Xenopus laevis zygote arrest 2 (zar2) encodes a zinc finger RNA-binding protein that binds to the translational control sequence in the maternal Wee1 mRNA and regulates translation.
ABSTRACT Zygote arrest (Zar) proteins are crucial for early embryonic development, but their molecular mechanism of action is unknown. The Translational Control Sequence (TCS) in the 3' untranslated region (UTR) of the maternal mRNA, Wee1, mediates translational repression in immature Xenopus oocytes and translational activation in mature oocytes, but the protein that binds to the TCS and mediates translational control is not known. Here we show that Xenopus laevis Zar2 (encoded by zar2) binds to the TCS in maternal Wee1 mRNA and represses translation in immature oocytes. Using yeast 3 hybrid assays and electrophoretic mobility shift assays, Zar2 was shown to bind specifically to the TCS in the Wee1 3'UTR. RNA binding required the presence of Zn(2+) and conserved cysteines in the C-terminal domain, suggesting that Zar2 contains a zinc finger. Consistent with regulating maternal mRNAs, Zar2 was present throughout oogenesis, and endogenous Zar2 co-immunoprecipitated endogenous Wee1 mRNA from immature oocytes, demonstrating the physiological significance of the protein-RNA interaction. Interestingly, Zar2 levels decreased during oocyte maturation. Dual luciferase reporter tethered assays showed that Zar2 repressed translation in immature oocytes. Translational repression was relieved during oocyte maturation and this coincided with degradation of Zar2 during maturation. This is the first report of a molecular function of zygote arrest proteins. These data show that Zar2 contains a zinc finger and is a trans-acting factor for the TCS in maternal mRNAs in immature Xenopus oocytes.
- SourceAvailable from: Amanda Charlesworth[Show abstract] [Hide abstract]
ABSTRACT: Poly(A) tail elongation after export of an messenger RNA (mRNA) to the cytoplasm is called cytoplasmic polyadenylation. It was first discovered in oocytes and embryos, where it has roles in meiosis and development. In recent years, however, has been implicated in many other processes, including synaptic plasticity and mitosis. This review aims to introduce cytoplasmic polyadenylation with an emphasis on the factors and elements mediating this process for different mRNAs and in different animal species. We will discuss the RNA sequence elements mediating cytoplasmic polyadenylation in the 3' untranslated regions of mRNAs, including the CPE, MBE, TCS, eCPE, and C-CPE. In addition to describing the role of general polyadenylation factors, we discuss the specific RNA binding protein families associated with cytoplasmic polyadenylation elements, including CPEB (CPEB1, CPEB2, CPEB3, and CPEB4), Pumilio (PUM2), Musashi (MSI1, MSI2), zygote arrest (ZAR2), ELAV like proteins (ELAVL1, HuR), poly(C) binding proteins (PCBP2, αCP2, hnRNP-E2), and Bicaudal C (BICC1). Some emerging themes in cytoplasmic polyadenylation will be highlighted. To facilitate understanding for those working in different organisms and fields, particularly those who are analyzing high throughput data, HUGO gene nomenclature for the human orthologs is used throughout. Where human orthologs have not been clearly identified, reference is made to protein families identified in man. WIREs RNA 2013, 4:437-461. doi: 10.1002/wrna.1171 Conflict of interest: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.WIREs RNA 07/2013; 4(4):437-61. · 6.15 Impact Factor
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ABSTRACT: Maternal mRNAs are translationally regulated during early development. Zar1 and its closely related homolog, Zar2, are both crucial in early development. Xenopus laevis Zygote arrest 2 (Zar2) binds to the Translational Control Sequence (TCS) in maternal mRNAs and regulates translation. The molecular mechanism of Zar1 has not been described. Here we report similarities and differences between Xenopus Zar1 and Zar2. Analysis of Zar sequences in vertebrates revealed two Zar family members with conserved, characteristic amino acid differences in the C-terminal domain. The presence of only two vertebrate Zar proteins was supported by analyzing Zar1 synteny. We propose that the criteria for naming Zar sequences is based on the characteristic amino acids and the chromosomal context. We also propose reclassification of some Zar sequences. We found that Zar1 is expressed throughout oogenesis and is stable during oocyte maturation. The N-terminal domain of Zar1 repressed translation of a reporter construct in immature oocytes. Both Zar1 and Zar2 bound to the TCS in the Wee1 and Mos 3' UTRs using a zinc finger in the C-terminal domain. However, Zar1 had much higher affinity for RNA than Zar2. To show the functional significance of the conserved amino acid substitutions, these residues in Zar2 were mutated to those found in Zar1. We show that these residues contributed to the different RNA binding characteristics of Zar1 compared to Zar2. Our study shows that Zar proteins have generally similar molecular functions in the translational regulation of maternal mRNAs, but they may have different roles in early development.Biochimica et Biophysica Acta 07/2013; · 4.66 Impact Factor
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ABSTRACT: In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3' untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3' UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 mRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumilio1 and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation.Biochemical and Biophysical Research Communications 04/2014; · 2.28 Impact Factor
Xenopus laevis zygote arrest 2 (zar2) encodes a zinc finger RNA-binding
protein that binds to the translational control sequence in the maternal
Wee1 mRNA and regulates translation
Amanda Charleswortha,b,c,n, Tomomi M. Yamamotoa, Jonathan M. Cooka, Kevin D. Silvaa,
Cassandra V. Kottera, Gwendolyn S. Carterb, Justin W. Holtb,1, Heather F. Lavenderb,
Angus M. MacNicolb,d,e, Yi Ying Wangd,2, Anna Wilczynskab,3
aDepartment of Integrative Biology, University of Colorado Denver, P.O. Box 173364, Denver, CO 80217, USA
bDepartment of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA
cCenter for Translational Neuroscience, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA
dDepartment of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA
eWinthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA
a r t i c l e i n f o
Received 19 March 2012
Received in revised form
12 June 2012
Accepted 17 June 2012
Available online 23 June 2012
a b s t r a c t
Zygote arrest (Zar) proteins are crucial for early embryonic development, but their molecular
mechanism of action is unknown. The Translational Control Sequence (TCS) in the 30untranslated
region (UTR) of the maternal mRNA, Wee1, mediates translational repression in immature Xenopus
oocytes and translational activation in mature oocytes, but the protein that binds to the TCS and
mediates translational control is not known. Here we show that Xenopus laevis Zar2 (encoded by zar2)
binds to the TCS in maternal Wee1 mRNA and represses translation in immature oocytes. Using yeast
3 hybrid assays and electrophoretic mobility shift assays, Zar2 was shown to bind specifically to the TCS
in the Wee1 30UTR. RNA binding required the presence of Zn2þand conserved cysteines in the
C-terminal domain, suggesting that Zar2 contains a zinc finger. Consistent with regulating maternal
mRNAs, Zar2 was present throughout oogenesis, and endogenous Zar2 co-immunoprecipitated
endogenous Wee1 mRNA from immature oocytes, demonstrating the physiological significance of
the protein–RNA interaction. Interestingly, Zar2 levels decreased during oocyte maturation. Dual
luciferase reporter tethered assays showed that Zar2 repressed translation in immature oocytes.
Translational repression was relieved during oocyte maturation and this coincided with degradation of
Zar2 during maturation. This is the first report of a molecular function of zygote arrest proteins. These
data show that Zar2 contains a zinc finger and is a trans-acting factor for the TCS in maternal mRNAs in
immature Xenopus oocytes.
& 2012 Elsevier Inc. All rights reserved.
Maternal effect genes encode proteins or RNAs found in the
cytoplasm of the oocyte that regulate development of the early
embryo after fertilization and prior to zygotic genome activation
(Farley and Ryder, 2008). Maternal effect genes were first char-
acterized in Drosophila in the 1980s, but mammalian maternal
effect genes were not described until 2000 (Li et al., 2010). One of
the earliest mammalian maternal effect factors reported, Zygote
arrest 1 (Zar1) (Wu et al., 2003a), was found in a subtractive
hybridization screen to identify mammalian maternal acting
genes. Female mice null for Zar1 are infertile because embryogen-
esis is blocked at the 1-cell stage. Since then, Zar1 has been
identified in many vertebrate species from frogs to humans
(Brevini et al., 2004; Michailidis et al., 2010; Uzbekova et al.,
2006; Wu et al., 2003b). A sequence related to Zar1 has also been
identified in many vertebrates, and called Zar2 (Xzar2 in Xenopus
laevis) or Zar1-like/Zar1l (Hu et al., 2010; Michailidis et al., 2010;
Misra et al., 2010; Nakajima et al., 2009; Sangiorgio et al., 2008).
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/developmentalbiology
0012-1606/$-see front matter & 2012 Elsevier Inc. All rights reserved.
nCorresponding author at: University of Colorado Denver, Campus Box 171,
P.O. Box 173364, Denver, CO 80217, USA.
Fax: þ1 303 556 4352.
E-mail addresses: firstname.lastname@example.org (A. Charlesworth),
email@example.com (T.M. Yamamoto),
firstname.lastname@example.org (J.M. Cook), email@example.com (K.D. Silva),
firstname.lastname@example.org (C.V. Kotter).
1Current address: University of Colorado School of Medicine, Anschutz
Medical Campus, Aurora, CO 80045, USA.
2Current address: NCTR, AR. USA.
3Current address: MRC Toxicology Unit, University of Leicester, Leicester, UK.
