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Molecular and Cellular Biology, March 2001, p. 1662-1671, Vol. 21, No. 5
Anatomy and Cell Biology, University of Iowa, Iowa
City, Iowa 52242
Received 30 August 2000/Returned for modification 11 October
2000/Accepted 7 December 2000
At the midblastula transition, the Xenopus
laevis embryonic cell cycle is remodeled from rapid
alternations between S and M phases to become the complex adult cell
cycle. Cell cycle remodeling occurs after zygotic transcription
initiates and is accompanied by terminal downregulation of maternal
cyclins A1 and B2. We report here that the disappearance of both cyclin
A1 and B2 proteins is preceded by the rapid deadenylation of their
mRNAs. A specific mechanism triggers this deadenylation. This mechanism
depends upon discrete regions of the 3' untranslated regions and
requires zygotic transcription. Together, these results strongly
suggest that zygote-dependent deadenylation of cyclin A1 and
cyclin B2 mRNAs is responsible for the downregulation of these
proteins. These studies also raise the possibility that zygotic control of maternal cyclins plays a role in establishing the adult cell cycle.
In Xenopus laevis, the
first 12 cell cycles following fertilization occur in the absence of
transcription. Maternal mRNAs and proteins that are synthesized and
stored in the growing oocyte control these early embryonic cell
divisions. Zygotic transcription begins upon completion of the 12th
cell cycle and, in Xenopus, is referred to as the
midblastula transition (MBT) (25). Prior to the MBT, gene
expression is controlled by posttranscriptional mechanisms including
regulation of the adenylation state of maternal mRNAs.
Polyadenylated mRNAs are recruited into polysomes and translated, while deadenylated mRNAs are released from polysomes and
translationally silenced (27).
In Xenopus, adenylation control regulates both progression
through meiosis (oocyte maturation) and the transition from the meiotic
to the mitotic cell cycle by regulating levels of cell cycle proteins,
including cyclins. The mRNAs encoding cyclins A1, B1, B2, and E1 are
synthesized during oogenesis and stored untranslated as
poly(A) The 12th cell division is completed approximately 6 h
postfertilization (p.f.) and is followed by remodeling of the cell
cycle between cell cycles 13 and 15 (24). The cell cycle
is remodeled from a rapid alternation between DNA synthesis and mitosis
to become a cell cycle containing gap phases and checkpoint controls. The mechanism of cell cycle remodeling is not well understood, but it
is accompanied by temporally specific degradation of maternal cyclin
A1, B2, and E1 proteins (14, 33). We define this event as
the terminal disappearance of the maternal cyclin protein.
Because cyclins A1 and B2 are normally degraded during each cell cycle
through the ubiquitin-proteasome degradation pathway (7),
we hypothesized that translational silencing of their mRNAs via
deadenylation leads to the terminal disappearance of these proteins at
the time of cell cycle remodeling. In this report, we show that the
disappearance of these cyclins is preceded by deadenylation of their
mRNAs. Deadenylation is mediated by sequence elements in the 3' UTRs of
these maternal mRNAs and requires zygotic transcription. Our results
show that deadenylation of cyclins correlates with cell cycle
remodeling, strongly suggesting that deadenylation regulates the early
embryonic cell cycle and initiates cell cycle remodeling. This is also
the first report of zygotic control of maternal gene expression in
Xenopus.
Chimeric genes and in vitro transcription.
Chimeric genes
pGbA1, pGbB1, pGbB2, and pGbE1 were constructed by inserting the 3' UTR
of the indicated cyclin mRNA downstream of the
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1662-1671.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Zygotic Regulation of Maternal Cyclin A1 and
B2 mRNAs
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
mRNAs until oocyte maturation (5,
35). In response to progesterone, cytoplasmic polyadenylation
elements (CPEs) in the 3' untranslated region (UTR) trigger
polyadenylation and translational activation of these mRNAs, allowing
accumulation of cyclin protein and progression through meiosis. After
fertilization, cyclin mRNAs are further adenylated and continuously
translated (35). However, periodic degradation of cyclin
A1, B1, and B2 proteins during each cell cycle allows progression
through the first 12 cell divisions (18).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-globin coding
sequence in the plasmid pGbORF/mosEDEN (1). Cyclin 3'
UTRs were generated by PCR, and the XbaI and EcoRV sites were added using the combination of primers and
DNA templates shown in Table 1. The
vector pGbORF/mosEDEN was digested with XbaI and
EcoRV, and a cyclin 3' UTR was inserted between these sites,
replacing mosEDEN (Fig. 1A).
TABLE 1.
Isolation of cyclin 3' UTRs by PCR
View larger version (22K):
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FIG. 1.
Structure of chimeric genes. (A) General structure of
the pGb-cyclin 3'-UTR genes. The T7 promoter (T7) and the restriction
sites XbaI (X) and EcoRV (EV) are
indicated. The -globin 5'-UTR, open reading frame, and partial
globin 3'-UTR sequences are indicated (GbORF). The cyclin 3' UTRs are
complete and contain the hexanucleotide AATAAA. (B)
Structure of the mutagenized chimeric genes. The black box represents
the nuclear polyadenylation signal, and the striped box corresponds to
the CPE. The restriction endonuclease recognition sites used in the
deletion analysis of pGbA1 are indicated (S = SpeI;
H = HpaI). For pGbB2Sn, mutated sequences S1 to S6
are indicated and have been replaced by the sequence indicated in
Materials and Methods.
