In vivo antisense oligodeoxynucleotide
mapping reveals masked regulatory elements
in an mRNA dormant in mouse oocytes.
A Stutz, J Huarte, P Gubler, B Conne, D Belin and J D Vassalli
Mol. Cell. Biol. 1997, 17(4):1759.
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MOLECULAR AND CELLULAR BIOLOGY, Apr. 1997, p. 1759–1767
0270-7306/97/$04.0010
Copyright q 1997, American Society for Microbiology
Vol. 17, No. 4
In Vivo Antisense Oligodeoxynucleotide Mapping Reveals
Masked Regulatory Elements in an mRNA
Dormant in Mouse Oocytes
ANDRÉ STUTZ,1* JOACHIM HUARTE,1 PASCALE GUBLER,1 BÉATRICE CONNE,2
DOMINIQUE BELIN,2 AND JEAN-DOMINIQUE VASSALLI1
Departments of Morphology1 and Pathology,2 University of Geneva Medical School, 1211 Geneva 4, Switzerland
In mouse oocytes, tissue-type plasminogen activator (tPA) mRNA is under translational control. The newly
transcribed mRNA undergoes deadenylation and translational silencing in growing oocytes, while readenylation and translation occur during meiotic maturation. To localize regulatory elements controlling tPA mRNA
expression, we identified regions of the endogenous transcript protected from hybridization with injected
antisense oligodeoxynucleotides. Most of the targeted sequences in either the 5* untranslated region (5*UTR),
coding region, or 3*UTR were accessible to hybridization, as revealed by inhibition of tPA synthesis and by
RNase protection. Two protected regions were identified in the 3*UTR of tPA mRNA in primary oocytes: the
adenylation control element (ACE) and the AAUAAA polyadenylation signal. These sequences were previously
shown to be involved in the translational control of injected reporter transcripts. During the first hour of
meiotic maturation, part of the ACE and the AAUAAA hexanucleotide became accessible to hybridization,
suggesting a partial unmasking of the 3*UTR of this mRNA before it becomes translationally competent. Our
results demonstrate that in vivo antisense oligodeoxynucleotide mapping can reveal the dynamics of regulatory
features of a native mRNA in the context of the intact cell. They suggest that specific regions in the 3*UTR of
tPA mRNA function as cis-acting masking determinants involved in the silencing of tPA mRNA in primary
oocytes.
structural features that allow their specific recognition. To
search for such structural features, a common approach explores the translational activity of exogenous reporter mRNAs,
designed so as to contain candidate regulatory regions. A severe limitation of this approach is that it does not directly
explore the relevant features of the endogenous, native
mRNAs: instances have been described in which the fates of
endogenous and injected RNAs are strikingly different (9). It
seems thus critical to develop alternative approaches.
In mouse oocytes, the maternal mRNA encoding the serine
protease tissue-type plasminogen activator (tPA) has been extensively studied (16–18, 48, 52). Newly transcribed tPA
mRNA is endowed with a long poly(A) tail, but the transcript
that accumulates, untranslated, during oocyte growth has an
unusually short poly(A) tail. Later, when meiotic maturation
resumes (i.e., following germinal vesicle [GV] breakdown
[GVBD]), tPA mRNA is subject to a process of cytoplasmic
polyadenylation, and the presence of a long poly(A) tail appears necessary and sufficient for translation to occur. Taken
together, these observations suggest that the transient translational silencing of tPA mRNA in primary oocytes occurs
through a reversible process of deadenylation. This process is
selective, in the sense that it occurs at a time when many other
oocyte mRNAs retain their poly(A) tail and are being actively
translated (2, 3, 25). An investigation of the cis determinants in
tPA mRNA involved in its translational control in primary and
maturing oocytes has suggested the importance of a ;40-nucleotide (nt)-long region in the 39 untranslated region (39UTR)
that is required both for deadenylation in primary oocytes and,
together with the canonical AAUAAA cleavage and polyadenylation signal, for readenylation following GVBD; this region
has been termed the adenylation control element (ACE) (18).
The experiments leading to this model were based upon the
injection of exogenous reporter transcripts, and the possible
Gene expression can be controlled at different points along
the path from transcription to biological function. For certain
genes, a very effective checkpoint is the translational status of
the mRNA. During germ cell development, for instance, a
subset of mRNAs are under strict translational control. In
somatic cells also, marked changes in the translational efficiency of individual mRNAs can occur depending on the physiological state of the cell. However, the mechanisms involved in
controlling translation of given mRNAs in germ cells or somatic cells have been elucidated in only a minority of cases (10,
11, 14, 15, 32).
Among the best-explored contexts in which translational
control plays a decisive part in regulating gene expression are
the meiosis of female gametes and early embryonic development. In oocytes from a wide variety of animal species, including clams, Drosophila melanogaster, Xenopus laevis, and mice,
certain maternal mRNAs are rendered dormant immediately
upon transcription or shortly thereafter; after remaining silent
for days or weeks, they undergo translational activation at later
stages of meiosis or after fertilization (10, 26, 51, 54, 58). Two
possibly distinct regulatory processes must be considered: an
initial silencing, by physical sequestration from the translational machinery, reduction in the length of the poly(A) tail, or
binding of masking proteins; and a later awakening by a reversal of the silencing process, for instance, through readenylation
or some other mechanism. The selectivity of both processes,
which affect only a subpopulation of mRNAs in the cell, indicates that mRNAs under translational control must possess
* Corresponding author. Mailing address: André Stutz, Department
of Morphology, CMU, 1 rue Michel Servet, 1211 Geneva 4, Switzerland. Phone: 41-22 70 25 218. Fax: 41-22 70 25 260. E-mail: Andre.stutz
@medecine.unige.ch.
1759
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Received 5 September 1996/Returned for modification 17 October 1996/Accepted 16 December 1996
1760
STUTZ ET AL.
MATERIALS AND METHODS
Oocyte collection, injection, and culture. Procedures for oocyte collection
from Swiss albino mice, injection, and culture have been previously described
(16, 17, 34, 48). For injection of maturing oocytes, primary oocytes were incubated in Dulbecco modified Eagle medium containing 5% fetal calf serum, 25 mg
of sodium pyruvate per ml, and 2.5 mg of polyvinylpyrrolidone (Pharmacia) per
ml. In all experiments, a volume of ;10 pl was injected in the cytoplasm of
oocytes. Oocytes were cultured either in the presence of 100 mg/ml dibutyryl
cyclic AMP (dibutyryl-cAMP) to prevent resumption of meiosis or in its absence
to allow meiotic maturation. For biosynthetic labelling during meiotic maturation, primary oocytes were cultured in modified Biggers medium (6, 24) for 16 h
in the presence of 200 mCi of Tran35S-label (1,079 Ci/mmol; ICN Biomedicals,
Inc.) per ml.
ODN synthesis and purification. ODNs were synthesized on an Applied Biosystems model 380A DNA synthesizer by the phosphoramidite method (local
oligonucleotide synthesis service) or purchased from Microsynth (Balgach, Switzerland). All ODNs were purified by extraction with n-butanol (36), extracted
once with phenol-chloroform, and precipitated with ethanol. The purified ODNs
were dissolved in 150 mM KCl at 1 mg/ml (1 A260 5 33 mg/ml).