Developmental Biology 369 (2012) 177–190
Both zygote arrest proteins have been implicated in progres-
sion of embryogenesis. Zar1 has been implicated in completion of
fertilization, activation of the zygotic genome and progression
past the 1-cell stage in mouse embryos (Wu et al., 2003a), and
Zar2 (aka Zar1l) has been implicated in epidermalization of
Xenopus embryos and progression past the 2-cell stage in mouse
embryos (Hu et al., 2010; Nakajima et al., 2009). Zar1 and Zar2
transcripts are predominantly expressed in ovary and testis, with
the highest levels in immature (germinal vesicle) oocytes (Brevini
et al., 2004; Hu et al., 2010; Michailidis et al., 2010; Pennetier
et al., 2004; Sangiorgio et al., 2008; Uzbekova et al., 2006; Wu
et al., 2003a; Wu et al., 2003b). Zar1 and Zar2 transcripts decline
by the 2-cell stage in mouse embryos (Hu et al., 2010; Wu et al.,
2003b), the 8-cell stage in pig embryos (Uzbekova et al., 2006),
the blastocyst stage in bovine embryos (Brevini et al., 2004), and
gastrulation in Xenopus embryos (Nakajima et al., 2009; Wu et al.,
2003b). In the chick embryo, Zar1 and Zar2 transcripts are
expressed for at least 7 days (Michailidis et al., 2010). Thus,
Zar1 and Zar2 likely play a role in early zygotic development in
many vertebrate species.
Zar1 and Zar2 share a highly conserved C-terminal domain
sequenced to date, which has led to the suggestion that Zar1
and Zar2 contain zinc fingers that regulate transcription. Indeed,
Zar1 is implicated in activation of the zygotic genome (Wu et al.,
2003a), and Zar2 is implicated in RNA synthesis, histone methyla-
tion and expression of nuclear reprogramming factors (Hu et al.,
2010). However, Zar1 and Zar2 have also been shown to localize
to the cytoplasm in mouse oocytes and embryos, and specifically
to P-bodies, suggesting a role for Zar proteins in RNA metabolism
(Hu et al., 2010; Misra et al., 2010; Wu et al., 2003a). While it is
clear that Zar1 and Zar2 have important roles in early develop-
ment, their molecular mechanism of action is still unclear.
Translational control of maternal mRNAs is critical to the
developing zygote prior to genomic activation (Farley and
Ryder, 2008). A plethora of developmentally regulated cis ele-
ments in the 30untranslated regions of mRNAs determine where,
when and to what extent an mRNA is translated (Colegrove-Otero
et al., 2005; MacNicol and MacNicol, 2010). In vertebrate meiosis,
mRNA translation is often correlated with elongation of the
poly(A) tail in a process called cytoplasmic polyadenylation
(Villalba et al., 2011). Polyadenylation and translation of maternal
Wee1 mRNA occur after the first meiotic metaphase in maturing
Xenopus oocytes (Charlesworth et al., 2000). The resulting Wee1
protein is implicated in the lengthening of the first embryonic
mitotic cell cycle (Murakami et al., 1999; Murakami and Vande
Woude, 1998) and in morphogenesis at gastrulation (Murakami
et al., 2004). Wee1 mRNA translation is under the control of at
least two different types of cis elements in the 30UTR, the
cytoplasmic polyadenylation element (CPE) and the newly
described Translational Control Sequence (TCS) (Charlesworth
et al., 2000; Wang et al., 2008). Both CPEs and TCSs repress
translation in immature oocytes and stimulate translation in
mature oocytes. Whereas the mechanism of translational regula-
tion by CPEs is well documented and is mediated by CPE-binding
protein (CPEB) (Villalba et al., 2011), the mechanism of transla-
tional regulation by TCSs has yet to be described and the
protein(s) that binds to the TCS has yet to be identified.
In a previous study (Charlesworth et al., 2006), we isolated two
proteins in a screen to identify the trans-acting factor that bound
to a new cis-element, the polyadenylation response element
(Charlesworth et al., 2002), now referred to as the Musashi
binding element (MBE) (Arumugam et al., 2010), in the 30UTR
of the Mos mRNA. One protein, Musashi (C36), bound to the MBE,
whereas the second protein (A8) did not. Here, we show that A8 is
the C-terminal of Xenopus laevis Zar2 and that it binds to the TCS
in the Mos and Wee1 mRNA 30UTRs via a zinc finger. Further-
more, we show that Zar2 represses translation in immature
Xenopus oocytes. We propose that Zar2 is a trans-acting factor
for the TCS.
Material and methods
Yeast 3 hybrid assay and plasmids
The yeast screen has been described previously (Charlesworth
et al., 2006). Yeast transformations and b-galactosidase expres-
sion colony lift and liquid culture assays were performed as
described in Yeast Protocols Handbook (Clontech). Yeast strains
and plasmids were kindly provided by Dr. Marvin Wickens,
University of Wisconsin, Madison.
All restriction enzymes, Klenow fragment and Quick Ligase
were obtained from New England Biolabs. DNA oligonucleotides
were synthesized by Integrated DNA Technologies.
pACT2-A8 (C-zar2) was isolated from a Xenopus oocyte cDNA
pIIIA MS2-2.1 Mos M1 48 has been described previously
(Charlesworth et al., 2006). pIIIA MS2-2.1 Mos WT 48 was made
from pIIIA MS2-2.1 Mos M1 48 using QuikChange. The disrupted
CPE (TTTGGT) was changed back to wild type CPE (TTTtaT). pIIIA
MS2-2.1 Mos DMBE was made from pIIIA MS2-2.1 Mos M1 48
using QuikChange (Stratagene). The disrupted CPE (TTTGGT) was
changed back to TTTtaT and 20 nt of the MBE (ATC CAT ATG TGA
ATA TAT AG) (Charlesworth et al., 2002) were deleted. pIIIA MS2-
2.1 Mos DTCS was made from pIIIA MS2-2.1 Mos M1 48 using
QuikChange by deleting the 7 nt TCS (TTTGTCT).
pIIIA MS2 IRE and pACT2 IRP have been described previously
(Bernstein et al., 2002).
pIIIA 2–2.1 b-globin and pIIIA 2–2.1 b-globin/TCS: PCR primers
were designed to amplify the last 81 bp of the b-globin and the
b-globin/TCS 30UTR sequence with a 50XhoI site and a 30NarI site,
using pGEMGST b-globin and pGEMGST b-globin/TCS (TTTGTCT).
The b-globin 30UTR was amplified using Platinum Pfx (Invitrogen)
and the PCR product digested with XhoI and NarI, and ligated into
XhoI/NarI digested pIIIA MS2-2.1.
pIIIA MS2-1 Wee WT: pGEM Wee79 EMSA (Charlesworth
et al., 2000) was digested with EcoRI/HindIII, blunted with
Klenow and ligated into pIIIA MS2-1 (Bernstein et al., 2002) that
was digested with SmaI.
pIIIA MS2-2.1 Wee DTCS: QuikChange mutagenesis was also
employed to delete the TCS elements (ATTGTCT and ATTATCT)
within the Wee1 30UTR to generate Wee1 DTCS.
(Requests for the above plasmids should be made to AMM.)
Cloning of full length zar2b and subsequent plasmids
pENTR zar2b: The 50end of zar2b was cloned using First Choice
RLM-RACE Kit (Ambion), according to manufacturer’s directions.
50RACE was performed on the mRNA from immature oocytes
from two different frogs to ensure the full length 50end was
identified. The inner 50RACE primer was 50-GTC GAC TGG CCA
AGG GCT GCG CGA C and the outer primer was 50-GGT AAT CCG
TGG GCT CAG AAG CCT T. EST sequences AW644676, BJ614564
and AW637792 were used to verify the integrity of sequence we
had identified and support the idea that we have the paralog of
zar2 that was previously reported (Nakajima et al., 2009). Once
the sequence from the 50end was confirmed, primers were
designed to amplify the full length zar2b, forward primer 50-CAC
CAT GGC GGG CTT TAT GTA TGC GC, reverse primer 50-TCA GAC
GAT GTA CTT GTA GCT GTA AGT GTT GT, using the high fide-
lity Pfu (Stratagene) according to manufacturer0s directions.
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
50Primers had CACC (underlined) for directional cloning into
pENTR (Invitrogen). The full length zar2b sequence has been
submitted to Genbank (Genbank ID: JQ776638).
pVL1393-FLAG-C-zar2 for baculovirus protein expression:
pVL1393 (Orbigen) was cut with BamHI/PstI. A duplex encoding
3xFLAG with BamHI and XbaI sites was synthesized (IDT). The
C-terminal 159–307 amino acids were amplified from pENTR
zar2b, forward primer 50-GCA TCT AGA ATG GCG GGC TTT ATG
TAT GCG and reverse primer, 50-GCT CTG CAG TCA GAC GAT GTA
CTT GTA GCT with XbaI and PstI sites (underlined). The 3xFLAG
and C-zar2 were digested with the appropriate restriction
enzymes and ligated into pVL1393.
pGEX 6P-3 zar2b, pGEX 6P-3 N-zar2 (amino acids 1–158),
pGEX 6P-3 C-zar2 (amino acids 159–307) and cysteine mutants:
Plasmids were made by inserting PCR products from template
pENTR zar2b into pGEX 6P-3 vector (GE Lifesciences). The forward
primer for full length and N-terminus was 50-GAT CGG ATC CAT
GGC GGG CTT TAT GTA T and the reverse primer for full length
was 50-CTA GGT CGA CTC AGA CGA TGT ACT TGT A, and for the
N-terminus was 50-CTA GGT CGA CTC ACT CCT TCA GCG GCT GTG
A, with BamHI and SalI sites (underlined). The forward primer for
C-terminus was 50-CGG GAT CCA GAG CGC CCT CCC CCG AG and
the reverse primer was 50-ATA AGA ATG CGG CCG CTC AGA CGA
TGT ACT TGT AGC, with BamHI and NotI sites (underlined). The
cysteine to alanine mutations were performed by QuikChange
mutagenesis methods with the following codon changes: C215A,
TGC-GCC; C242A, TGT-GCT; C259A, TGC-GCC; C267A, TGC-
GCC; C287A, TGC-GCC.