XS was constructed by deleting the
XbaI/SpeI fragment from pGbA1 and recircularizing
the vector. Similarly, pGbA1XH was generated by deletion of the
XbaI/HpaI fragment from pGbA1. The
XbaI and HpaI sites were blunt ended, and the
vector was recircularized (Fig. 1B).
Substitution mutants of the GbB2 construct were made using PCR-based
linker scanning mutagenesis (3). Six substitution mutants
of 30 nucleotides (nt), each covering the complete 180-nt cyclin B2 3'
UTR in the context of pGbB2, were constructed. The deleted sequences
were replaced by the sequence GATCTGAGTCTCTAAGCTAGCTAATAC. The nuclear polyadenylation signal or hexanucleotide
(AAUAAA) was not mutated, as it is required for cytoplasmic
polyadenylation (8). The two CPEs were mutated one at a
time. The remaining CPE should still mediate polyadenylation of the
injected mRNA (Fig. 1B). The pGbORF/mosEDEN chimeric gene was provided
by H. B. Osborne (Universite de Rennes I, Rennes, France)
(29).
For in vitro transcription, plasmids were linearized with
EcoRV. 32P-labeled, capped mRNAs were
synthesized according to the recommendations of the manufacturer (Stratagene).
Embryos and microinjections.
Embryos were obtained according
to standard protocols (16) and maintained in 0.1× Marc's
modified Ringer's medium at 23°C. For Western blot analysis,
five embryos were collected at the times specified, and protein was
extracted as described previously (14). For mRNA
microinjections, two-cell embryos were injected with 18.4 nl of in
vitro-transcribed capped mRNA in water (50,000 cpm/µl; 0.25 to 1 fmol/µl), with the addition of 50 ng of 
-amanitin per embryo when
indicated. For adenylation, Northern blotting, and poly(A) tail
analyses, five embryos were collected at the specified times and
processed for RNA extraction as described previously (13)
or by following the TRIZOL protocol (Gibco-BRL). The cell cycle stages
of the embryos were determined according to the work of Nieuwkoop and
Faber (26). In addition, where indicated, the detection of
the zygote-specific transcript GS17 (20) was performed.
Immunoanalyses. One embryo equivalent was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, which were successively probed with each of the indicated antibodies as described previously (15). Bands were visualized by enhanced chemiluminescence. Polyclonal antibodies to Xenopus cyclins were kindly provided by J. L. Maller (University of Colorado Health Sciences Center, Denver).
Poly(A) tail analysis.
Total RNA was extracted from embryos
injected with radiolabeled, capped chimeric mRNAs and resolved by
electrophoresis on 4% polyacrylamide-urea gels as described previously
(2). Fixed and dried gels were analyzed by autoradiography
or phosphorimaging (Molecular Dynamics). The number of replicate
experiments performed is indicated in the figure legends. For
quantification, poly(A) mRNA was defined as the
radioactive signal present in a window ranging from A0 to A20 as
defined by the RNA size markers. The total signal was defined as the
signal ranging from A0 to the top of the signal.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The disappearance of cyclin A1 and B2 proteins is zygote
dependent.
Cyclins A1 and B2 disappear after the initiation of
transcription at the MBT (15), suggesting that their
disappearance may be mediated by zygotically expressed genes. To
determine if zygotic transcription is necessary for cyclin
disappearance, we injected one-cell embryos with the transcriptional
inhibitor -amanitin. Injected embryos were sampled every 30 min
between 5 and 11 h p.f., and cyclin levels were assessed by
Western blot analysis. The same membrane was probed successively with
specific antibodies against cyclins A1, B1, B2, and E1. Inhibition of
transcription was assayed by Northern blot analysis for the
zygote-specific transcript GS17 (20). As shown previously
(15), the majority of cyclin E1 disappeared by 7 h
p.f. (stage 9), independent of the activation of zygotic transcription
(Fig. 2). In contrast (Fig. 2, lower
panels), the degradation of cyclins A1 and B2 strictly depended upon
zygotic transcription. In control embryos, cyclin A1 was degraded after
8.5 h (stage 9.5/10), whereas in -amanitin-injected embryos, it
accumulated until at least 11 h p.f. Likewise, cyclin B2, which
normally disappears approximately 10 h p.f., also accumulated until at least 11 h p.f. in the presence of -amanitin. Cyclin B1 was detected through 11 h p.f. both in the presence and in the
absence of -amanitin (Fig. 2, second row), indicating that this
protein is not zygotically regulated and may not play a role in cell
cycle remodeling. These data show that a zygotic product is necessary
for the terminal disappearance of maternal cyclin A1 and B2 proteins.
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Deadenylation of maternal cyclin A1 and B2 mRNAs precedes
disappearance of their encoded proteins.