Sequences of ODNs used for injection are written in the 59-to-39 orientation;
the numerical designations refer to the position of the 39-most nucleotide in the
tPA cDNA (27), while as or s indicates antisense or sense orientation: as-ODN
103, CTCTC TTCAT TTTGC TCCCC GTTTC (positions 79 to 103); as-ODN
1429, CAGAA AGCTC ACACT CTGTC CAGTC (positions 1405 to 1429);
as-ODN 1703, TTCTG CCCAC AGCCG AGGCC CCAG (positions 1680 to
1703); as-ODN 1837, CTTCT GTAGA AGAGG AAGAG (positions 1818 to
1837); as-ODN 2044, CCTAG AGTTA TGGAA GGTTG G (positions 2024 to
2044); as-ODN 2058, CCTCT TTTTA AAATC CTAG (positions 2039 to 2058);
as-ODN 2196, GTAAC TATAA AAACA CATTC (positions 2176 to 2196);
as-ODN 2309 (23-mer), GTCCC AAGAG TTGAG GAGTG TGG (positions
2287 to 2309); s-ODN 2309, CCACA CTCCT CAACT CTTGG GAC (positions
2287 to 2309); as-ODN 2309 (18-mer), GTCCC AAGAG TTGAG GAG (positions 2292 to 2309); as-ODN 2309 (13-mer), GTCCC AAGAG TTG (positions
2297 to 2309); as-ODN 2309 (10-mer), GTCCC AAGAG (positions 2300 to
2309); as-ODN 2351, GTTTA TAAAG AAAAA GACAT TTA (positions 2329
to 2351); as-ODN 2378, CATAC AGTTC TCCCA ACCAT CTATA GAG
(positions 2351 to 2378); as-ODN 2391, CAATT ATTAA AATCA TACAG
(positions 2371 to 2391); as-ODN 2405, CTAGT GTTAT TCATC AATTA
TTAAA ATC (positions 2378 to 2405); as-ODN 2427, AATAG ATTAA
AATAT AAATA TAC; as-ODN 2438, GTAAA ATCTA AAATA GATTA
FIG. 1. Schematic representation of tPA mRNA and localization of sequences complementary to the as-ODNs used in this study. The open box
represents the coding region of tPA mRNA. The lines represent the 59 and 39
UTRs; the bottom line represents the 455 39-most nt of tPA mRNA. Solid and
open circles within a shaded ellipse represent UA-rich sequences that are part of
the ACE (18). The other solid circle, the hexagon, and the lozenge correspond
to other CPE-like sequences. The solid rectangle represents the AAUAAA
cleavage and polyadenylation signal. Numbered represent as-ODNs; numbering
refers to the position of the 39-most complementary nucleotide in tPA cDNA.
Ellipses, rectangles, and the polygon highlight as-ODNs studied in detail in Fig.
3 to 6.
AAAT (positions 2415 to 2438); as-ODN 2442, CAAAG TAAAA TCTAA
AATAG; as-ODN 2464, GTATA ATACA AAGTT ATAGT AAC (positions
2442 to 2464); as-ODN 2484, GAATT TATTA TTTAA G (positions 2468 to
2484); as-ODN 2504, AAAGT GTGAA AAATA CCTCT G (positions 2483 to
2504).
Plasmid constructions and in vitro transcription. To prepare the chimeric
mRNA-1 (Ch-1 [18]), a SmaI-ApaLI fragment containing 235 bp of mouse
b-actin 39UTR cDNA (positions 1638 to 1873) was subcloned into pBluescript
KS downstream of the reporter urokinase-type plasminogen activator (uPA)
cDNA insert (B1 mRNA [52]). Chimeric mRNA-2 (Ch-2 [18]) contains a reporter uPA cDNA insert plus the 455 39-terminal nt of mouse tPA cDNA (B2
mRNA [52]). Both plasmid constructs were linearized with BamHI and transcribed with either T3 or SP6 polymerase.
Capped chimeric mRNAs used in translation assays were prepared by using
[32P]UTP at 0.5 mCi/ml and a total concentration of UTP of 500 mM (16). When
indicated, the transcripts were polyadenylated in vitro as described previously
(52). The purified RNAs were dissolved in 150 mM KCl at ;100 ng/ml before
injection.
RNA analysis and translation assays. RNA extractions were performed as
described previously (16). Size analysis of transcripts was performed by electrophoresis in 6% polyacrylamide–urea gels and autoradiography of the dried gels.
For translation assays, cultured oocytes were collected in groups of three, lysed
in 0.25% Triton X-100–1 mg of bovine serum albumin per ml, and assayed by
zymography (17).
RNase H digestion has been described previously (52). For RNase protection
assays (4, 5), a 455-bp DraI fragment (positions 2054 to 2504 of tPA cDNA) was
cloned in the antisense orientation into the HincII site of pSP64 (16). The
plasmid was linearized with EcoO109 to generate a 326-nt-long cRNA probe.
Lyophilized RNA extracted from 10 oocytes was mixed with 5 3 105 cpm of
gel-purified radiolabelled probe in 30 ml of hybridization buffer [80% formamide,
0.4 M NaCl, 40 mM piperazine-N,N9-bis (2-ethanesulfonic acid (PIPES; pH 6.4),
2 mM EDTA]. The RNAs were denatured at 908C for 2 min and annealed
overnight at 408C. The samples were digested for 1 h at 258C with 20 mg of RNase
A in a buffer containing 10 mM Tris-HCl (pH 7.4), 4 mM EDTA, and 300 mM
NaCl. Samples were subsequently adjusted to 0.5% sodium dodecyl sulfate
(SDS) and digested with 10 mg of proteinase K per ml for 20 min at 378C.
Samples were extracted twice with phenol-chloroform, and the RNA was precipitated with ethanol in the presence of 10 mg of carrier yeast tRNA. The RNAs
were resuspended in sample buffer, denatured for 2 min at 908C, resolved by
polyacrylamide-urea gel electrophoresis, and visualized by autoradiography.
Poly(A) test. The length of the poly(A) tail of tPA mRNA was measured by the
poly(A) test of Sallés and Strickland (35). Total RNA purified from mouse
oocytes was annealed with poly(dT)12–18 in the presence of T4 DNA ligase; an
oligo(dT)17 anchor [59-CGAAT TCTCG AGGAT CCGTC GAC(T)17] was then
added to the reaction. Reverse transcription was followed by PCR amplification
with a tPA 59-specific ODN (s-2309; see above) and with an adapter oligonucleotide (59-CGAAT TCTCG AGGAT CCGTC GAC); the reaction mixture was
labelled with 5 mCi of [32P]dATP (3,000 Ci/mmol; Amersham). The amplification
products were electrophoresed on 6% denaturing urea–polyacrylamide gels.
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role of the ACE in the endogenous transcript could only be
inferred.
cis-acting elements responsible for the translational control
of an endogenous mRNA must be recognized by the cellular
machinery; they may form specialized structures through intramolecular base pairing and/or interact with other cellular
macromolecules. We reasoned that because of such structural
features, the regulatory cis-acting elements may be unable to
hybridize with antisense oligonucleotides. It has previously
been shown that injection of DNA or RNA fragments complementary to portions of oocyte mRNAs results in the degradation of the target mRNA in the duplex region, by an
RNase H-like activity (23, 34, 56) or an RNase III-like activity
(21, 48), respectively. In the case of tPA mRNA, antisensemediated amputation of the RNA upstream of these 39UTR
sequences prevents its translational activation, most likely because the ACE and the AAUAAA sequence are indispensable
for readenylation following resumption of meiosis (48). This
can be monitored by an enzyme assay sensitive enough to be
performed on individual oocytes (17). Thus, regions of tPA
mRNA susceptible to or protected from hybridization with
injected antisense oligodeoxynucleotides (as-ODNs) should be
easy to identify. Assuming that protection from as-ODN-directed cleavage is due to structural features of the mRNA in
the targeted region, in vivo ODN mapping could be a unique
tool to localize cis-acting elements involved in regulating the
function of an endogenous mRNA, in the context of the live
cell. Using this novel approach to explore structural aspects of
tPA mRNA in mouse oocytes, we identify here dynamic regulatory elements in the 39UTR of this maternal mRNA under
meiosis-related translational control.