pXen N-MS2: PCR primers were designed to amplify the MS2
coding sequence from pJC5 (kindly provided by Jeff Coller, Case
Western Reserve University, OH) (Gray et al., 2000) with a 50NcoI
site and a 30ClaI site, forward primer 50-GAT CCC ATG GCT TCT
AAC TTT ACT CAG TTC and reverse primer 50-GAT CAT CGA TGC
GTA GAT GCC GGA GTT TGC TGC. Digested PCR product was
ligated into NcoI/ClaI digested pXen1, replacing the GST coding
pXen MS2-Xp54: PCR primers were designed to amplify the
Xp54 coding sequence from Xp54 in the MSP vector (Minshall
et al., 2001) (kindly provided by Nancy Standart, University of
Cambridge, UK) with a 50KpnI site and a 30BamHI site, forward
primer 50-GAT CGG TAC CCA TGA GCA CCG and reverse primer 50-
GAT CGG ATC CTT AAG GTT TGT. Digested PCR product was
ligated into KpnI/BamHI digested pXen N-MS2.
pXen N-zar2-MS2: pXen1 was digested with NcoI and ClaI to
remove GST coding sequence, then treated with Klenow to blunt
ends and self-ligated make pXen DGST. MS2 coding sequence was
amplified from pJC5 using primers with a 50XmaI site and a 30
XbaI site, forward primer 50-CTA GCC CGG GCT ATG GCT TCT AAC
TTT ACT CAG TTC and reverse primer 50-GAT CTC TAG AGT TAG
TAG ATG CCG GAG TTT GCT G. Digested PCR product was then
ligated into XmaI/XbaI digested pXen DGST to make pXen C-MS2.
Amino acids 1–158 of zar2b was amplified from pENTR-zar2b
with a 50KpnI site and a 30BamHI site, forward primer 50-GAT
CGG TAC CAT GGC GGG CTT and reverse primer 50-GAT CGG ATC
CGC TCT CTT CAG, appropriately digested, and ligated into KpnI/
BamHI digested pXen C-MS2.
pXen rluc: PCR primers were designed to amplify Renilla
luciferase (rluc) coding sequence (plasmid kindly provided by
Nancy Standart) with a 50NcoI site and a 30ClaI site, forward
primer 50-CAT GCC ATG GCT TCG AAA GTT TAT GAT CCA and
reverse primer 50-GAT CAT CGA TTT ATT GTT CAT TTT TGA GAA
CTC G. Digested PCR product was then ligated into NcoI/ClaI
digested pXen1, replacing the GST coding sequence. This plasmid
was linearized with EcoRI prior to in vitro transcription.
pXen fluc: The firefly luciferase (fluc) coding sequence was
amplified from JC18 (kindly provided by Jeff Coller) (Gray et al.,
2000) using primers with a 50NcoI site and a 30XhoI site, forward
primer 50-GAT CCC ATG GAA GAC GCC AAA AAC ATA AAG and
reverse primer 50-GAT CCT CGA GTT ACA ATT TGG ACT TTC CGC C.
The PCR product was inserted into pXen1 using NcoI/XhoI,
replacing the GST coding sequence.
pXen fluc-2x-SL (pXen fluc with stem–loops): An Nde I site
was inserted into the b-globin 30UTR of pXen fluc, 59 nucleotides
upstream of the polyadenylation hexanucleotide, using Quik-
Change site directed mutagenesis (Stratagene) to make pXen
fluc-NdeI. A DNA duplex (IDT) containing the sequence of two
MS2 stem–loops (2?-SL) (Bardwell and Wickens, 1990) with 50
and 30NdeI sites was digested with NdeI and ligated into NdeI
digested pXen fluc-NdeI. This plasmid was linearized with SacI
prior to in vitro transcription.
All plasmids were sequenced to verify integrity using the
University of Colorado Cancer Center DNA Sequencing and Ana-
lysis Core. Requests for the latter plasmids should be made to AC.
For in vitro transcription, all plasmids were linearized with PstI
unless otherwise noted. 50capped RNA was synthesized in vitro
with SP6 mMessage mMachine transcription kit (Ambion). RNA
quality was assessed using gel electrophoresis.
Insect cell protein expression and purification
FLAG-tagged C-terminus Xenopus Zar2 (FLAG-C- Zar2) protein
was prepared from baculovirus infected Sf9 insect cells. Infections
and cell culture were performed by Lori Sherman, Protein Produc-
tion/Mab/Tissue Culture Core Manger, University of Colorado
Cancer Center. About 3 ml of pelleted cells were suspended in
lysis buffer (50 mM Tris HCl pH 7.7, 150 mM KCl, 0.1% Triton
X-100, complete protease inhibitors (Roche)) and lysed by soni-
cating 20 s twice at 30% output (Virsonic 50, Virtis). After
centrifugation at 14,000?g for 15 min at 4 1C, supernatant was
collected and incubated with 3 ml of anti-FLAG agarose (Sigma)
overnight at 4 1C. Beads were washed with lysis buffer supple-
mented with 0.35 M NaCl for 1.5 h and protein was eluted with
3xFLAG peptide overnight. After quality and quantity of eluate
were checked by SDS-PAGE, protein was concentrated with
Vivaspin2 columns (GE Healthcare) at 5000?g until a concentra-
tion of 0.4 mg/ml was reached.
Bacterial protein expression and purification
GST-C-Zar2 protein was purified from E. coli BL21 (DE3)
(Novagen). The protein expression was induced with 0.5 mM
IPTG overnight at 25 1C. Cell pellets were suspended into GST
lysis buffer (PBS, 0.1% Triton X-100, Complete EDTA-free protease
inhibitor (Roche)) and lysed by sonicating 20 s, 3 times at
30% output (Virsonic 50, Virtis) on ice. After centrifugation,
supernatant was incubated with glutathione sepharose beads
(GE Healthcare) for 1.5 h at room temperature. Beads were
washed with wash buffer (PBS, 0.5 M NaCl, 1% Triton X-100) for
30 min at 4 1C. GST-C-Zar2 was eluted from beads with elution
buffer (20 mM reduced glutathione, 10 mM Tris–HCl pH 8.0,
150 mM NaCl, 1 mM DTT, 0.1% Triton X-100), dialysed against
dialysis buffer (50 mM Tris–HCl pH 7.4, 300 mM NaCl, 1 mM DTT,
0.01% TritonX-100) using Slide-A-Lyzer 10,000 MWCO (Thermo
Scientific), and concentrated with Vivaspin2 (GE Healthcare). For
the Zn2þrequirement study, 1 mM EDTA was added to lysis and
wash buffers at the time of purification, but not to elution or
Electrophoretic mobility shift assays (EMSA)
RNA probes were the last 50 nt of the Wee1 30UTR labeled at
the 50end with Cy5 (Integrated DNA Technologies) and were heat
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
denatured immediately prior to use. 80 fmol of probe was
combined with 400–800 ng protein (or as described in figure
legends) in a 20 ml reaction containing 10 mM HEPES pH 7.7,
100 mM KCl, 1 mM MgCl2, 10 mM DTT, 20 mM ZnCl2, 50 mg/ml
tRNA, 0.1 mg/ml BSA, 5% glycerol, 0.25% NP40 and incubated
20 min at room temperature. 0.5 ml of heparin (200 mg/ml) was
added for a further 20 min. 5 ml of 5? loading buffer (0.15 g/ml
Ficoll 400, 0.25% Orange G, 1xTBE) was added to the binding
reaction and 5 ml of this loaded onto a 6% RNA retardation gel
(Novex, Invitrogen). Gels were run according to manufacturer0s
instructions. For the Zn2þrequirement study, ZnCl2was elimi-
nated from the binding buffer. Anti-FLAG (Sigma) and anti-C-Zar2
antibodies used for the supershift assay were added for the last
10 min of the binding reaction. For competition assays, proteins
were preincubated for 20 min at room temperature with unla-
beled RNA (IDT). The gel was imaged directly with the Odyssey
(LiCor) in the 700 nm channel.
Oocyte isolation, culture and microinjection
Adult female Xenopus laevis were housed and sacrificed
according to internationally recognized guidelines and with the
approval of the University of Colorado Denver Institutional
Animal Care and Use Committee. Xenopus laevis (Nasco) oocytes
were isolated and cultured as has been described (Machaca and
Haun, 2002). All incubations were carried out in 0.5? L-15
(MediaTech, Inc.) with penicillin (100 mg/ml) and streptomycin
(50 mg/ml). Dumont stage VI (Dumont, 1972) oocytes were
selected and injected with 23 nl of the appropriate MS2 fusion
mRNA: approximately 1 ng of MS2, 20 ng of MS2-Xp54, and 5 ng,
20 ng or 50 ng of N-zar2-MS2. Oocytes were incubated overnight
(?16 h) at 18 1C, then injected with ?100 pg of fluc-2?-SL or
fluc mRNA, together with ?5 pg of rluc mRNA as a loading
control, in 23 nl of nuclease free water. Injections were performed
using a Drummond NanoInject II microinjector, and media was
supplemented with 2.5% Ficoll 400 during injections. Appropriate
samples were then induced to mature with 2 mM progesterone
and all samples were collected 2–3 h after progesterone treated
samples underwent GVBD, as indicated by the appearance of a
white spot. Immature samples were time-matched to progester-
one-treated samples (?11 h after luciferase injection).