During each of the first
12 cell cycles, A and B cyclins are continuously synthesized and
periodically degraded. Therefore, we reasoned that the terminal
disappearance of cyclin A1 and B2 proteins may result from a block in
the translation of their mRNAs. Deadenylation triggers translational
silencing of numerous mRNAs in Xenopus oocytes and embryos
(21, 30, 42), and deadenylated mRNAs are unstable in
blastula-stage embryos (2, 13). Therefore, we asked if
deadenylation of cyclin mRNAs precedes or accompanies disappearance of
cyclin A1 and B2 proteins. To determine the adenylation status of
cyclin mRNAs, we performed a reverse transcriptase PCR-based PAT
(34). PAT for cyclin mRNAs was performed on total
RNA isolated from an embryo time course (Fig.
3). A PCR product is present only if the
mRNA tested contains a poly(A) tail long enough (12 residues) to serve
as a template for hybridization with the oligo(dT) anchor. The expected
size of the PCR product for a cyclin A1 mRNA with a minimal poly(A)
tail of 12 residues is around 510 nt. The cyclin A1 PCR product
migrated as a diffuse band from approximately 500 to 700 nt in 1-h
embryos, which corresponds to a poly(A) tail of approximately 200 A's.
These results agree with previous measurements of poly(A) tail length
for the cyclin A1 mRNA in Xenopus embryos prior to the MBT,
as measured by the oligo(dT)-RNase H method (35). The PCR
product size remained constant until 6 h p.f., when smaller
products appeared, indicating shortening of the poly(A) tail. By 8 h, the product migrated as a discrete band around 500 nt, corresponding
to cyclin A1 mRNA with the smallest detectable poly(A) tail. The
product size remained constant until 10 h p.f. We and others have
shown previously that maternal cyclin A1 is undetectable on Northern
blots by 11 h after fertilization (unpublished data) and in stage
10.5 (midgastrula) embryos (17), suggesting that
deadenylation leads to destabilization of the mRNA.
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The 3' UTRs of cyclin mRNAs specify deadenylation at the MBT.
Sequences controlling cytoplasmic adenylation behavior have primarily
been localized to the 3' UTR of mRNAs. Therefore, we asked if the 3'
UTRs of maternal cyclins could confer specific deadenylation on
chimeric mRNAs at the MBT. We took advantage of known CPEs in the
cyclin A1, B1, and B2 3' UTRs (37) and a potential CPE in
the cyclin E1 3' UTR to generate chimeric mRNAs that would be
polyadenylated in vivo after their injection into two-cell embryos.
Chimeric mRNAs contained the 5' UTR and the coding sequence of
-globin followed by the complete 3' UTR of one of the cyclins A1,
B1, B2, and E1 (Fig. 1A). Radiolabeled, capped,
poly(A) mRNAs were injected into two-cell
embryos. The poly(A) tail size of the injected mRNAs was determined 5 min after injection and every 1 or 2 h thereafter by denaturing
gel electrophoresis (Fig. 4).
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mRNA by 8 h. At 9 h p.f., the
majority of these mRNAs were poly(A). In
contrast, GbB1 mRNA slowly decreased in size and never accumulated as
poly(A) mRNA. GbE1 mRNA remained polyadenylated
until at least 10 h p.f., similar to the adenylation behavior of
maternal cyclin E1 mRNA as determined by the PAT (Fig. 3). These
results show that the 3' UTRs of cyclin A1 and B2 mRNAs confer specific
and rapid deadenylation on chimeric mRNAs in blastula-stage embryos.
Deadenylation mediated by the cyclin A1 and B2 3' UTRs is under
zygotic control.
Cyclin A1-B2 mRNA deadenylation occurs
after the onset of zygotic transcription (Fig. 3, GS17), and the
terminal disappearance of cyclin A1 and B2 proteins requires zygotic
transcription (Fig. 2). Therefore, if deadenylation is directly
responsible for protein disappearance, inhibition of zygotic
transcription should also block deadenylation. To test this prediction,
we injected chimeric mRNAs along with the transcriptional inhibitor
-amanitin. In the presence of -amanitin, the expression of the
zygotic transcript GS17 is completely abolished (Fig.
5A), and yet the embryos remain viable
for several hours (36). Following injection, the GbA1 and
GbB2 mRNAs acquired a poly(A) tail of about 200 A's both in -amanitin-treated and in control embryos (Fig. 5).This shows that
-amanitin does not affect in vivo polyadenylation. Strikingly, deadenylation of GbA1 and GbB2 mRNAs was completely abolished in
-amanitin-treated embryos (Fig. 5A, compare GbA1 control and -amanitin-treated embryos at 8 h). As quantitated in Fig. 5C, in the presence of -amanitin, the percentage of polyadenylated mRNA
peaked at approximately 5 h and then remained constant throughout the time course of the experiment for both GbA1 and GbB2. In
comparison, in buffer-injected embryos the percentage of polyadenylated
mRNA dropped dramatically between 6 and 8 h for GbA1 and between 8 and 10 h for GbB2. To ensure that -amanitin is not simply
stabilizing the poly(A) tail independently of transcription inhibition,
we coinjected -amanitin with the polyadenylated GbORF/mosEDEN mRNA (29). The embryonic deadenylation element (EDEN) contained
in this mRNA triggers its rapid deadenylation after injection into fertilized eggs. By 4 h p.f., the mRNA accumulated as
poly(A) RNA either in the absence or in the
presence of -amanitin (Fig. 6). Thus,
-amanitin does not block deadenylation in a manner independent
of transcription inhibition. These experiments demonstrate that a
zygotic product is required both for protein downregulation of cyclins
A1 and B2 (Fig. 2) and for the rapid deadenylation triggered by the 3'
UTRs of their mRNAs (Fig. 5).
