (to Y. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB045628.
** To whom correspondence should be addressed: Div. of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. Tel.: 81-11-706-4454; Fax: 81-11-706-4456; E-mail: myama@sci.hokudai.ac.jp.
Published, JBC Papers in Press, March 29, 2001, DOI 10.1074/jbc.M010528200
2 M. Yamashita (2000) GenBankTM accession number AB040435.
3 S. Nakahata and M. Yamashita, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MPF, maturation-promoting factor; MAPK, mitogen-activated protein kinase; GVBD, germinal vesicle breakdown; CPE, cytoplasmic polyadenylation element; CPEB, CPE-binding protein; UTR, untranslated region; hb, hunchback; NRE, nanos response element; MBS-H, modified Barth's saline buffered with Hepes; RT, reverse transcription; PCR, polymerase chain reaction; GST, glutathione S-transferase; ORF, open reading frame; bp, base pair(s); kb, kilobase(s); RACE, rapid amplification of cDNA ends; XPum, Xenopus Pumilio.
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