Luciferase assay and statistical analysis
Two pools of 5 oocytes were collected from each sample and
lysed in duplicate in 50 ml/oocyte of Passive Lysis Buffer (Pro-
mega). A 10 ml portion of cleared lysate was analyzed using the
Dual-Luciferase Reporter Assay System (DLR) (Promega) on a
Synergy HT plate reader (BioTek). The ratio of firefly to Renilla
luciferase activity (DLR ratio) was calculated for each experimen-
tal point. The mean DLR ratio was calculated for immature oocyte
samples injected with MS2 alone and all immature samples were
normalized to this value, resulting in MS2 alone values being
arbitrarily set to 1 and all other values being expressed as a
change in translation relative to MS2 alone. Progesterone-treated
samples were similarly normalized to the mean value of proges-
terone-treated MS2 alone. This experiment was performed inde-
pendently with the same parameters on multiple frogs (n¼4–5)
and normalized results were pooled. One-way analysis of var-
iance and post hoc Bonferroni multiple comparison test were
performed using GraphPad Prism 5 software to analyze difference
among the means of the normalized results. Differences with P-
value less than 0.01 were considered statistically significant.
Additionally, a Mann–Whitney U test was performed to analyze
differences among medians of the normalized results with similar
Pools of 10 oocytes were collected from each sample and lysed
in 10 ml/oocyte of NP-40 lysis buffer (1% Igepal CA-630, 20 mM
Tris pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA) supplemen-
ted with protease and phosphatase inhibitors (HALT, Pierce). 5 or
10 ml of cleared lysate (0.5 or 1 oocyte equivalent respectively)
was loaded onto a NuPage 4–12% Bis-Tris polyacrylamide gel
(Novex). For stages I–VI and in vitro matured oocytes, oocytes
were lysed in NP-40 lysis buffer and total protein amount was
measured by BCA protein assay kit (Pierce). 3.75 mg total protein
per lysate was loaded onto the gel. Electrophoresis was performed
using MOPS-SDS running buffer (Invitrogen), then transferred to
0.45 mm Immobilon-FL PVDF membrane (Millipore) using an
XCell II Blot Module (Invitrogen) according to NuPage technical
guide protocol. Membranes were probed with MS2 antibody
(TetraCore), GST antibody (Santa Cruz Biotechnology), b-Tubulin
antibody (Sigma), or Zar2 antibodies (Charlesworth). Secondary
antibodies were 1:20,000 IR Goat anti-rabbit IR Dye 800 CW and
Goat anti-mouse IR Dye 680LT (LiCor). Membranes were imaged
on an Odyssey infrared imager and data was analyzed using
Odyssey 2.1 software (LiCor).
Custom Zar2 antibody preparation
Anti-C-Zar2 antibodies were raised in rabbit against peptides
encoding C-terminus (amino acids 267–286) of Zar2 and anti-N-
Zar2 antibodies were raised against N-terminus (amino acids
29–44) and purified by peptide column (Proteintech). To test
antibody specificity, 1.5 mg/ml of the immunizing peptide was
incubated 1 h, 25 1C with the primary antibody before adding to
the transfer membrane.
RNA immunoprecipitation and analysis
Pools of 50 oocytes were lysed in 500 ml homogenization
buffer (150 mM NaCl, 50 mM Tris pH 7.5, 0.5% NP40, 2% BSA)
supplemented with 10 mM ribonucleoside vanadyl complex
(NEB), HALT protease inhibitors, RNaseOUT (Invitrogen) and
1 mM DTT. Lysates were clarified by centrifugation 2?5 min,
14,000?g, 4 1C. 4 mg of antibody was added and incubated for
6 h, 4 1C. 30 ml of a 1:1 slurry of protein A/G PLUS-agarose (Santa
Cruz Biotechnology) was added and samples were rotated for
30 min. Agarose beads were washed twice with homogenization
buffer and twice with NP40 lysis buffer. 5 oocyte equivalent of
beads was used to analyze immunoprecipitation efficiency by
western blot. RNA was extracted from the beads using RNA STAT-
60 (Tel-Test) according to manufacturer0s directions.
The amount of immunoprecipitated RNA was determined by
semi-quantitative PCR. cDNA was synthesized from 40 oocyte
equivalents of RNA using iScript (Bio-Rad). 1 ml of cDNA was
amplified using Platinum Taq (Invitrogen). Annealing was per-
formed at 60 1C. The following gene-specific primers and cycles
were used: Mos (f) 50-GAG AAT CAC AGT TCC ACA GCA ACC, Mos
(r) 50-AGA CAG TTC CCC CAA CAG AAG C, 30 cycles; Wee1a (f)
50-TGC CGG AAG CAG ACA GAG TTG G, Wee1a (r) 50-TTA GCG GCT
TTC AAC TCC CTC TCA, 25 cycles; Protein Phosphatase Inhibitor 2
(PPI2) (f) 50-CGT GTC ATT AGC AAG CCA GAG AC, PPI2 (r) 50-GCA
ATC AAG TGT CTG GCG AGT C, 40 cycles. The cycle number was
determined by running 1/100 and 1/1000 oocyte equivalent of
cDNA from total RNA and making sure there was a difference in
the amount of PCR product between these two amounts of cDNA.
Also, these standards were used to normalize the exposure of the
gels so all the positive controls look the same and the immuno-
precipitated mRNAs can be directly compared.
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
Analysis of RNA destabilization
Total RNA was extracted from pools of 20 oocytes using
Tri ReagentsSolution (Ambion) according to manufacturer0s
directions. A LiCl precipitation step was included for further
purification. cDNA was synthesized from 2 mg of total RNA using
Superscript III (Invitrogen) and oligo dT(12–18)according to manu-
facturer0s directions. PCR was performed in duplicate (only one
set of bands is shown) using One TaqTMHot Start (New England
BioLabs) according to manufacturer0s directions. Forward primer
was fluc 50-TCT TCC CGA CGA TGA CGC, reverse primer was b-
globin 50-AGA CTC CAT TCG GGT GTT CTT GAG G. Annealing was
carried out at 56 1C and the reaction proceeded for 26 cycles.
Quantitative range was determined by using dilutions of the
Isolation of a protein that interacts with the TCS
A previous study screened an unfertilized Xenopus laevis egg
library to identify proteins that bound to the MBE in the 30UTR of
the Mos mRNA (Charlesworth et al., 2006). One of the proteins
that was isolated was Musashi and we showed that Musashi
bound to the MBE in the Mos 30UTR. Another protein, designated
A8, was isolated in that screen, and we characterize A8 in this
study. The screen was performed with the Mos M1 48 Mos UTR
where the last 48 nucleotides of the Mos UTR with a mutation
(M1) in the CPE (UUUAAU to UUUggU) were used to prevent
recovery of CPEB. As A8 was recovered on Mos M1 48 this
demonstrated that A8 did not bind to the CPE. The yeast three
hybrid assay was further used to determine where A8 interacted
with the Mos UTR. To show that A8 interaction was not an artifact
of binding to the mutant CPE, the CPE was restored to make Mos
WT 48. A8 still interacted with Mos WT 48 showing that the
interaction was not an artifact of the mutant CPE. The interaction
did not appear as strong for unknown reasons. Next we tested if
A8 interacted with the MBE in the Mos 30UTR. The MBE was
deleted from the Mos 30UTR in the RNA hybrid to make Mos
DMBE as shown in Fig. 1A. Removal of the MBE (Mos DMBE) did
not prevent interaction with A8 (Fig. 1B), indicating that A8 does
not interact with the MBE and likely interacts with a different
element within the 30UTR of Mos mRNA. The only place left for
this element to be located was 30of the polyadenylation hexanu-
cleotide. Accordingly, when these seven nucleotides at the very 30
end of the UTR were deleted to make Mos DTCS, interaction with
A8 was abrogated. Sequence analysis revealed that these seven
nucleotides were remarkably similar to the TCSs in the Wee1 30
UTR. This suggested that Mos has a TCS (MacNicol and MacNicol,
2010) and that A8 was a TCS-interacting protein. The Wee1 30UTR
has three maturation-type CPEs (Charlesworth et al., 2000) and
two TCSs between the first two CPEs (Wang et al., 2008). To test if
A8 interacted with other TCSs, the Wee1 30UTR was used in
the yeast three hybrid assay, either with the TCSs intact (wild
type, Wee WT) or with the TCSs deleted (Wee DTCS) (Fig. 1A).
A8 interacted with Wee WT, but not when the TCSs were deleted
(Wee DTCS), indicating that the TCS is necessary for interaction
relative βgal activity
MS2 Mos WT 48
MS2 Mos M1 48
βg/TCS Wee ΔTCS
MS2 Wee WT
Fig. 1. Characterization of a TCS binding protein by yeast three hybrid analysis. (A) Cartoon of the RNA hybrids used: white oval, MBE; square, CPE; hexagon,
polyadenylation hexanucleotide; red oval, TCS; X, disrupted CPE; dotted line, deleted sequences; bold line, Iron Response Element (IRE). (B) Yeast three hybrid colony lift
assay for lacZ expression. Blue color indicates RNA–protein interaction. Yeast were transformed with pACT2-A8 and pIIIA plasmids containing the indicated RNA hybrids
and three independent colonies streaked for each analysis. A8 interacts with sequences 30of the polyadenylation hexanucleotide in the Mos 30UTR, the TCS region in the
Wee1 30UTR, and a TCS inserted into the b-globin 30UTR. (C) Yeast three hybrid liquid culture assay for lacZ expression. Yeast were transformed with the indicated RNA
and protein hybrids. Chart shows b-galactosidase activity. The IRE/Iron Response Protein (IRP) interaction serves as a positive control (SenGupta et al., 1996). More
b-galactosidase activity was observed when the TCS was present in the b-globin 30UTR.