MOL. CELL. BIOL.
VOL. 17, 1997
MAPPING OF mRNA REGULATORY ELEMENTS
RESULTS
Effects of different as-ODNs on tPA synthesis in mouse
oocytes. Translation of tPA mRNA during meiotic maturation
requires readenylation of the dormant transcript (18, 52). Sequences in the 39-most region of tPA mRNA, the ACE and the
AAUAAA hexanucleotide, are necessary to direct readenylation and translation (18). Amputation of this 39-most region by
antisense RNA-targeted cleavage of the duplexed portion of
the mRNA prevents its translational activation after GVBD
(48). This can be monitored by analyzing lysates of oocytes
collected at the end of meiotic maturation, using a zymographic assay that reveals tPA activity (17). As expected, injection into fully grown primary oocytes of as-ODNs complementary to sequences in the 39UTR of tPA mRNA upstream
of the 39-most regulatory region resulted in inhibition of tPA
production during meiotic maturation. The effects of two such
as-ODNs (2309 and 2351 [Fig. 1]) are compared in Fig. 2.
While both could completely inhibit tPA production, the doses
required differed by a factor of 10, presumably because of a
difference in the predicted melting temperatures (Tm) of the
DNA-RNA hybrids (Fig. 2A and B). To verify this hypothesis,
progressively shorter 2309-derived as-ODNs were tested: when
injected at a concentration of 1 mg/ml, a 13-mer (Tm 5 408C)
completely inhibited tPA production, whereas a 10-mer (Tm 5
328C) was only partly effective (Fig. 2C). Overall, all as-ODNs
complementary to accessible regions of tPA mRNA and having
a predicted Tm of 408C or above inhibited tPA production by
more than 90% when injected at a concentration of 1 mg/ml.
Eighteen different as-ODNs targeted to the translation initiation site, the coding region, and the 39UTR (Fig. 1) were
injected into primary oocytes. After 20 h of culture under
conditions allowing meiotic maturation, the secondary oocytes
were analyzed for tPA expression by SDS-polyacrylamide gel
electrophoresis (PAGE) and zymography. Most of the asODNs (103, 1429, 1703, 1837, 2044, 2058, 2196, 2309, 2351,
2378, 2391, 2405, and 2464) prevented the production of tPA
(Fig. 2 and 3 and data not shown). By contrast, five as-ODNs
(2427, 2438, 2442, 2484, and 2504) complementary to the ACE,
the AAUAAA hexanucleotide, and the 39 end of the mRNA,
respectively, did not affect tPA production (Fig. 3 and data not
shown).
The proportion of as-ODN-injected oocytes undergoing
meiotic maturation when cultured under conditions allowing
resumption of meiosis was similar to that of uninjected oocytes. Furthermore, the extent and overall pattern of protein
synthesis, analyzed by [35S]methionine labelling, SDS-PAGE,
and autoradiography, were similar in oocytes injected with
sense-ODN (2309), as-ODNs, or 150 mM KCl (data not
shown). Thus, as-ODN-mediated inhibition of tPA synthesis
appeared specific and did not reflect a generalized decrease in
protein synthesis or an arrest of meiotic maturation.
We conclude that different as-ODNs complementary to distinct regions of tPA mRNA have contrasting effects on tPA
synthesis: while some ODNs specifically prevented production
of the protein, others, complementary to putative regulatory
sequences in the 39UTR of tPA mRNA, did not. Interestingly,
as-ODNs 2058, 2196, and 2391, targeted to 39UTR UA-rich
sequences similar to sequences present in the ACE, did not
cause inhibition of tPA production. Further experiments were
designed to investigate the mechanism of action of the as-ODNs.
Effects of different as-ODNs on tPA mRNA in mouse oocytes. Control experiments verified that the as-ODNs used
could induce cleavage by RNase H at the expected complementary sites of a synthetic tPA mRNA in vitro (data not
shown). Furthermore, total RNA purified from primary oocytes was hybridized in vitro to six as-ODNs (2405, 2427, 2438,
2464, 2484, and 2504) complementary to sequences in the
39UTR of tPA mRNA; following addition of RNase H, the
oocyte RNA was analyzed by RNase protection by using a
32
P-labelled murine tPA cRNA probe. The six as-ODNs induced the generation of protected fragments of the predicted
sizes (Fig. 4A and B, lanes 8 to 10; Fig. 4C, lanes 6 to 8),
confirming their intrinsic ability to hybridize to complementary
sequences on purified oocyte tPA mRNA and to promote its
cleavage by RNase H. By contrast, RNase protection analysis
FIG. 3. Effects of different as-ODNs on tPA expression. Primary oocytes injected with the indicated as-ODNs (1-mg/ml concentrations) were allowed to mature to
secondary oocytes, lysed in pools of three, and assayed in triplicate by zymography. Lanes 1, 2, and 3, control (C) lysates from three, two, and one uninjected oocytes,
respectively.
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FIG. 2. as-ODN-mediated inhibition of tPA expression. Primary oocytes injected with the indicated as-ODNs were allowed to mature to secondary oocytes,
lysed in pools of three, and assayed in triplicate by zymography. (A and B) Lanes
1 to 9, as-ODNs 2309 and 2351 were injected at the indicated concentrations;
lanes 10 to 12, control (C) lysates from three, two, and one uninjected oocytes
(A) or three, two, and one oocytes injected with 150 mM KCl (B), respectively.
(C) Lanes 1 to 12, as-ODN 2309 (23-mer) or as-ODNs consisting of the 18, 13,
and 10 59-most nt of as-ODN 2309 were injected at 1 mg/ml; lanes 13 to 15,
control (C) lysates from three, two, and one uninjected oocytes. Tm values
correspond to the computed DNA-DNA duplex melting temperatures for the
individual as-ODNs.
1761
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STUTZ ET AL.
of total RNA extracted from oocytes that had been injected
with the six as-ODNs revealed that in the intact primary oocyte, as-ODNs 2427, 2438, and 2484 did not promote cleavage
of tPA mRNA (Fig. 4B, lanes 4 and 5; Fig. 4C, lanes 1 and 3),
while as-ODNs 2405, 2464, and 2504 were as effective in vivo as
in vitro (Fig. 4B, lanes 2, 3, 6, and 7; Fig. 4C, lane 5).
We conclude that as-ODN-mediated inhibition of tPA synthesis is due to cleavage of tPA mRNA. The lack of effect of
as-ODNs 2427, 2438, and 2484 on tPA synthesis correlates with
their failure to induce mRNA cleavage in vivo and contrasts
with their effects on purified deproteinized tPA mRNA in
vitro. as-ODN 2504 induces amputation of the 39 end of tPA
mRNA but fails to inhibit tPA synthesis during oocyte meiotic
maturation; this finding confirms that sequences downstream
of the AAUAAA hexanucleotide are not required for cytoplasmic polyadenylation and translational activation after
GVBD (18, 52).
Since as-ODNs 2427, 2438, and 2484 can promote tPA
mRNA cleavage in vitro but not in vivo, it follows that the
complementary regions, i.e., the ACE and the AAUAAA sequence, may be inaccessible to hybridization in the context of
the intact oocyte; this could be due to secondary structure of
the RNA and/or to the presence of an RNA-binding protein(s).