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Specific regions in the 3' UTRs are required for zygote- mediated deadenylation. Our results show that the 3' UTRs of cyclins A1 and B2 specify the deadenylation that precedes the disappearance of these cyclin proteins. The ability of the cyclin A1 and B2 3' UTRs to confer the maternal deadenylation behavior on a chimeric mRNA implies that all of the sequence information required is contained in the 3' UTR. Several sequence-dependent mechanisms of deadenylation have been described previously for pre-MBT Xenopus embryos (29, 43). To determine if deadenylation at the MBT is due to a known or novel mechanism, we performed substitution and deletion analyses to localize the necessary regions in the 3' UTRs.
To delimit the necessary region in the 491-bp cyclin A1 3' UTR, 309 or 200 nt were deleted as depicted in Fig. 1B (pGbA1XH and pGbA1XS).
The chimeric mRNAs transcribed from these plasmids were injected into
two-cell embryos, and changes in the poly(A) tail length were assayed
by denaturing gel electrophoresis as for Fig. 4. The deletions left the
two CPEs and the nuclear polyadenylation signal (the hexanucleotide
AAUAAA) that are required for polyadenylation (8) intact, and so adenylation of the chimeric transcripts occurred as previously observed (Fig. 6A, 1.5 to 5 h p.f.).
Deletion of the first 309 or 200 nt of the 3' UTR in the context of
GbA1 greatly reduced deadenylation (Fig. 6A, GbA1XH and GbA1XS, 7 to 15 h p.f.). Deadenylation of GbA1 was evident by 7 h,
while deletion mutants GbA1XH (309 nt) and GbA1XS (200 nt)
retained a poly(A) tail until at least 15 h following
fertilization. For both of the deletion mutants, a gradual shortening
of the poly(A) tail was evident after 7 h, but fully deadenylated
transcripts did not accumulate (Fig. 6). The percentage of chimeric
mRNAs that are adenylated at each time point is shown in Fig.
7. The maximum percentage of
polyadenylated transcripts peaks at 6 h p.f. for full-length GbA1
and both of the deletion mutants. The percentage remains constant for
both deleted mRNAs, while the full-length GbA1 is rapidly deadenylated
beginning at 7 h. The minimum percentage of adenylated transcripts
is seen by 10 h and is comparable to the proportion of
polyadenylated mRNA at the first time point. These data show that the
sequence information necessary for deadenylation of the cyclin A1 mRNA
is contained in the first 200 nt of its 3' UTR. This 200-nt region
contains two AU-rich sequences, UUAUUUAUU and
UUAUUUAUAA, that are known to mediate deadenylation in
Xenopus embryos before the MBT, i.e., before the
initiation of zygotic transcription (43, 46, 47).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In X. laevis, regulation of poly(A) tail length allows for tight translational control during early development from oocyte maturation until the MBT. Our results, along with other recent reports (31, 41), show that control of poly(A) tail length continues after the MBT. We have shown that rapid deadenylation of maternal cyclin A1 and B2 mRNAs coincides with the onset of zygotic transcription and precedes the disappearance of their encoded proteins (Fig. 2 and 3). The 3' UTR of cyclin A1 or cyclin B2 is sufficient to confer the same deadenylation behavior on a chimeric mRNA, while neither the cyclin B1 nor the cyclin E1 3' UTR triggers rapid deadenylation (Fig. 4). Further confirming the specificity of deadenylation, we have localized the necessary sequence information to within 60 nt for the cyclin B2 3' UTR and to within 200 nt for the cyclin A1 3' UTR (Fig. 5). However, further analyses are needed to characterize the elements sufficient for zygotic deadenylation of these maternal transcripts.
The requirement for zygotic transcription for both disappearance of
protein (Fig. 2) and deadenylation of mRNA (Fig. 5) strongly supports a
model in which the deadenylation triggered by the cyclin A1 and B2 3'
UTRs leads to translational repression and/or mRNA destabilization,
resulting in terminal downregulation of these maternal gene products.
There is a difference in timing of the terminal disappearance of cyclin
A1 and B2 proteins (9 and 11 h, respectively), but our data show
little difference in the deadenylation kinetics of GbB2 and GbA1 mRNAs.