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
with A8 (Fig. 1B). It should be noted that the Wee DTCS construct
still contains two intact CPEs, further showing that A8 does not
interact with CPE sequences. To test if the TCSs are sufficient for
interaction with A8, we inserted the Mos TCS into b-globin 30UTR
(bg/TCS). A8 did not interact with wild type b-globin 30UTR (bg),
but did interact with bg/TCS, as assessed by both colony lift assay
(Fig. 1B) and liquid culture assay (Fig. 1C). These data demon-
strate that a TCS is necessary and sufficient for interaction
Identification of A8
BLASTs(NCBI) analysis showed that the amino acid sequence
of A8 was similar to the entire C-terminal half (i.e. amino acids
159–307) of mouse Zygote arrest1 (Zar1) (Wu et al., 2003a).
To ensure that the most complete and accurate 50end of A8 was
identified for analysis and cloned for mechanistic studies, 50RACE
from two individual frogs was performed. The full-length
sequence of A8 was 1128 nt, predicting a protein of 307 amino
acids. Upon comparison, the full-length sequence of A8 was more
similar to zar2 (Nakajima et al., 2009), which was identified
during the timeframe of this study, than to Xenopus laevis Zar1
(Genbank ID: AY283176) (Wu et al., 2003b) (Fig. 2A). We propose
we have cloned the paralog of zar2 arising from the pseudote-
traploid nature of Xenopus laevis. Further, we suggest that zar2
(Genbank ID: AB190316) (Nakajima et al., 2009) be designated as
zar2a and our sequence from this point forward be designated as
zar2b (Genbank ID: JQ776638). According to this proposed
nomenclature, all experiments in this study were performed with
zar2b. In addition to the observed C-terminal conservation
between Zar1 and Zar2 proteins, an area of homology in the
N-terminal was also observed. In the subsequent experiments,
we show that the C-terminal half of Zar2 binds RNA and the
N-terminal half regulates mRNA translation, as summarized in
- - - - M Y P A Y N P Y S - Y R Y L N P R N K G M S W R Q - K N Y L A S Y G - - - - - D T G D Y C D
M A G F V Y S P Y N A Y Q G Y G G N F G Q N P H R P Q A F P K N K Q A A W K S N K S S E P P D Y L D
M A G F M Y A P Y N V Y Q G Y G G N F G Q N P H V A Q P L A K N K Q A A W K S N K A S E P T D Y L D
N Y Q R A Q L K A I L S Q V N P N L T P R L C R A N T R D V G V Q V N P R Q D A S V Q C S L G P R T
H F Q R A Q L K A I L S Q V N P N L T P R L R K V N T K E M G V Q V N P R V D T G V Q C S L G P R T
N F Q R A Q L K A I L S Q V N P N L T P R L R K A N T K E I G V Q V N P R V D T G V Q C S L G P R T
L L R R R P G A L R K P P P E Q G S P A S P T K T V R F P R T I A V Y S P V A A G R L A P F Q D E G
L R R - - - - - - - - P P P P P S S P V K P T D C A R F S R P V A V Y S P V V D R R L F S L P - Q G
L R R - - - - - - - - P P P P P C S P V K A A D C V R F T R P L A V Y S P V V D R R L F S L P - Q G
V N L E E K - - - G E A V R S E G S E G G R Q E G K - - - - - - - - Q G D G E I K E Q M K M D K T D
G R L P K K S L P A P D S Q S Q P L K D R G P S P E - - - - - - - - E K E P E T K E A L E K S P V P
G R L P K K - - - S P D S Q S Q P L K E R A P S P E D K E R E K V S E K E P D T K D E L E K R P V P
- - E E E A A P A Q T R P K F Q F L E Q K Y G Y Y H C K D C N I R W E S A Y V W C V Q E T N K V Y F
G A E E P N E E E P N K P A F Q F L E Q K Y G Y F H C K D C K T R W E S A Y V W C I S G S N K V Y F
G T E E P G N E E Q T K S A F Q F L E Q K Y G Y F H C K D C K T R W E S A Y V W C I S G S N K V Y F
260270280 290 300
K Q F C R T C Q K S Y N P Y R V E D I M C Q S C K Q T R C A C P V K L R H V D P K R P H R Q D L C G
K Q L C R K C Q K G F N P Y R V E V I Q C Q V C A K T R C S C P Q K K R H I D L K R P H R Q E L C G
K Q L C R K C Q K G F N P Y R V E A I Q C Q V C A K T R C S C P Q K Q R H I D L K R P H R Q E L C G
R C K G K R L S C D S T F S F K Y I I
R C K N K K L S C D N T Y S Y K Y I V
R C K N K R L S C D N T Y S Y K Y I V
translation control domainRNA binding domain
Fig. 2. A8 is the C-terminal of Zar2. (A) Alignment of Xenopus laevis Zar1 and Zar2 amino acid sequences was performed using MacVector 11.1.2. A blue box and arrow
below the sequences designates the start of A8. Shaded areas indicate identical amino acid residues. Residues targeted by antibodies are highlighted in yellow and labeled
below the sequence. Cysteine pairs conserved in both Zar1 and Zar2 proteins are highlighted in red. Cysteines targeted for mutation are indicated below the residue of
interest in bold text. Genbank accession numbers are: Zar1, AAP37038; Zar2a, BAH36746; and zar2b (Zar2b), JQ776638. For purposes of discussion, the putative loop 1 is
between C215 and C242, and the putative loop 2 is between C259 and C287. (B) Proposed schematic of Zar protein regions and functions. Shaded areas (in grey) represent
regions of high sequence homology between Zar1 and Zar2 while red lines indicate conserved cysteine pairs of interest. The box and arrow designate the boundary of the
A8, depicted as a blue line.
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
Expression of Zar2 during oogenesis and meiotic maturation
The data in Fig. 1 suggested that Zar2 might bind to maternal
mRNAs that are regulated during oocyte maturation. To further
this line of study, it was essential to determine if Zar2 protein is
present in Xenopus oocytes. To do this we raised antibodies to
peptides in the C-terminal and N-terminal domains. An antibody
specific to a peptide in the C-terminal domain that had the most
mismatches between Zar2 and Zar1 (amino acids 267–286)
(Fig. 2A) was developed. To test that the antibody recognized
Zar2, we expressed GST-Zar2 in immature oocytes. The C-term-
inal Zar2 antibody recognized a band at the same size as the GST
antibody (Fig. 3A). To show specificity we blocked the antigen
binding sites on the C-terminal Zar2 antibody by incubating with
the immunizing peptide before western blot. This treatment
prevented the C-terminal Zar2 antibody from recognizing GST-
Zar2, demonstrating that the antibody was specific. Recognition
of Zar2 and not Zar1 by the C-terminal antibody was verified by
western blot of recombinant proteins (Fig. 3D). The C-terminal
antibody is used for western blot. We also raised an antibody for
immunoprecipitation against a peptide in the N-terminal of Zar2
that was predicted to be on the surface of the protein, amino acids
29–44 (Fig. 2A). These amino acids are not conserved at all
between Zar2 and Zar1. We analyzed the N-terminal antibody
in a similar fashion to the C-terminal antibody (Fig. 3B). The
N-terminal antibody recognized GST-Zar2 and this recognition
was blocked by the immunizing peptide. We use the N-terminal
antibody mainly for immunoprecipitation (Fig. 6), however, it is
also useful for western blot. To test if the antibodies could detect
endogenous Zar2 we western blotted lysates from immature and
progesterone-stimulated mature oocytes (Fig. 3C). Both Zar2
antibodies recognized a band that migrated about 39 kDa
and for both antibodies this band was more intense in imma-
ture oocytes compared to mature oocytes. We conclude that
endogenous Zar2, which has a predicted molecular weight of
35 kDa, migrates at 39 kDa on a polyacrylamide gel, similar to
mouse Zar1 (Wu et al., 2003a). We have expressed different
truncations of Zar2 with different tags in several eukaryotic cell
IV V VII IIIIIE
single oocyteconstant protein
IV V VI I II IIIE
– + – + – +
+ 29-44 peptide
– + – +– +
+ 267-286 peptide
Fig. 3. Zar2 protein expression during oogenesis and oocyte maturation. (A–D). Characterization by western blot of the antibodies used in this study. A and B Lysates from
uninjected (–) or GST-Zar2-expressing (þ) immature oocytes were analyzed by the purified C-terminal Zar2 antibody (A), or N-terminal antibody (B), or GST antibody as
indicated. The immunizing peptide 267–286 (A), or 29–44 (B), was used to block specific antibody sites. Both antibodies recognize GST-Zar2. (C) Lysates from uninjected
immature (I) or progesterone-stimulated mature (P) oocytes were analyzed with both Zar2 antibodies. A common band at 39 kDa is seen that is endogenous Zar2. (D)
Lysates from immature oocytes expressing GST-C-Zar2 or GST-C-Zar1 were analyzed by anti C-Zar2 or GST antibodies. The C-Zar2 antibody does not recognize GST-C-Zar1.
(E–G). Endogenous Zar2 expression. Western blots using Zar2 C-terminal antibody (solid arrowhead) and b-Tubulin (open arrowhead) as a loading control. Representative
blots are shown. Similar results were obtained in oocytes from 4–5 different frogs. (E) Pools of oocytes were lysed and protein equivalent to a single oocyte from stage I to
VI or a single in vitro matured oocyte (egg) (E) was loaded. (F) 3.75 mg of total protein was loaded. (G) Pools of oocytes were lysed at different times after progesterone
stimulation and protein equivalent to 0.5 oocyte was loaded.