Differential effects of an as-ODN on translation of injected
and endogenous mRNAs. To ascertain that the lack of effect of
as-ODN 2438 on tPA production was due to a particular feature of the endogenous tPA mRNA in oocytes, we prepared a
synthetic chimeric transcript (Fig. 5A, Ch-2), containing the
coding region of uPA mRNA linked to the 455 39-most nt of
tPA mRNA. When injected in oocytes that are allowed to
undergo meiotic maturation, this chimeric transcript undergoes translational activation, like the endogenous tPA mRNA
(52); uPA can be visualized by SDS-PAGE and zymography,
thus allowing translation of both exogenous (uPA-encoding)
and endogenous (tPA-encoding) mRNAs to be monitored in
the same oocytes (Fig. 5B, lanes 1 to 3). Coinjection of the
chimeric transcript with as-ODN 1837, complementary to a
sequence present in endogenous tPA mRNA but not in Ch-2
transcript, resulted in the specific inhibition of tPA production
(lanes 4 to 6), while coinjection with as-ODN 2351, complementary to both mRNAs, abolished expression of both uPA
and tPA (lanes 7 to 9). When Ch-2 transcript was injected
together with as-ODN 2438, also complementary to both chimeric and endogenous mRNAs, uPA production was prevented while tPA production was unaffected (lanes 10 to 12).
As a control, we prepared another synthetic chimeric transcript
(Fig. 5, Ch-1 A1), containing the same uPA coding region but
a different 39UTR. This transcript was polyadenylated before
injection, to ensure its translation in both primary and maturing oocytes (18). Coinjection of as ODN 2438 with Ch-1 A1
did not decrease uPA production (Fig. 5C, lanes 4 to 6), confirming that the effect of this as-ODN on translation of Ch-2 is
due to its interaction with the 39UTR fragment of tPA mRNA
in the chimeric mRNA.
Taken together, these experiments show that the hybrid
between as-ODN 2438 and its complementary sequence in
Ch-2 is a target for efficient inhibition of reporter uPA synthesis in the oocyte. The lack of effect of as-ODN 2438 on tPA
synthesis indicates that this as-ODN cannot hybridize with its
complementary sequence in the endogenous tPA mRNA. We
conclude that the sequence complementary to as-ODN 2438,
i.e., a part of the ACE region, is protected from hybridization
in the 39UTR of endogenous tPA mRNA, because of RNARNA and/or RNA-protein interactions. In earlier studies, it
has been shown that the ACE present in Ch-2 is responsible
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FIG. 4. Effects of different as-ODNs on tPA mRNA. (A) Schematic diagram
of the fragments resulting from RNase H digestion of tPA mRNA in the presence of as-ODNs, as revealed in RNase protection assays (4) using the indicated
cRNA riboprobe. The oblique 59 portion of the riboprobe represents the vectorderived sequence. The lengths of the protected fragments are indicated. The
shaded ellipse represents the ACE region; the solid rectangle represents the
AAUAAA sequence. (B) RNase protection analysis of tPA mRNA collected
from primary (GV) and maturing (BD [breakdown]) oocytes that had been
injected with the indicated as-ODNs. Uninjected primary oocytes (lane 1) and
primary oocytes injected with the indicated as-ODNs (lanes 2 to 7) were lysed
after 12 h of culture in the presence or absence of dibutyryl-cAMP, to prevent
(GV) or allow meiotic maturation (BD). In each lane, total RNA prepared from
pools of 10 oocytes was analyzed. Lanes 8 to 11, total RNA from uninjected
primary oocytes was exposed in vitro to RNase H in the presence or absence of
the indicated as-ODN and analyzed by RNase protection. (C) Same as for panel
B except that for lanes 2 and 4, as-ODNs 2427 and 2484 were injected after
resumption of meiosis, i.e., 3 h after GVBD. Lanes 1, 2, 6, and 9 are from one
experiment; lanes 3, 4, 5, 7, and 8 are from another. n.i., not injected.
MOL. CELL. BIOL.
VOL. 17, 1997
1763
for the translational control of this reporter transcript (18),
suggesting that it must also become involved in RNA-RNA
and/or RNA-protein interactions after injection in oocytes; in
the present work, Ch-2 was mixed with as ODNs at the time of
injection, so that complementary sequences could hybridize
before any such interactions could occur.
Stage-dependent effects of as-ODNs on oocyte tPA expression. The 39UTR of tPA mRNA is implicated in both the
deadenylation and translational repression of injected reporter
transcripts during oocyte growth and in their readenylation and
translational activation during meiotic maturation (18, 52). It is
thus possible that changes in RNA secondary structure or
RNA-protein interactions involving this 39UTR occur upon
resumption of meiosis. Such changes in endogenous tPA
mRNA could perhaps be revealed by the oligonucleotide mapping approach. The half-life of unmodified 59-radiolabelled
ODNs injected in mouse oocytes is less than 30 min (data not
shown), a result consistent with other reports of studies using
end-labelled or internally labelled ODNs injected in Xenopus
oocytes (56). Thus, as-ODNs injected in primary oocytes or
after GVBD might yield different results.
Ten as-ODNs (2058, 2196, 2309, 2391, 2405, 2427, 2438,
2442, 2464, and 2484) were injected either in arrested primary
oocytes (GV) or in oocytes having resumed meiosis (3 h after
GVBD). All oocytes were then placed under conditions allowing meiotic maturation and analyzed for tPA production 20 h
later. For eight of these ODNs, similar results were obtained
for both injection times: as-ODNs 2058, 2196, 2309, 2391,
2405, and 2464 prevented tPA production (Fig. 6, lanes 1 to 3
and 13 to 15, and data not shown), while as-ODNs 2438 and
2442 did not (lanes 7 to 12). By contrast, as-ODNs 2427 and
2484, complementary to a part of the ACE and to the polyadenylation signal AAUAAA, respectively, did not inhibit tPA
expression when injected in primary oocytes but completely
prevented tPA production when injected after GVBD (lanes 4
to 6 and 16 to 18). This stage-specific effect was verified by
RNAse protection: injection of as-ODNs 2427 and 2484 after
GVBD resulted in cleavage of all tPA mRNA molecules, while
the same ODNs injected in primary oocytes had only a limited
effect on this transcript (Fig. 4C, lanes 1 to 4).
The effects of as-ODNs 2427 and 2484 in maturing oocytes
demonstrate the intrinsic capacities of these ODNs to induce
cleavage of their complementary sequences in vivo as well as in
FIG. 6. Stage-specific effects of as-ODNs complementary to the ACE and the AAUAAA-containing region on tPA expression. The indicated as-ODNs were
injected either in primary oocytes (GV) or 3 h after GVBD (BD). After 20 h of culture under conditions allowing meiotic maturation, secondary oocytes were lysed
in pools of three and assayed in triplicate by zymography. Lane 19, lysates from three control (C) secondary oocytes obtained from either noninjected primary oocytes
(GV) or 150 mM KCl-injected maturing oocytes (BD).
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FIG. 5. Effects of as-ODNs on expression of endogenous and injected chimeric mRNAs. (A) mRNA constructs, comprising the first 2,040 nt of uPA
mRNA and the 39UTR of either tPA (Ch-2) (52) or b-actin (Ch-1) (18) mRNA.