This difference may stem from the earlier degradation of cyclin A1 in
the embryonic cell cycle. Cyclin B2 is degraded at the end of M phase
(38), whereas cyclin A1 is degraded 15 min before
(14). After the MBT, the cell cycle becomes asynchronous
and lengthens as S phase is extended and gap phases are added. The
delay between cyclin A1 and B2 protein degradation may reflect this
change. Furthermore, after the MBT, cyclin A1 mRNA is rapidly degraded
while cyclin B2 mRNA is more stable (data not shown), which may permit
sufficient cyclin B2 protein synthesis to result in the levels of
cyclin B2 observed. We do not believe that the inhibition of
deadenylation by -amanitin results from nonspecific stabilization of
the poly(A) tail, since deadenylation mediated by the EDEN of c-mos
still occurs in these embryos (Fig. 6).
The transcription inhibition in post-MBT cell divisions initially
disrupts the cell cycle and ultimately triggers apoptosis in what would
be midgastrula embryos (36). First, we observed that, in
the presence of -amanitin, downregulation of cyclin A1 and B2
proteins is prevented. This suggests that the cell cycle is blocked in
a phase where cyclin A1 and B2 proteins are stable (before or at the
beginning of M phase [14]), and yet the downregulation of cyclin E1 protein still occurs (Fig. 1), showing that not all processes are nonspecifically disrupted at this time. As cyclin protein
degradation is a cell cycle stage-specific event, it is not unexpected
that such stabilization occurs. If deadenylation is also linked to a
specific cell cycle stage, it is possible that blocking cell cycle
progression would also block cyclin A1 and B2 deadenylation. In this
case, we could say that the block in deadenylation is a consequence of
the block in the cell cycle. However, to our knowledge, deadenylation
has never been linked directly to a specific cell cycle stage. The
other possibilities are that a required component of the cyclin A1-B2
deadenylation machinery is zygotically produced or that this machinery
is present in an inactive state before the onset of zygotic
transcription and requires an upstream zygotic transcription-dependent
event to be activated. Clarification of this issue must await a more complete delineation of the mechanism.
Despite the identical timing and the common requirement for zygotic
transcription, it is not clear whether cyclin A1 deadenylation and B2
deadenylation are mediated by the same mechanism. Sequence comparison
of the necessary regions of the cyclin A1 and B2 3' UTRs did not lead
to the identification of common elements. We believe that the
deadenylation conferred by the cyclin B2 3' UTR is due to a novel
sequence element while that conferred by the cyclin A1 3' UTR may be
mediated by the AU-rich elements (AREs) present in the first 200 nt of
its 3' UTR. In Xenopus, AREs mediate deadenylation just
after fertilization and therefore do not require zygotic transcription
(43). Thus, if cyclin A1 mRNA deadenylation is ARE
mediated, a specific mechanism must prevent rapid deadenylation from
fertilization to the MBT. In this case, a zygotic factor would be
required to inhibit the protective mechanism after the onset of zygotic
transcription (a model is shown in Fig.
8B).
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Figure 8 depicts two models in which a newly synthesized zygotic factor triggers cyclin A1 and B2 deadenylation. These models are based on current knowledge of deadenylation in Xenopus oocytes and/or early embryos and on our results, which are consistent with either model. In the first model, a zygotic factor acts as a positive regulator of deadenylation by targeting the mRNA containing the deadenylation element to the poly(A) exoribonuclease (PARN), which is already present in the embryos (19). Sequence-specific deadenylation is usually mediated by RNA binding proteins. Thus, it is likely that the zygotic factor is an RNA binding protein. Two other alternatives are that the zygotic component is a novel PARN transcribed at the MBT and that the factor is a newly transcribed RNA. This last possibility is exemplified in Caenorhabditis elegans, where the 21 nt let-7 RNA is complementary to 3'-UTR sequences of its target mRNAs and inhibits their expression (32).
In the second model, the deadenylation activity and the specificity factor are present in the pre-MBT embryo but cannot act on the mRNAs because of the presence of a sequence-specific protective factor. In this model, these RNAs should bear a protective element in addition to the deadenylation element. Zygotic transcription would be necessary to inactivate this protective factor. One prediction of this model is that mutagenesis of the protective element alone should lead to the early deadenylation of the chimeric reporter mRNA. In the set of substitutions and deletions that we performed, we never observed such behavior. This may mean that the protective element does not exist or that it was untouched in our set of chimeric mRNAs. Since the CPE and hexanucleotides were always present in our mRNAs to allow in vivo polyadenylation, future work will test if these sequences act as protective elements against zygotic factor-mediated deadenylation. Another possibility is that the deadenylation element and protective element cannot be uncoupled.
Deadenylation has been shown previously to be dependent on and/or facilitated by a 5' 7-methylguanosine cap and, consistent with this, dependent on active translation of the deadenylated mRNA (6, 11). These requirements also hold true for deadenylation mediated by PARN (6). We do not know if active translation of the chimeric transcripts is necessary for deadenylation, but since these transcripts are capped, contain a coding sequence, and are polyadenylated upon injection into two-cell embryos, it is likely that they are translated. Nevertheless, it must be kept in mind that cap-independent deadenylation does occur (4).