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
types and Zar2 consistently migrated slightly larger than pre-
dicted (data not shown), which suggests post-translational mod-
ification. The C-terminal antibody recognized an additional band
that migrated at 32 kDa (Fig. 3), however, the 32 kDa band was
not detected by the N-terminal antibody (Fig. 3C), suggesting that
amino acids 29–44 were not present and that the 32 kDa band is
therefore not an unmodified version of Zar2. A similar smaller
band was also seen with antibodies to mouse Zar1, and it was
proposed that this might be a degradation product (Wu et al.,
2003a). Although it has been shown that the 50end of the Zar1
(Uzbekova et al., 2006), isolation of the 50end of zar2 did not
reveal any evidence of alternative 50ends in our hands. At this
time, we do not know if the smaller band recognized by the
C-terminal Zar2 antibody is a degradation product, a product of
alternative splicing or a cross-reacting protein that is not Zar2.
The C-terminal antibody also recognizes a band at 20 kDa. As this
band follows the same pattern as Tubulin expression (Fig. 3E–G),
we think it is an unrelated cross-reacting band.
Using the C-terminal antibody, Zar2 protein levels during
oogenesis were characterized. The total amount of Zar2 per
oocyte increased during stages I–IV of oogenesis (Dumont,
1972), as the oocytes grow in size. Zar2 reached maximum levels
at stage IV, even though the oocyte continues to grow in size from
stage IV to stage VI, as seen by the increase in Tubulin (Fig. 3E).
However, it was stage I oocytes that had the greatest concentra-
tion of Zar2 relative to Tubulin (Fig. 3F). Interestingly, the 32 kDa
band showed a similar profile. These data suggest that Zar2 might
play a role in early oogenesis.
Next, we characterized Zar2 levels during meiotic maturation.
Levels of Zar2 gradually declined during oocyte maturation
(Fig. 3G). In mature oocytes, there was a 60–80% (n¼5) decrease
in the amount of endogenous Zar2 compared to immature
oocytes. Interestingly, the 32 kDa band showed a similar profile.
Thus Zar2 protein is present in immature oocytes, consistent with
a role in maternal mRNA regulation.
Zar2 binds specifically to the TCS
As A8 (the C-terminal domain of Zar2) interacted with TCSs in
the Wee1 and Mos 30UTRs (Fig. 1), we examined whether Zar2
directly binds to the TCS by the use of electrophoretic mobility
shift assays (EMSA). In these experiments 50 nt of the Wee1
30UTR, very close to the poly(A) tail, were used as the labeled
probe (Fig. 4A). The cis elements are indicated. There are three
CPEs and two TCSs. The TCSs overlap with the CPEs. The nucleo-
tides of the TCS mutation (UU to gg) that disrupt polyadenylation
and translation control (Wang et al., 2008) are marked in red.
The C-terminal of Zar2 was FLAG-tagged (FLAG-C-Zar2) and
expressed in Sf9 cells. The binding of FLAG-C-Zar2 to the Wee1
30UTR was determined. Fig. 4B shows that a specific complex
forms when FLAG-C-Zar2 is purified from baculovirus infected Sf9
cells but not with proteins mock purified from uninfected cells.
Also there was no specific complex formed when the FLAG elution
buffer was used in the EMSA. This shows that the specific
complex was not formed from endogenous insect proteins or
from FLAG peptides. The specific complex could be supershifted
by antibodies to FLAG and Zar2, but not by antibodies to Tubulin,
showing that the complex contained FLAG-C-Zar2 (Fig. 4B).
To show that Zar2 was targeting the TCS in the Wee1 30UTR,
we used competition assays with unlabeled RNA specifying the
same 50 nt as the labeled Wee1 probe. The labeled probe could be
competed with a 50-fold excess of unlabeled Wee1 UTR, but it
could not be competed when the TCSs in the unlabeled RNA were
disrupted (red UU mutated to gg) (Fig. 4C). It should be noted that
mutation of TCS1 also disrupts CPE1, however there are two other
CPEs present in the unlabeled probe and neither of them com-
petes with the labeled probe for Zar2 binding. These results
indirectly show that Zar2 binds to the TCS. To directly show that
Zar2 binds to the TCS, we used labeled probes where the TCSs had
the same mutations as in the competition study. For these
experiments, purified bacterially expressed Zar2 proteins were
used. First, we verified that GST-tagged Zar2 expressed in E. coli
binds to the Wee1 30UTR. Fig. 4D shows that GST-C-Zar2 bound
to the Wee1 30UTR, but GST alone or the N-terminal of Zar2
(GST-N-Zar2) did not bind, even though equivalent amounts of
protein were used (Fig. 4D lower panel). Next, we asked if GST-C-
Zar2 could bind to mutant Wee1 UTRs. When either TCS1 or TCS2
was disrupted, the binding of Zar2 was unchanged (Fig. 4E).
However, when both TCSs were disrupted, binding of Zar2 to
the Wee1 30UTR was markedly reduced. The number of CPEs was
the same in the TCS1 mutant versus the TCS1&2 mutant showing
that the change in binding was not due to Zar2 binding to the
CPEs. Increasing amounts of Zar2 protein showed a dose response
with respect to the amount of complex formed, verifying the
specificity of this reaction. Because only one specific complex is
seen, these data show that Zar2 can bind to either TCS, but two
Zar2s do not bind to both TCSs at the same time.
Zar2 contains a zinc finger
The conserved C-terminus of Zar1 has been speculated to
contain an atypical PHD domain, a fungal C6 domain, or a FYVE
domain based on conserved cysteine residues (Sangiorgio et al.,
2008; Uzbekova et al., 2006; Wu et al., 2003a). All of these
domains are zinc fingers, but they are found in proteins with
different functions, such as histone modification, phospholipid
binding and DNA binding (Matthews and Sunde, 2002; Todd and
Andrianopoulos, 1997). Also, it is not known which cysteines in
Zar1 form the putative zinc finger(s). To test whether the
C-terminal of Zar2 contained a zinc finger, we purified the protein
from bacteria in the presence of EDTA to remove pre-incorporated
Zn2þand then performed the EMSA either with or without adding
back Zn2þto the binding buffer. EDTA treatment also removed
Mg2þthat was essential for Zar2 RNA binding, but activity was
recovered when Mg2þwas added back to the binding buffer (data
not shown). In decreased Zn2þconditions (but normal Mg2þ
conditions), the binding of Zar2 to the Wee1 30UTR was
dramatically reduced (Fig. 5A), demonstrating that Zar2 binds to
RNA using a zinc finger. To substantiate this result, we mutated
one cysteine in each conserved pair of cysteines illustrated in
Fig. 2. The ability of the mutant proteins to bind the Wee1 30UTR
was assessed by EMSA. Mutating any of these four pairs of
cysteines abolished binding of Zar2 to the Wee1 30
(Fig. 5B), even though equivalent amounts of protein were used
(Fig. 5B, lower panel). These results demonstrate that Zar2
binding to RNA is dependent on the structural integrity of a zinc
Endogenous maternal mRNAs are targets for Zar2 in Xenopus oocytes
Our previous results assessed the in vitro interaction of
recombinant Zar2 with synthetic RNA. To test whether these
interactions actually occurred in the immature Xenopus oocyte,
oocytes (Fig. 6A) using the N-terminal Zar2 antibody (Figs. 2
and 3). RNA was extracted from the immunoprecipitate, and the
presence of the endogenous Wee1 or Mos mRNA was assessed by
semi-quantitative RT-PCR. The PCR conditions were optimized to
be able to distinguish a 10-fold difference in total RNA (total
1/100 vs total 1/1000). Endogenous Wee1 mRNA co-precipitated
with endogenous Zar2 indicating that Zar2 interacts with Wee1
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
FLAG-C-Zar2FLAG-C-Zar2 + 50x Wee WT
FLAG-C-Zar2 + 50x Wee mt
FLAG-C-Zar2 + a-C-Zar2
FLAG-C-Zar2 + a-Tubulin
FLAG-C-Zar2 + a-FLAG
WT TCS mt1TCS mt2TCS mt1&2
Wee1 UTR probe
Fig. 4. Zar2 binds to TCSs in Wee1 30UTR in vitro. (A) Diagram showing the Wee1 UTR probe used in these studies: box, CPE; red oval, TCS; hexagon, polyadenylation
hexanucleotide. The red ‘‘U’’ s were mutated to ‘‘g’’ in the TCS mutants. (B) EMSA showing that Sf9-expressed FLAG-C-Zar2 forms specific complexes with Wee1 30UTR that
can be supershifted with specific antibodies to FLAG or C-Zar2. The RNA probe (the last 50 nt of the Wee1 30UTR) was incubated with mock purified, FLAG peptide, or
FLAG-C-Zar2 proteins. Antibodies against bTubulin, FLAG or C-terminal Zar2 were added where indicated. (C) EMSA showing that specific complex formation can be
competed with unlabelled RNA containing TCSs. The Wee1 RNA probe was incubated FLAG-C-Zar2 protein, and the unlabeled RNAs were used to compete for labeled probe
binding. A 50-fold molar excess of wild type (WT) or TCS-disrupted (mt) Wee1 30UTR was used. (D) Upper panel, EMSA showing bacterially expressed GST-C-Zar2, but not
GST-N-Zar2, binds to Wee1 30UTR. The Wee1 probe was incubated with GST alone, GST-N-Zar2 or GST-C-Zar2 proteins. Lower panel, western blot with GST antibodies
showing that equivalent amounts of proteins were expressed. (E) EMSA showing that Zar2 directly binds to the TCSs in the Wee1 UTR. Wee1 probes with mutations in TCS
1 (TCS mt1), TCS 2 (TCS mt2) or both TCSs (TCS mt1&2) were used. These mutant probes were incubated with decreasing amounts of GST-C-Zar2 from 15 mg to 24 ng in a
5-fold dilution series. Below each gel is a diagram of the probe that was used showing which TCSs or CPEs were present. Mutating either TCS alone did not affect binding,
but mutating both TCSs markedly reduced binding of GST-C-Zar2.