The shaded ellipse and the solid rectangle represent the ACE and the AAUAAA
sequence, respectively; arrows represent the as-ODNs. (B and C) Ch-2 mRNA or
in vitro-polyadenylated Ch-1 mRNA (Ch-1 A1) was injected (100 ng/ml) in
primary oocytes alone or with as-ODNs (1 mg/ml; molar excess of as-ODN
approximately 103 over chimeric mRNA). Oocytes were cultured for 20 h to
allow meiotic maturation, lysed in groups of three secondary oocytes, and assayed in triplicate by zymography. (B) Ch-2 mRNA was injected alone (lanes 1
to 3) or together with the indicated as-ODNs (lanes 4 to 12). (C) Ch-1 A1 was
injected alone (lanes 1 to 3) or together with as-ODN 2438 (lanes 4 to 6). Lanes
7 to 9, lysates from three, two, and one control uninjected secondary oocytes.
MAPPING OF mRNA REGULATORY ELEMENTS
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STUTZ ET AL.
MOL. CELL. BIOL.
FIG. 7. Timing of the changes in accessibility to as-ODNs hybridization,
polyadenylation, and translation of tPA mRNA during meiotic maturation. (A)
Effects of as-ODNs 2427 and 2484 on tPA mRNA cleavage, determined by
RNase protection analysis of tPA mRNA collected either from noninjected (n.i.)
oocytes (lane 1) or from primary (GV) and maturing (BD [breakdown]) oocytes
that had been injected with the indicated as-ODNs (lanes 2 to 7). Pools of
as-ODN-injected primary oocytes were divided in two groups and incubated in
either the presence (1) or absence (2) of dibutyryl-cAMP. Blocked primary
oocytes (lanes 2 and 5) and oocytes that had resumed meiosis within 1 h after
injection (lanes 3 and 6) were lysed after a total of 2 h of culture. Maturing
oocytes injected immediately after GVBD were lysed 1 h after injection (lanes 4
and 7). Lanes 2, 4, and 7, correspond to 25 oocytes; lanes 1, 3, 5, and 6
correspond to 15 oocytes. The size of the undigested probe is indicated (325 nt).
(B) Time course of poly(A) tail elongation. Oocytes were collected before (GV)
or at the indicated times after GVBD. Total RNA was extracted and subjected
to a poly(A) test (35). The poly(A) tail lengths (in nucleotides) were determined
by comparison with a sequencing ladder loaded on the same gel. Each lane
corresponds to approximately one oocyte. (C) Time course of tPA accumulation.
Duplicate samples of 10 oocytes were collected before (GV) or at the indicated
times after GVBD and assayed by zymography. The zymogram was allowed to
develop for 42 h. (D) Quantitative representation of the results illustrated in
panels A to C. The percentage of cleaved tPA mRNA molecules following
injection of as-ODN 2427 (lozenges) and as-ODN 2484 (squares) was determined from the autoradiogram in panel A, using the ImageQuant program.
Poly(A) tail elongation (triangles) was expressed as a percentage of maximal
elongation, i.e., that achieved 18 h after GVBD; poly(A) tail length was determined by estimating the size of the longest abundant amplification product (B).
tPA activity (X) was quantitated from the zymogram shown in panel C by
measuring the surface of the proteolytic zones. A calibration curve was established by using serial dilutions of a lysate prepared from 10 oocytes (lysed 8 h
after GVBD) and analyzed in the same zymographic assay (not shown); tPA
activity 8 h after GVBD was defined as 100%.
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vitro. This finding reinforces the view that a part of the ACE
and the AAUAAA region of tPA mRNA are protected from
hybridization in primary oocytes. In maturing oocytes, before
tPA synthesis starts, these regions become at least transiently
accessible to as-ODN hybridization. We conclude that because
of the short half-life of as-ODNs in oocytes, the ODN mapping
approach can be used to study at least some changes in mRNA
structural features occurring upon resumption of meiosis. In
the context of the present experiments, this approach reveals a
change affecting both the ACE and the AAUAAA region of
tPA mRNA, at the time these cis-acting determinants are required for the readenylation and translational activation of
dormant tPA mRNA.
Early changes in tPA mRNA accessibility following GVBD.
Previous studies have shown that elongation of the poly(A) tail
of tPA mRNA can be demonstrated by Northern blot hybridization 3 h after GVBD, while translation is detected 1 h later
(16, 17). As shown above, tPA mRNA is accessible to as-ODNs
2427 and 2484 injected 3 h after GVBD but not the same
as-ODNs injected in primary oocytes. This change in accessibility of tPA mRNA could thus be a cause or a consequence of
polyadenylation and/or translation. To explore this issue, we
compared the timing of tPA mRNA polyadenylation and translational activation with the change in accessibility to injected
as-ODNs (Fig. 7).
As-ODNs 2427 and 2484 were injected either in arrested
primary oocytes, in oocytes before GVBD, or immediately
after GVBD. All oocytes were cultured for 1 or 2 h after
injection and then lysed. Total RNA was extracted and submitted to an RNase protection assay (Fig. 7A). Whereas both
as-ODNs induced only limited cleavage of tPA mRNA in primary oocytes (lanes 2 and 5), injection before (lanes 3 and 6)
or just after (lanes 4 and 7) GVBD resulted in cleavage of most
or all tPA mRNA molecules within 1 h after resumption of
meiosis, indicating a very early change in accessibility of both
complementary regions. Groups of uninjected oocytes were
cultured in parallel. Their tPA mRNA was subjected to a test
VOL. 17, 1997
MAPPING OF mRNA REGULATORY ELEMENTS
DISCUSSION
The mechanisms responsible for translational control are
only poorly understood, in part because of a lack of information on the structure of mRNA as it exists in the cytoplasm, i.e.,
as a component of ribonucleoproteins (RNPs). While in vitro
studies have proven extremely useful to start investigating
these issues, it is mandatory to develop in vivo approaches that
can help probe, in the context of the intact cell, the structures
and molecular events that modulate the rate of translation of
specific mRNAs. In this respect, oocytes provide a remarkable
experimental tool (reviewed in reference 11). They contain a
pool of maternal mRNAs that are transcribed during oocyte
growth and translated only at later developmental stages, i.e.,
after resumption of meiosis or following fertilization (28–31);
these maternal mRNAs are thus a paradigm of transcripts
under translational control. An additional advantage of oocytes is that they can be quite easily microinjected, so that the
fate of endogenous or exogenous transcripts can be studied
under conditions of experimental manipulation in a live cell.
While it is possible that translational control of maternal
mRNAs involves mechanisms distinct from those operative in
somatic cells, a more detailed description of this process in
oocytes should nevertheless be useful for understanding the
modulation of protein synthesis in general.
In vitro synthesis of reporter mRNAs from recombinant
vectors provides reagents to identify cis-acting elements that
can place a given injected transcript under translational control. However, injected mRNAs may not mimic all aspects of
the metabolism of endogenous transcripts (9). Hence, the features of endogenous mRNAs that are relevant in the control of
their translation must also be explored. Here again, oocytes
could be of great experimental value, if structural features of
endogenous maternal mRNAs could be probed in intact cells.
To this end, we have injected as-ODNs complementary to
different parts of an endogenous oocyte mRNA to identify
regions accessible to and regions protected from hybridization.
Two different types of tests were performed on injected oocytes: the translation of the target mRNA, which encodes the
serine protease tPA, was monitored by a highly sensitive zymography enzymatic assay, and the structure of the target
mRNA was investigated by RNase protection.