In addition to AREs, other 3'-UTR sequences specifying deadenylation in
Xenopus include the EDEN (21, 29), CPE-mediated deadenylation (31), and tra-2 and GLI element
(TGE)-mediated deadenylation (40). EDENs are UGUA
rich and, along with their binding protein, EDEN-BP, mediate rapid
deadenylation of c-mos, Eg-2, and Eg-5 just after fertilization
(29). The difference in timing and the absence of
UGUA-rich elements in both the cyclin A1 and cyclin B2 3' UTR suggest
that this mechanism is not involved in their deadenylation. UA-rich
CPE-mediated deadenylation occurs after the MBT in gastrula-stage
embryos and was recently described for maternal lamin B1. This mRNA is
adenylated and translationally activated at the MBT and is deadenylated
and translationally repressed in gastrula stages (31). In
this case, deadenylation does not confer degradation, as the lamin B1
mRNA is stable until late in embryogenesis (stage 22). It is not clear
whether CPE binding protein is involved in this process. Our data show
that the CPEs are not sufficient for deadenylation of cyclin A1 and B2
mRNA at the MBT, as chimeric mRNAs containing CPEs are not
deadenylated (Fig. 6 and 7, GbB2S2, GbA1XH, and GbA1XS). As
for the EDEN-mediated deadenylation, the difference in timing and the
lack of TGE-like sequences in cyclin A1 or cyclin B2 3' UTR suggest
that TGE-mediated deadenylation is not involved in cyclin A1 and B2 deadenylation.
Another example of posttranscriptional control in post-MBT embryos is that mediated by the 3' UTR of Xwnt8, a zygotic mRNA. The 3' UTR of Xwnt8 induces translational repression and degradation of a heterologous transcript at gastrulation, probably by promoting deadenylation (41). The element mediating translational repression has not been identified, but the timing of Xwnt8 downregulation correlates well with that of cyclin B2, raising the possibility that a similar zygotic mechanism is involved. On the other hand, there is no obvious sequence or structural similarity between the Xwnt8 and cyclin B2 3' UTRs.
Non-sequence-specific or default deadenylation also occurs in
Xenopus, during both oocyte maturation and embryogenesis.
Default deadenylation acts on mRNAs devoid of CPE (9, 42)
and is unable to trigger the complete deadenylation of an injected
poly(A)+ mRNA even after 12 h p.f.
(2). The slow and incomplete deadenylation of GbB2S2,
GbA1XH, and GbA1XS resembles the kinetics of default deadenylation, suggesting that this mechanism may also act on CPE-containing mRNAs when the majority of CPEB protein has been destroyed (12). The default deadenylation described
previously for the Xenopus embryo is maternally driven
(28), distinguishing it from the zygote-dependent
deadenylation described in the current report. This does not rule out
the possibility that common components may be involved in the two processes.
The elimination of maternal cyclin A1 and B2 mRNAs and proteins may mediate the transition from the rapid embryonic cell cycle to the highly regulated adult cell cycle. As cyclin A1 disappears, cyclin A2 is upregulated (17). Despite the fact that cyclin A1-Cdk1 and cyclin A2-Cdk1/Cdk2 have roles in both the S and M phases, it is likely that the substrates of Cdk1 and Cdk2 differ (10, 39, 44). The dependence of cyclin A1-B2 deadenylation on zygotic transcription suggests that this mechanism may control the accumulation of cell cycle regulators in somatic cells. This probability is supported by the recent report that, in the adult mammalian cell cycle, cyclins B1 and A2 are controlled at the level of mRNA stability (45). Identification of the sequences and factors involved will allow us to define the role of this novel regulatory mechanism in cell cycle remodeling and in the adult cell cycle. In addition, the zygotic transcription-mediated deadenylation activity that we describe may also silence other maternal mRNAs whose expression is required for early development but detrimental for later stages.
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ACKNOWLEDGMENTS |
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We thank J. Sible, H. B. Osborne, and D. L. Weeks for critical reading of the manuscript and H. B Osborne for pGbORF/mosEDEN and Paul Krieg for pGS17.
This work was supported by a Basil O'Connor award (F 498-758) from the March of Dimes Birth Defects Foundation, a DERC Pilot and Feasibility award (DERC-NIH DK252295), and a Carver Trust Medical Research Initiative grant from the Roy J. Carver Charitable Trust.
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FOOTNOTES |
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* Corresponding author. Mailing address: 1-572 BSB, Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7501. Fax: (319) 335-7198. E-mail: rebecca-hartley{at}uiowa.edu.
Present address: Life Sciences Group, Bio-Rad Laboratories,
Hercules, CA 94547.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. |
Audic, Y.,
F. Omilli, and H. B. Osborne.
1998.
Embryo deadenylation element-dependent deadenylation is enhanced by a cis element containing AUU repeats.
Mol. Cell. Biol.