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
mRNA in immature oocytes. More Wee1 mRNA was immunopre-
cipitated than is found in 1/100 of an oocyte (we estimate 1/30).
As we originally found Zar2 because it interacted with the Mos 30
UTR, we tested if endogenous Mos mRNA co-precipitated with
Zar2. Fig. 6B shows that Mos mRNA also co-precipitates with
Zar2. The Mos mRNA that was immunoprecipitated was more
than 1/1000 but less than 1/100 of an oocyte (we estimate 1/200).
In contrast, the protein phosphatase inhibitor 2 mRNA, a negative
control (Charlesworth et al., 2006), showed much lower affinity
and the amount of PPI2 mRNA that was immunoprecipitated
was close to that found in 1/1000 of an oocyte. These data show
that endogenous Zar2 preferentially co-immunoprecipitates with
Wee1 and Mos mRNAs.
Zar2 represses translation in immature oocytes and repression is
relieved during maturation
The TCS confers translational repression in immature oocytes
and translational activation in meiotically maturing oocytes
(Wang et al., 2008). If Zar2 is a trans-acting factor for the TCS,
then it too should repress translation in immature oocytes and/or
stimulate translation in maturing oocytes. To test this, we used
the tethered assay, where viral MS2 coat protein fusions bind to
MS2 stem–loops in the reporter RNA, thus tethering the MS2 coat
proteins to the RNA ((Gray et al., 2000), reviewed recently in
Minshall et al., 2010). This method separates effects on transla-
tion regulation from changes in RNA binding. The firefly luciferase
whereas Renilla luciferase did not contain MS2 stem–loops and
was used as a loading control (Fig. 7A). Since the C-terminal
domain of Zar2 is the RNA binding domain (Figs. 1 and 4),
we hypothesized that the N-terminal contains the translational
activation domain. Therefore, we replaced the C-terminal domain
of Zar2 with MS2 coat protein (Fig. 7A). MS2-Xp54 was used as a
positive control for translational repression (Minshall et al., 2001).
Oocytes were injected with mRNA encoding the indicated MS2
fusion protein and incubated overnight to allow expression.
Oocytes were then injected with the luciferase translation repor-
ter constructs. Progesterone was added to half the oocytes and all
oocytes were at meiosis II. Oocytes were lysed and the amount
of luciferase protein accumulated was measured. Firefly luciferase
is reported relative to Renilla luciferase (Fig. 7B). Oocytes not
expressing a fusion protein (–) showed no significant difference in
translation of firefly luciferase mRNA from oocytes expressing
MS2, verifying that the binding of MS2 to the reporter mRNA does
not stimulate or repress translation, as expected (Gray et al.,
2000). Also, MS2-Xp54 repressed translation of firefly luciferase
by about 60% compared to MS2 alone (in agreement with
Minshall et al., 2001). MS2 and MS2-Xp54 were expressed at
equivalent levels (Fig. 7C). When N-Zar2-MS2 was tethered to the
luciferase reporter, it repressed translation in immature oocytes
by up to 3377.5%. This decrease in translation was not due to
destabilization of the firefly RNA reporter as assessed by semi-
quantitative PCR (Fig. 7F). Increasing amounts of injected RNA
resulted in increasing amounts of N-Zar2-MS2 fusion protein that
accumulated (Fig. 7C) and increasing amounts of repression,
showing that the observed repression is due specifically to the
presence of the N-Zar2-MS2 fusion protein. At the highest dose
prot A/G beads
prot A/G beads
Fig. 6. Endogenous Zar2 interacts with endogenous mRNAs in immature oocytes.
(A) Western blot showing immunoprecipitation of Zar2. Immature oocytes were
lysed and Zar2 immunoprecipitated with the Zar2 N-terminal antibody (a-Zar2)
(see Fig. 2, aa 29–44 and Fig. 3) and protein A/G-agarose beads. As a specificity
control, an anti-GST antibody (a–GST) was used to mock immunoprecipitate. As a
positive control, total cell lysate was run on the gel. Only samples that contained
lysate, beads and N-Zar2 antibodies immunoprecipitated Zar2. (B) Semi-quanti-
tative PCR showing selective co-precipitation of maternal mRNAs. PCR conditions
were adjusted for each mRNA so that a 1/100 and 1/1000 oocyte equivalent of
total RNA showed a 10-fold difference. Wee1 and Mos mRNAs were present in
Zar2 immunoprecipitates and were not detected in the mock immunoprecipita-
Fig. 5. Zar2 binds RNA via a zinc finger. (A) EMSA showing reduced specific
complex formation in reduced Zn2þconditions. The Wee1 probe was incubated
with GST-C-Zar2 protein purified from bacteria in the presence of EDTA. Binding
reactions were performed in buffer with (þZn2þ) or without (– Zn2þ) zinc
chloride. (B) Upper panel, EMSA showing no specific complex formation with
mutant C-Zar2 proteins that contained cysteine to alanine mutations within the
predicted zinc finger domain. A series of cysteine to alanine mutations were made
in the C-terminal domain of Zar2 (as shown in Fig. 2). Mutant proteins were
bacterially expressed, purified and mixed with the Wee1 RNA probe. Lower panel,
western blot showing equivalent amounts of mutant proteins were used in
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
Relative luciferase activity
Relative luciferase Activity
β-globin 3’ UTR
MS2 Fusion Proteins
Fig. 7. N-terminal of Zar2 represses translation in immature oocytes. (A) Cartoon representing the tethered assay constructs used in this experiment. Right panel shows the MS2
fusion proteins. Left panel shows the luciferase reporter mRNAs, the luciferase coding region is fused to the b-globin UTR. The firefly stem–loop reporter contained two MS2 stem–
loops (fluc-2x-SL) or no stem–loops (fluc). Renilla luciferase (rluc) had no stem–loops. (B) Bar chart showing N-Zar2-MS2 represses translation when tethered to the reporter mRNA.
Oocytes were injected with RNA encoding MS2 fusion proteins or not injected (–), and incubated overnight. N-zar2-MS2 RNA was injected at 5 ng, 20 ng and 50 ng. Oocytes were
then injected with a mixture of firefly with stem–loops (fluc-2x-SL) and Renilla (rluc) reporter constructs. Half the oocytes were stimulated with progesterone (P) and all oocytes
were harvested when the progesterone-stimulated oocytes had reached meiosis II. Relative luciferase activity was calculated as described in materials and methods. Bars show mean
relative luciferase activity normalized to MS2 alone for immature (black) and progesterone-treated (white) oocytes. Error bars represent S.D. and differences in mean were
considered significant with po0.01 (**) as analyzed by one way ANOVA. MS2-Xp54 shows repression of translation in both immature and mature oocytes. N-Zar2-MS2 represses
translation in immature oocytes and this repression is relieved upon maturation (n¼5). (D) Bar chart showing N-Zar2-MS2 does not repress translation when not tethered. Oocytes
were injected with RNA encoding MS2 fusion proteins and incubated overnight. N-zar2-MS2 RNA was injected at 50 ng. Oocytes were then injected with a mixture of firefly without
stem–loops (fluc) and Renilla (rluc) reporter constructs. Oocytes were harvested when control oocytes had reached meiosis II (n¼4). (C and E) Western blots showing expression of
MS2 fusion proteins at the end of the experiments. MS2, MS2-Xp54 and N-Zar2-MS2 (50 ng) were expressed at equivalent levels. N-Zar2 levels were markedly lower in progesterone
treated samples. (F) Left panel, semi-quantitative PCR to show that the firefly reporter (fluc-2x-SL) is stable in immature oocytes. The detection limits of the PCR are demonstrated by
the lower product formed when ½ or ¼ of the cDNA was added to the PCR reaction. Reporter RNA levels were not lower when tethered to N-Zar2-MS2 than when tethered to MS2
alone. Right panel, ethidium bromide stained gel of total RNA after extraction from oocytes showing equal recovery of rRNA.
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
tested, N-Zar2-MS2 protein expression was similar to MS2 and
MS2-Xp54 (Fig. 7C). Data were analyzed using one-way ANOVA
and differences in means with a p-valueo0.01 (nn) were con-
sidered significant. When 20 ng or 50 ng of N-zar2-MS2 RNA was
Mann–Whitney U tests were performed to assess differences in
median values with similar conclusions. Repression of translation
was specific to N-Zar2-MS2 being tethered to the RNA, as no
repression was seen when the stem–loops were omitted from the
firefly luciferase reporter (fluc) (Fig. 7D), even though proteins
were expressed at similar levels (Fig. 7E).
Next, we determined whether the translational control exerted
by Zar2 changed during meiotic maturation of Xenopus oocytes.
Accumulation of luciferase in MS2-, or MS2-Xp54-expressing
oocytes, or oocytes not expressing fusion proteins (–), did not
change during maturation (Fig. 7B). However, N-Zar2-MS2-
mediated repression was relieved during oocyte maturation
(Fig. 7B). This change was observed at all levels of protein
expression and was determined to be significantly different
(p-value o0.01)(nn) using one-way ANOVA and Mann–Whitney
U test. Because N-Zar2-MS2 binds to luciferase through MS2 coat
protein: stem–loop interactions, RNA binding does not change
during maturation. Rather, relief of repression could be due to loss
of N-Zar2-MS2, as protein levels were markedly lower in proges-
terone treated samples (Fig. 7C). There was a 70–80% (n¼5)
reduction in N-Zar2-MS2 in progesterone-treated oocytes, which
is comparable to the reduction in endogenous Zar2 during oocyte
maturation (Fig. 3G).