In previous studies, investigators have injected as-ODNs in
oocytes to inhibit translation of targeted mRNAs as a means to
explore the functions of specific gene products (12, 22, 23, 33,
34, 37, 50, 56). However, there can be limitations to this approach: in a detailed study on Xenopus oocytes, it was concluded that the high dose of as-ODN required to destroy a
targeted mRNA was frequently toxic for oocytes and embryos,
probably at least in part because of nonspecific effects due to
hybridization to other RNAs (57). In our experiments, these
limitations do not appear relevant, for the following reasons:
first, although the concentration of as-ODNs in injected mouse
oocytes (;10 pg/;320 pl) is comparable to the concentration
found to be toxic in Xenopus oocytes (50 ng/;1,250 nl) (57),
mouse oocytes are incubated at a temperature 158C higher
than are Xenopus oocytes, and this should afford a higher
specificity of as-ODN hybridization; second, we did not observe signs of nonspecific effects, since resumption of meiosis
and first meiotic division occurred normally. Furthermore, neither the general pattern of protein synthesis nor the translation
of an irrelevant reporter mRNA was qualitatively or quantitatively disturbed in the presence of ODNs. Finally, the interpretation of our experiments would not be invalidated by putative nonspecific effects that might have escaped our notice,
since we have examined both the translation and the as-ODNdirected cleavage of the target mRNA. We are thus confident
that the use of as-ODNs in our study is not subject to the
limitations discussed in the functional studies mentioned
above.
Injection in oocytes of as-ODNs complementary to sequences in tPA mRNA yielded four types of effects, depending
on the targeted region and the developmental stage at which
the injection was performed.
(i) as-ODNs complementary to the 59 region (comprising the
initiation codon), the coding region, and most of the 39UTR
prevented tPA synthesis, and this could be accounted for by
cleavage of the mRNA in the hybridized region. This set of
results indicates that most of the tPA mRNA sequence is
probably accessible to hybridization of as-ODNs. In accordance with our previous observations (48), amputation of the
39UTR upstream of the AAUAAA sequence precluded translational activation of tPA mRNA following resumption of
meiosis.
(ii) An as-ODN complementary to the 39 end of tPA mRNA,
downstream of the AAUAAA polyadenylation signal, directed
the amputation of the mRNA, but it did not prevent translation during meiotic maturation. This finding indicates that the
39 end of tPA mRNA is accessible to hybridization. Since the
regulatory sequences (ACE and AAUAAA) are not removed,
translational activation of the amputated mRNA can proceed.
(iii) Injection in primary oocytes of as-ODNs complementary to two specific portions of the 39UTR neither prevented
translation nor induced cleavage of the mRNA. Control experiments, using these as-ODNs injected together with a naked
chimeric reporter transcript containing the appropriate target
sequences, demonstrated their capacity to inhibit reporter expression in the oocytes; their lack of effect on the endogenous
tPA mRNA is thus unlikely to be due to properties of the
ODNs themselves, and it most likely reflects intrinsic properties of this mRNA. Hence, these two specific regions of tPA
mRNA appear to be protected from as-ODN hybridization.
They contain the determinants previously shown to be required for translational control of an injected reporter transcript, i.e., the ACE and the AAUAAA cleavage and polyadenylation signal (52).
(iv) In contrast, injection in maturing oocytes of two asODNs complementary respectively to part of the ACE and to
the AAUAAA region caused cleavage of the RNA and prevented its translation. We conclude that these two regions,
which are protected from hybridization in primary oocytes,
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for poly(A) tail length (Fig. 7B); incipient polyadenylation was
detected 1 h after GVBD (lane 2), and elongation of the
poly(A) tail continued in the following hours (lanes 3 to 6).
Lysates of uninjected oocytes were also assayed for tPA enzymatic activity (Fig. 7C); an increase in tPA content was detectable only at 4 h after GVBD, and accumulation of the enzyme
progressed at a constant rate over the following 4 h. These
results were quantitated and combined in a graph (Fig. 7D)
that compares the timing of the changes affecting the structure
and expression of tPA mRNA.
The change in tPA mRNA accessibility to as-ODN hybridization is thus an early event following GVBD, for both the
ACE and the AAUAAA polyadenylation signal. This change
occurs concomitantly with the start of poly(A) tail elongation,
and it cannot be determined which of these two events occurs
first; however, it is noteworthy that polyadenylation continues
long after unmasking of the as-ODN target sequences. In conclusion, translation of tPA mRNA is a late event, and hence
changes in accessibility and polyadenylation cannot be a consequence of translation.
1765
1766
STUTZ ET AL.
the mRNA could be a direct consequence of the presence of
such a masking factor; alternatively, the factor may be involved
in the deadenylation process, and the resulting shortened
poly(A) tail would be insufficient for translation. Although the
available evidence does not allow us to distinguish between
these two possibilities, it appears unlikely that silencing of tPA
mRNA is exclusively related to the binding of a masking protein or nucleic acid sequence: if this were the case, an as-ODN
that promotes cleavage upstream of the 39UTR regulatory
regions (without affecting the coding sequence) would free the
mRNA and allow its translation. Such amputated mRNAs are
not translated in primary oocytes, perhaps because they lack a
poly(A) tail. Despite these remaining uncertainties concerning
the mechanism of maternal mRNA silencing, it is tempting to
consider that the masked configuration of parts of the 39UTR
of tPA mRNA in primary oocytes is related to the dormancy of
the mRNA in these cells.
The concept of masked mRNAs, first proposed by Spirin
(38–40), refers to mRNAs that are not translated in vivo but
can be efficiently translated in vitro provided that they have
previously been subjected to deproteinization (19, 43, 44).
Masking thus appears to involve binding of specific proteins
that recognize particular regulatory sequences within the
mRNA and/or help condense the mRNA in a compact messenger ribonucleoprotein, inaccessible to initiation factors, ribosomes, etc. Dormant transcripts in oocytes are considered
typical examples of masked mRNAs (1, 8, 9, 13, 42–45, 53).
Standart et al. (43) have localized a masked region of a dormant oocyte mRNA in in vitro studies. Wolffe and collaborators have demonstrated a causal role for masking proteins in
directing translational repression of Xenopus oocyte dormant
mRNAs in vivo (9, 20, 49) and provided evidence for sequenceselective interactions involved in mRNA masking through
packaging by Y-box family proteins, in a manner comparable
to the packaging of DNA by histones (8, 55). In the context of
our analysis of endogenous tPA mRNA in intact cells, it remains to be demonstrated whether a protected region of this
mRNA is indeed a cis-acting masking element responsible for
the dormancy of this transcript.
The translational activation of certain dormant oocyte
mRNAs is related to their cytoplasmic polyadenylation, and a
long poly(A) tail is thought to be necessary and sufficient for
translation (26, 51, 54). Interestingly, one of the 39UTR sequences required for polyadenylation and translation of tPA
mRNA in oocytes undergoing meiotic maturation, the ACE, is
also involved in the deadenylation and silencing of this mRNA
in growing and primary oocytes (18). as-ODN mapping shows
that the ACE is differently protected in primary and in maturing oocytes, in accord with the view that it plays presumably
different roles at these two developmental stages. A challenge
for future studies will be to understand how the same protected
region can act as a masking element in primary oocytes and a
signal for sequence-specific polyadenylation during meiotic
maturation.
ACKNOWLEDGMENTS
We thank F. Silva for technical assistance, G. Andrey for help with
software, K. Kariko for a computer modeling of tPA mRNA secondary
structure, A. Nichols and A. Vaezi for helpful discussions and comments, and A. Wohlwend, P. Herrera, and R. Madani for critical
reading of the manuscript.