18:6879-6884 |
| 2. | Audic, Y., F. Omilli, and H. B. Osborne. 1997. Postfertilization deadenylation of mRNAs in Xenopus laevis embryos is sufficient to cause their degradation at the blastula stage. Mol. Cell. Biol. 17:209-218[Abstract]. |
| 3. | Barnhart, K. M. 1999. Simplified PCR-mediated, linker-scanning mutagenesis. BioTechniques 26:624-626[Medline]. |
| 4. |
Brewer, G.
1998.
Characterization of c-myc 3' to 5' mRNA decay activities in an in vitro system.
J. Biol. Chem.
273:34770-34774 |
| 5. | Chevalier, S., A. Couturier, I. Chartrain, R. Le Guellec, C. Beckhelling, K. Le Guellec, M. Philippe, and C. C. Ford. 1996. Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication. J. Cell Sci. 109:1173-1184[Abstract]. |
| 6. | Dehlin, E., M. Wormington, C. G. Korner, and E. Wahle. 2000. Cap-dependent deadenylation of mRNA. EMBO J. 19:1079-1086[CrossRef][Medline]. |
| 7. | Deshaies, R. J. 1995. The self-destructive personality of a cell cycle in transition. Curr. Opin. Cell Biol. 7:781-789[CrossRef][Medline]. |
| 8. |
Fox, C. A.,
M. D. Sheets, and M. Wickens.
1989.
Poly(A) addition during maturation of frog oocytes: distinct nuclear and cytoplasmic activities and regulation by the sequence UUUUUAU.
Genes Dev.
3:2151-2162 |
| 9. |
Fox, C. A., and M. Wickens.
1990.
Poly(A) removal during oocyte maturation: a default reaction selectively prevented by specific sequences in the 3' UTR of certain maternal mRNAs.
Genes Dev.
4:2287-2298 |
| 10. |
Furuno, N.,
N. den Elzen, and J. Pines.
1999.
Human cyclin A is required for mitosis until mid prophase.
J. Cell Biol.
147:295-306 |
| 11. | Gao, M., D. T. Fritz, L. P. Ford, and J. Wilusz. 2000. Interaction between a poly(A)-specific ribonuclease and the 5' cap influences mRNA deadenylation rates in vitro. Mol. Cell 5:479-488[CrossRef][Medline]. |
| 12. | Hake, L. E., and J. D. Richter. 1994. CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation. Cell 79:617-627[CrossRef][Medline]. |
| 13. |
Harland, R., and L. Misher.
1988.
Stability of RNA in developing Xenopus embryos and identification of a destabilizing sequence in TFIIIA messenger RNA.
Development
102:837-852 |
| 14. | Hartley, R. S., R. E. Rempel, and J. L. Maller. 1996. In vivo regulation of the early embryonic cell cycle in Xenopus. Dev. Biol. 173:408-419[CrossRef][Medline]. |
| 15. | Hartley, R. S., J. C. Sible, A. L. Lewellyn, and J. L. Maller. 1997. A role for cyclin E/Cdk2 in the timing of the midblastula transition in Xenopus embryos. Dev. Biol. 188:312-321[CrossRef][Medline]. |
| 16. | Heasman, J., S. Holwill, and C. Wylie. 1991. Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules. Methods Cell Biol. 36:213-230[Medline]. |
| 17. |
Howe, J. A.,
M. Howell,
T. Hunt, and J. W. Newport.
1995.
Identification of a developmental timer regulating the stability of embryonic cyclin A and a new somatic A-type cyclin at gastrulation.
Genes Dev.
9:1164-1176 |
| 18. |
King, R. W.,
R. J. Deshaies,
J. M. Peters, and M. W. Kirschner.
1996.
How proteolysis drives the cell cycle.
Science
274:1652-1659 |
| 19. | Korner, C. G., M. Wormington, M. Muckenthaler, S. Schneider, E. Dehlin, and E. Wahle. 1998. The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes. EMBO J. 17:5427-5437[CrossRef][Medline]. |
| 20. | Krieg, P. A., and D. A. Melton. 1985. Developmental regulation of a gastrula-specific gene injected into fertilized Xenopus eggs. EMBO J. 4:3463-3471[Medline]. |
| 21. | Legagneux, V., F. Omilli, and H. B. Osborne. 1995. Substrate-specific regulation of RNA deadenylation in Xenopus embryos and activated egg extracts. RNA 1:1001-1008[Abstract]. |
| 22. | Minshull, J., J. J. Blow, and T. Hunt. 1989. Translation of cyclin mRNA is necessary for extracts of activated xenopus eggs to enter mitosis. Cell 56:947-956[CrossRef][Medline]. |
| 23. | Minshull, J., R. Golsteyn, C. S. Hill, and T. Hunt. 1990. The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J. 9:2865-2875[Medline]. |
| 24. | Newport, J., and M. Dasso. 1989. On the coupling between DNA replication and mitosis. J. Cell Sci. Suppl. 12:149-160. |
| 25. | Newport, J., and M. Kirschner. 1982. A major developmental transition in early Xenopus embryos. I. Characterization and timing of cellular changes at the midblastula stage. Cell 30:673-686[CrossRef]. |
| 26. | Nieuwkoop, P. D., and J. Faber. 1967. Normal table of Xenopus laevis (Daudin). North-Holland Publishing Co., Amsterdam, The Netherlands. |