This work was supported by grants from the Swiss National Science
Foundation.
REFERENCES
1. Ahringer, J., and J. Kimble. 1991. Control of the sperm-oocyte switch in
Caenorhabditis elegans hermaphrodites by the fem-3 39 untranslated region.
Nature 349:346–348.
Downloaded from http://mcb.asm.org/ on February 27, 2014 by PENN STATE UNIV
become at least partly accessible when meiotic maturation has
resumed, i.e., before or at the time of mRNA undergoes polyadenylation, and before translational activation. This result
also confirms the capacity of these as-ODNs to hybridize with
their target sequences in tPA mRNA in vivo. It thus supports
the view that the lack of effect of these as-ODNs in primary
oocytes is not due to their selective degradation or sequestration (by other oocyte macromolecules) but rather to protection
of the complementary sequences before meiotic maturation.
Taken together, these results show that as-ODNs can be
used to discriminate, in the intact cell, between accessible and
protected regions of an endogenous mRNA. The 39UTR of
tPA mRNA is very UA rich and contains, in addition to the
ACE, three other sequences similar to cytoplasmic polyadenylation elements (CPEs) identified in certain Xenopus maternal mRNAs. Multiple CPEs often contribute to the proper
poly(A) addition of maternal mRNAs (46). These three CPElike sequences were accessible to as-ODN hybridization in
both primary and maturing oocytes. This finding suggests that
they are inoperative in the translational control of tPA mRNA
in oocytes or that they may operate at a later developmental
stage. The presence of inoperative CPE-like sequences is reminiscent of a mouse maternal mRNA (19G [33]) that contains
two CPE-like sequences but is not elongated during oocyte
maturation. CPEs thus appear to require a specific context in
order to influence the fate of the mRNA. The antisense ODN
mapping of the 39UTR of oocyte tPA mRNA suggests that the
determinant that plays a prominent role in translational control of this mRNA may be the ACE rather than the other
related sequences.
The sequences that we have found to be protected in primary oocytes are within the two regions that were previously
shown to be necessary for the translational control of injected
transcripts (18). The present results suggest that in the endogenous transcript in the primary oocyte, these regions have a
different configuration than the bulk of the tPA mRNA sequence. This reinforces the view that the ACE and the
AAUAAA region are cis-acting determinants involved in the
translational control of endogenous tPA mRNA. Interestingly,
because of the short half-life of the injected as-ODNs, it was
possible to compare the accessibilities of these regions in primary and in maturing oocytes. Thus, as-ODN mapping can be
used to demonstrate dynamic changes in the structure of an
endogenous mRNA. While a part of the ACE was protected at
both developmental stages, another part of the ACE and the
AAUAAA region became accessible during meiotic maturation. This finding suggests structural changes affecting these
important determinants of the 39UTR, at the time they are
required for the cytoplasmic polyadenylation of dormant maternal transcripts, through a mechanism involving specific
ACE-binding proteins, CPE-binding protein (CPEB) (47)
and/or cleavage and polyadenylation specificity factor (CPSF)
(7) or a CPSF-like factor.
The results obtained by as-ODN mapping have implications
for the mechanism of translational control of tPA mRNA in
primary oocytes. Protection from as-ODN hybridization may
be due to specific structural features of the target regions, such
as RNA-protein or RNA-RNA interactions implicated in
translational control. These putative interactions affecting the
39UTR regulatory regions could be involved in the silencing of
this mRNA. Previous experiments have provided evidence that
sequence-specific and reversible cytoplasmic deadenylation is
instrumental in silencing newly transcribed tPA mRNA (18).
The present findings suggest that the deadenylated transcript
that has accumulated in fully grown primary oocytes is associated with a masking factor(s) (38, 40, 41). The silent state of
MOL. CELL. BIOL.
VOL. 17, 1997
1767
31. Rosenthal, E. T., and F. H. Wilt. 1986. Patterns of maternal messenger RNA
accumulation and adenylation during oogenesis in Urechis caupo. Dev. Biol.
117:55–63.
32. Rouault, T. A., R. D. Klausner, and J. B. Harford. 1995. Translational
control of ferritin, p. 335–362. In J. W. B. Hershey, M. B. Mathews, and N.
Sonenberg (ed.), Translational control. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
33. Sallés, F. J., A. L. Darrow, M. L. O’Connell, and S. Strickland. 1992.
Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation. Genes Dev. 6:1202–1212.
34. Sallés, F. J., W. G. Richards, J. Huarte, J.-D. Vassalli, and S. Strickland.
1993. Microinjecting antisense sequences into oocytes. Methods Enzymol.
225:351–361.
35. Sallés, F. J., and S. Strickland. 1995. Rapid and sensitive analysis of mRNA
polyadenylation states by PCR. PCR Methods Appl. 4:317–321.
36. Sawadogo, M., and M. W. Van Dyke. 1991. A rapid method for the purification of deprotected oligodeoxynucleotides. Nucleic Acids Res. 19:674.
37. Sheets, M. D., M. Wu, and M. Wickens. 1995. Polyadenylation of c-mos
mRNA as a control point in Xenopus meiotic maturation. Nature 374:511–
516.
38. Spirin, A. S. 1995. Masked and translatable messenger ribonucleoproteins in
higher eukaryotes, p. 319–334. In J. W. B. Hershey, M. B. Mathews, and N.
Sonenberg (ed.), Translational control. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
39. Spirin, A. S. 1966. On “masked” forms of messenger RNA in early embryogenesis and in other differentiating systems. Curr. Top. Dev. Biol. 1:1–38.
40. Spirin, A. S. 1994. Storage of messenger RNA in eukaryotes: envelopment
with protein, translational barrier at 59 side, or conformational masking by 39
side. Mol. Reprod. Dev. 38:107–117.
41. Standart, N. 1992. Masking and unmasking of maternal mRNA. Semin. Dev.
Biol. 3:367–379.
42. Standart, N., and M. Dale. 1993. Regulated polyadenylation of clam maternal mRNAs in vitro. Dev. Genet. 14:492–499.
43. Standart, N., M. Dale, E. Stewart, and T. Hunt. 1990. Maternal mRNA from
clam oocytes can be specifically unmasked in vitro by antisense RNA complementary to the 39-untranslated region. Genes Dev. 4:2157–2168.
44. Standart, N., and T. Hunt. 1990. Control of translation of masked mRNAs
in clam oocytes. Enzyme 44:106–119.
45. Standart, N., and R. Jackson. 1994. Translational regulation. Y the message
is masked? Curr. Biol. 4:939–941.
46. Stebbins Boaz, B., and J. D. Richter. 1994. Multiple sequence elements and
a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development. Mol. Cell. Biol. 14:5870–5880.
47. 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 oocytes maturation in Xenopus. EMBO J. 15:2582–2592.
48. Strickland, S., J. Huarte, D. Belin, A. Vassalli, R. J. Rickles, and J.-D.
Vassalli. 1988. Antisense RNA directed against the 39 noncoding region
prevents dormant mRNA activation in mouse oocytes. Science 241:680–684.
49. Tafuri, S. R., and A. P. Wolffe. 1993. Selective recruitment of masked maternal mRNA from messenger ribonucleoprotein particles containing
FRGY2 (mRNP4). J. Biol. Chem. 268:24255–24261.
50. Tong, Z. B., L. M. Nelson, and J. Dean. 1995. Inhibition of zona pellucida
gene expression by antisense oligonucleotides injected into mouse oocytes.