| 27. | Osborne, H. B., and J. D. Richter. 1997. Translational control by polyadenylation during early development. Prog. Mol. Subcell. Biol. 18:173-198[Medline]. |
| 28. | Paillard, L., V. Legagneux, and H. B. Osborne. 1996. Poly(A) metabolism in Xenopus laevis embryos: substrate-specific and default poly(A) nuclease activities are mediated by two distinct complexes. Biochimie 78:399-407[Medline]. |
| 29. | Paillard, L., F. Omilli, V. Legagneux, T. Bassez, D. Maniey, and H. B. Osborne. 1998. EDEN and EDEN-BP, a cis element and an associated factor that mediate sequence-specific mRNA deadenylation in Xenopus embryos. EMBO J. 17:278-287[CrossRef][Medline]. |
| 30. | Paris, J., and M. Philippe. 1990. Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development. Dev. Biol. 140:221-224[CrossRef][Medline]. |
| 31. | Ralle, T., D. Gremmels, and R. Stick. 1999. Translational control of nuclear lamin B1 mRNA during oogenesis and early development of Xenopus. Mech. Dev. 84:89-101[CrossRef][Medline]. |
| 32. | Reinhart, B. J., F. J. Slack, M. Basson, A. E. Pasquinelli, J. C. Bettinger, A. E. Rougvie, H. R. Horvitz, and G. Ruvkun. 2000. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403:901-906[CrossRef][Medline]. |
| 33. |
Rempel, R. E.,
S. B. Sleight, and J. L. Maller.
1995.
Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase.
J. Biol. Chem.
270:6843-6855 |
| 34. | Salles, F. J., W. G. Richards, and S. Strickland. 1999. Assaying the polyadenylation state of mRNAs. Methods 17:38-45[CrossRef][Medline]. |
| 35. |
Sheets, D. M.,
C. A. Fox,
T. Hunt,
G. V. Woude, and M. Wickens.
1994.
The 3'-untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation.
Genes Dev.
8:926-938 |
| 36. | Sible, J. C., J. A. Anderson, A. L. Lewellyn, and J. L. Maller. 1997. Zygotic transcription is required to block a maternal program of apoptosis in Xenopus embryos. Dev. Biol. 189:335-346[CrossRef][Medline]. |
| 37. | Stebbins-Boaz, B., L. E. Hake, and J. D. Richter. 1996. CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus. EMBO J. 15:2582-2592[Medline]. |
| 38. | Stewart, E., H. Kobayashi, D. Harrison, and T. Hunt. 1994. Destruction of Xenopus cyclins A and B2, but not B1, requires binding to p34cdc2. EMBO J. 13:584-594[Medline]. |
| 39. | Strausfeld, U. P., M. Howell, P. Descombes, S. Chevalier, R. E. Rempel, J. Adamczewski, J. L. Maller, T. Hunt, and J. J. Blow. 1996. Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts. J. Cell Sci. 109:1555-1563[Abstract]. |
| 40. |
Thompson, S. R.,
E. B. Goodwin, and M. Wickens.
2000.
Rapid deadenylation and poly(A)-dependent translational repression mediated by the Caenorhabditis elegans tra-2 3' untranslated region in Xenopus embryos.
Mol. Cell. Biol.
20:2129-2137 |
| 41. | Tian, Q., T. Nakayama, M. P. Dixon, and J. L. Christian. 1999. Post-transcriptional regulation of Xwnt-8 expression is required for normal myogenesis during vertebrate embryonic development. Development 126:3371-3380[Abstract]. |
| 42. |
Varnum, S. M., and W. M. Wormington.
1990.
Deadenylation of maternal mRNAs during Xenopus oocyte maturation does not require specific cis-sequences: a default mechanism for translational control.
Genes Dev.
4:2278-2286 |
| 43. |
Voeltz, G. K., and J. A. Steitz.
1998.
AUUUA sequences direct mRNA deadenylation uncoupled from decay during Xenopus early development.
Mol. Cell. Biol.
18:7537-7545 |
| 44. | Walker, D. H., and J. L. Maller. 1991. Role for cyclin A in the dependence of mitosis on completion of DNA replication. Nature 354:314-317[CrossRef][Medline]. |
| 45. | Wang, W., M. C. Caldwell, S. Lin, H. Furneaux, and M. Gorospe. 2000. HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation. EMBO J. 19:2340-2350[CrossRef][Medline]. |
| 46. | Xu, N., C. Y. Chen, and A. B. Shyu. 1997. Modulation of the fate of cytoplasmic mRNA by AU-rich elements: key sequence features controlling mRNA deadenylation and decay. Mol. Cell. Biol. 17:4611-4621[Abstract]. |
| 47. | Zubiaga, A. M., J. G. Belasco, and M. E. Greenberg. 1995. The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol. Cell. Biol. 15:2219-2230[Abstract]. |
Molecular and Cellular Biology, March 2001, p. 1662-1671, Vol. 21, No. 5
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1662-1671.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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