J. Biol. Chem. 270:849–853.
51. Vassalli, J. D., and A. Stutz. 1995. Translational control. Awakening dormant mRNAs. Curr. Biol. 5:476–479.
52. Vassalli, J.-D., J. Huarte, D. Belin, P. Gubler, A. Vassalli, M. L. O’Connell,
L. A. Parton, R. J. Rickles, and S. Strickland. 1989. Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes. Genes Dev. 3:2163–2171.
53. Walker, J., M. Dale, and N. Standart. 1996. Unmasking mRNA in clam
oocytes: role of phosphorylation of a 39 UTR masking element-binding
protein at fertilization. Dev. Biol. 173:292–305.
54. Wickens, M., J. Kimble, and S. Strickland. 1995. Translational control of
developmental decisions, p. 411–450. In J. W. B. Hershey, M. B. Mathews,
and N. Sonenberg (ed.), Translational control. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
55. Wolffe, A. P. 1994. Structural and functional properties of evolutionarily
ancient Y-box family of nucleic acid binding proteins. J. Biol. Chem. 271:
22706–22712.
56. Woolf, T. M., C. G. Jennings, M. Rebagliati, and D. A. Melton. 1990. The
stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos. Nucleic
Acids Res. 18:1763–1769.
57. Woolf, T. M., D. A. Melton, and C. G. Jennings. 1992. Specificity of antisense
oligonucleotides in vivo. Proc. Natl. Acad. Sci. USA 89:7305–7309.
58. Wormington, M. 1994. Unmasking the role of the 39 UTR in the cytoplasmic
polyadenylation and translational regulation of maternal mRNAs. Bioessays
16:533–535.
Downloaded from http://mcb.asm.org/ on February 27, 2014 by PENN STATE UNIV
2. Bachvarova, R., E. M. Cohen, V. De Leon, K. Tokunaga, S. Sakiyama, and
B. V. Paynton. 1989. Amounts and modulation of actin mRNAs in mouse
oocytes and embryos. Development 106:561–565.
3. Bachvarova, R. F. 1992. A maternal tail of poly(A): the long and the short of
it. Cell 69:895–897.
4. Belin, D. 1996. The RNase protection assay. Methods Mol. Biol. 58:131–136.
5. Belin, D. 1994. The use of riboprobes for the analysis of gene expression.
Methods Mol. Biol. 31:257–272.
6. Biggers, J. D., W. J. Whitten, and D. G. Whittingham. 1971. The culture of
mouse embryos in vitro, p. 86–116. In G. C. Daniel (ed.), Methods in
mammalian embryology. Academic Press, New York, N.Y.
7. Bilger, A., C. A. Fox, E. Wahle, and M. Wickens. 1994. Nuclear polyadenylation factors recognize cytoplasmic polyadenylation elements. Genes Dev.
8:1106–1116.
8. Bouvet, P., K. Matsumoto, and A. P. Wolffe. 1995. Sequence-specific RNA
recognition by the Xenopus Y-box proteins. An essential role for the cold
shock domain. J. Biol. Chem. 270:28297–28303.
9. Bouvet, P., and A. P. Wolffe. 1994. A role for transcription and FRGY2 in
masking maternal mRNA within Xenopus oocytes. Cell 77:931–941.
10. Curtis, D., R. Lehmann, and P. D. Zamore. 1995. Translational regulation in
development. Cell 81:171–178.
11. Davidson, E. H. 1986. Gene activity in early development, 3rd ed. Academic
Press, New York, N.Y.
12. Gebauer, F., W. Xu, G. M. Cooper, and J. D. Richter. 1994. Translational
control by cytoplasmic polyadenylation of c-mos mRNA is necessary for
oocyte maturation in the mouse. EMBO J. 13:5712.
13. Goodwin, E. B., P. G. Okkema, T. C. Evans, and J. Kimble. 1993. Translational regulation of tra-2 by its 39 untranslated region controls sexual identity
in C. elegans. Cell 75:329–339.
14. Gray, N. K., and M. W. Hentze. 1994. Regulation of protein synthesis by
mRNA structure. Mol. Biol. Rep. 19:195–200.
15. Hershey, J. W. B., M. B. Mathews, and N. Sonenberg. 1996. Translational
control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
16. Huarte, J., D. Belin, A. Vassalli, S. Strickland, and J.-D. Vassalli. 1987.
Meiotic maturation of mouse oocytes triggers the translation and polyadenylation of dormant tissue-type plasminogen activator mRNA. Genes Dev.
1:1201–1211.
17. Huarte, J., D. Belin, and J.-D. Vassalli. 1985. Plasminogen activator in
mouse and rat oocytes: induction during meiotic maturation. Cell 43:551–
558.
18. Huarte, J., A. Stutz, M. L. O’Connell, P. Gubler, D. Belin, A. L. Darrow, S.
Strickland, and J.-D. Vassalli. 1992. Transient translational silencing by
reversible mRNA deadenylation. Cell 69:1021–1030.
19. Kelso Winemiller, L. C., and M. M. Winkler. 1991. ’Unmasking’ of stored
maternal mRNAs and the activation of protein synthesis at fertilization in
sea urchins. Development 111:623–633.
20. Matsumoto, K., F. Meric, and A. P. Wolffe. 1996. Translational repression
dependent on the interaction of the Xenopus Y-box protein FRGY2 with
mRNA. J. Biol. Chem. 271:22706–22712.
21. Melton, D. A. 1985. Injected anti-sense RNAs specifically block messenger
RNA translation in vivo. Proc. Natl. Acad. Sci. USA 82:144–148.
22. Morgan, R., M. Edge, and A. Colman. 1993. A more efficient and specific
strategy in the ablation of mRNA in Xenopus laevis using mixtures of
antisense oligos. Nucleic Acids Res. 21:4615–4620.
23. O’Keefe, S. J., A. A. Kiessling, and G. M. Cooper. 1991. The c-mos gene
product is required for cyclin B accumulation during meiosis of mouse eggs.
Proc. Natl. Acad. Sci. USA 88:7869–7872.
24. Paleos, G. A., and R. D. Powers. 1981. The effect of calcium on the first
meiotic division of the mammalian oocyte. J. Exp. Zool. 217:409–416.
25. Paynton, B. V., and R. Bachvarova. 1994. Polyadenylation and deadenylation
of maternal mRNAs during oocyte growth and maturation in the mouse.
Mol. Reprod. Dev. 37:172–180.
26. Richter, J. D. 1995. Dynamics of poly(A) addition and removal during
development, p. 481–503. In J. W. B. Hershey, M. B. Mathews, and N.
Sonenberg (ed.), Translational control. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
27. Rickles, R. J., A. L. Darrow, and S. Strickland. 1988. Molecular cloning of
complementary DNA to mouse tissue plasminogen activator mRNA and its
expression during F9 teratocarcinoma cell differentiation. J. Biol. Chem.
263:1563–1569.
28. Rosenthal, E. T., M. Bahr, and F. H. Wilt. 1993. Regulation of maternal
messenger RNA translation during oogenesis and embryogenesis in Urechis
caupo. Dev. Biol. 155:297–306.
29. Rosenthal, E. T., and J. V. Ruderman. 1987. Widespread changes in the
translation and adenylation of maternal messenger RNAs following fertilization of Spisula oocytes. Dev. Biol. 121:237–246.
30. Rosenthal, E. T., T. R. Tansey, and J. V. Ruderman. 1983. Sequence-specific
adenylations and deadenylations accompany changes in the translation of
maternal messenger RNA after fertilization of Spisula oocytes. J. Mol. Biol.
166:309–327.
MAPPING OF mRNA REGULATORY ELEMENTS