Taken together, these results show that Zar2 repressed trans-
lation in immature oocytes and that repression was relieved in
The closely related Zar1 and Zar2 proteins are implicated in
early zygotic progression, but their mechanism of action is
unknown. Histone modification, transcriptional regulation and
RNA metabolism have all been proposed. Here we showed that
Xenopus Zar2 binds maternal mRNAs and regulates translation.
Zar2 is a trans-acting factor for the TCS
The maternal Wee1 mRNA has been shown to contain two TCS
cis-elements that repress translation in immature oocytes and
activate translation in maturing oocytes (Wang et al., 2008). This
study provides evidence that Zar2 is a trans-acting factor for the
TCS. Several criteria are necessary to support this conclusion: Zar2
should bind to the TCS, repress translation in immature oocytes,
and activate translation during meiosis. In support of these
criteria we show the following pieces of evidence. (1) Zar2
associates with Wee1 and Mos mRNAs in vivo. In yeast cells, a
Zar2 fusion protein interacts with Mos and Wee1 30UTRs (Fig. 1).
The TCS was necessary and sufficient for Zar2 interaction with the
RNA hybrid. Moreover, endogenous Wee1 and Mos mRNAs can be
co-immunoprecipitated with endogenous Zar2 from immature
oocytes (Fig. 6). (2) Zar2 binds directly to the TCS in vitro. Zar2
forms a specific complex with Wee1 RNA and labeled probe
binding in the specific complex was competed by unlabeled
RNA that contained a TCS (Fig. 4). Furthermore, when the TCSs
were disrupted, the specific complex showed markedly reduced
binding (Fig. 4E). (3) Zar2 represses translation in immature
oocytes. This is an important criterion, as this is the translational
activity that the TCS confers to RNA reporters (Charlesworth et al.,
2000; Wang et al., 2008). In this study, we show that Zar2
repressed translation of a luciferase reporter in immature oocytes
(Fig. 7). This translational repression is about 33% at the max-
imum Zar2 dose that was injected. This is comparable to the TCS
in the Wee1 30UTR that contributes about 33% repression in
immature oocytes (Wang et al., 2008). Thus, we propose that Zar2
is a trans-acting factor for the TCS.
The TCS also confers translational activation in maturing
oocytes (Wang et al., 2008). The activation of mRNA translation
by Zar2 during meiosis has not been established in this study.
However, Zar2-mediated repression was relieved during meiosis.
One reason for the relief of repression and not activation may be
that N-Zar2-MS2 is degraded during meiosis so N-Zar2-MS2
could not repress, nor could it activate translation. Interestingly,
the endogenous Zar2 protein is partially degraded during
meiosis (Fig. 3), so degradation might be part of the mechanism
of action of Zar2. Degradation of Zar2 might appear to be contra-
dictory for a role in embryogenesis. However, it is known that
approximately 80% of CPEB is degraded during meiosis (Mendez
et al., 2002), and the remaining CPEB plays an important role in
translating cyclin B1 mRNA during the first mitotic cell cycles
TCS-containing mRNAs may be controlled by more than one
trans-acting factor. For example, Zar2 might repress translation
of TCS-containing mRNAs during oogenesis. After degradation
of Zar2 during meiosis, another protein may load onto the TCS-
containing mRNA to activate translation. Precedent for protein
switching in the regulation of translational control comes from
analysis of CPEB4 protein, which replaces CPEB1 during Xenopus
meiotic maturation (Igea and Mendez, 2010). We are initiating
studies to assess TCS-directed translational activation during
Zar2 and transcriptional regulation
Mouse Zar2 (Zar1l) co-localizes with P-body components,
RAP55 (aka Lsm14a) and LIN28, to cytoplasmic foci in 2-cell
embryos (Hu et al., 2010). This suggests that Zar2 is found in
P-bodies, which are sites of mRNA translational repression and
degradation (Balzer and Moss, 2007; Parker and Sheth, 2007;
Yang et al., 2006). That Zar2 may be a component of P-bodies is
consistent with our results demonstrating that Xenopus Zar2
binds mRNA and represses translation. The original proposal that
Zar1 regulated transcription was based on the observation that in
embryos from Zar1-null mice, the zygotic genome was not
activated (Wu et al., 2003a). More specifically, the embryos did
not activate transcription of Transcription Requiring Complex
(TRC), a marker of embryonic genomic activation (Wu et al.,
2003a). Consistent with this is the observation that a dominant
negative Zar2/Zar1l also disrupted general transcription (Hu et al.,
2010). One explanation for these observations could be that
zygotic transcription is activated by the protein product of a
Zar2 target mRNA. However, it should be noted that although we
have shown that Xenopus Zar2 binds to RNA, this does not
necessarily mean that the Zar family of proteins are not transcrip-
tional regulators as well as translational regulators. ChIP analysis
of Zar2 in human breast cancer cells shows that Zar2 interacts
with the BRCA2/Zar2 bidirectional promoter (Misra et al., 2010).
precedent. Wilms0Tumor 1 protein and TFIIIA contain zinc fingers
that bind both RNA and DNA, and regulate gene expression both
transcriptionally and post-transcriptionally (Hall, 2005; Morrison
et al., 2008; Burdach et al., 2012).
Zar2 contains a new type of zinc finger
Central to the predicted function of the Zar proteins are the
conserved cysteines in the C-terminal domain. There are 12
A. Charlesworth et al. / Developmental Biology 369 (2012) 177–190
conserved cysteines and different investigators have suggested
that different cysteines may form different types of zinc fingers,
however, these cysteines are not recognized by protein motif-
predicting software. Zar proteins contain pairs of cysteines (C–X2–
C) similar to zinc knuckles that are found in zinc fingers with
interleaved pairs of cysteines/histidines, in a cross-brace topol-
ogy, such as FYVE (C4–C4) (phospholipid binding), PHD (C4–HC3)
(chromatin modification) and RING (C4–HC3) (ubiquitination)
domains (Matthews and Sunde, 2002). However, motifs such as
PHD domains and FYVE domains require specific amino acids
relative to the cysteines, which Zar proteins do not have. Also,
Zar proteins tend to have more amino acids between the cysteine
pairs in the loops (22–33 aa) than PHD or FYVE domains
(10–18 aa) (Bienz, 2006; Gillooly et al., 2001) (see Fig. 2 legend
for putative loop positioning). Furthermore, loop 1 is separated
from loop 2 by four amino acids in FYVE and PHD domains and
thirteen in Zar proteins. Ring fingers do tend to have more amino
acids in the loops, between the pairs of cysteines/histidines
(30–40), similar to Zar proteins but the two loops in RING fingers
are spaced only 2–3 amino acids apart (Budhidarmo et al., 2012).
Zar proteins also have conserved cysteines and histidines that are
not in closely spaced pairs, similar to other zinc fingers such as
binuclear clusters and classical zinc fingers (DNA binding), CCCH
proteins (mRNA stability, AU-rich element (ARE) binding protein),
RANBP2 proteins (splicing) and the zinc finger of CPEB (maternal
mRNA regulation) (Brown, 2005; Burdach et al., 2012; Hake et al.,
1998; Hall, 2005; Kutateladze, 2006; Loughlin et al., 2009).
However, the spacing of the cysteines and histidines that are
not in CxxC pairs in Zar proteins do not match the spacing of any
of these zinc fingers. Thus, the cysteines in Zar2 share similarities
with all these types of zinc fingers, but lack various characteristics
that would firmly place Zar2 in any one of these categories. We
show here for the first time that a Zar protein requires Zn2þfor a
biological function. We showed that Zar2 binding to the Wee1
RNA probe was markedly reduced in the absence of Zn2þ
(Fig. 5A). Moreover, we identified four pairs of cysteines that are
important for RNA binding (Fig. 5B), supporting predictions that
Zar proteins contain zinc fingers. We further propose that Zar
proteins have a new type of functional zinc finger motif.
Physiological role of Zar2 and target mRNAs
Although Zar2 regulates translation during oocyte maturation,
we have been unsuccessful in showing a role for Zar2 in oocyte
maturation. However, it should be noted that Zar2 might not
regulate meiosis because it is the downstream protein products of
the target mRNAs of Zar2 that will determine the physiological
role of Zar2. Although the Wee1 mRNA is translated during oocyte
maturation (Charlesworth et al., 2000), the resulting Wee1
protein is implicated in the first embryonic mitotic cell cycle,
and not meiosis (Murakami et al., 1999; Murakami and Vande
Woude, 1998). Indeed, studies in mice with disrupted Zar pro-
teins have shown embryonic defects around the 1- and 2-cell
stage rather than meiotic defects (Hu et al., 2010; Wu et al.,
2003a). Wee1 protein is also important at gastrulation (Murakami
et al., 2004), which occurs later during Xenopus development.
Moreover, in Xenopus embryos, it has been shown that Zar2 plays
a role in production of secreted factors involved in epidermaliza-
tion (Nakajima et al., 2009), which also occurs later in develop-
ment. We propose that Zar2 represses TCS-containing maternal
mRNAs until they are required for embryogenesis. Also, we expect
that Zar2 regulates translation of maternal mRNAs other than
Mos and Wee1.
Zar2 may also play a role repressing maternal mRNAs during
oogenesis. The amount of Zar2 increases during oogenesis as the
oocytes grow in size, reaching a maximum at stage IV (Fig. 3).
However, the concentration of Zar2 relative to Tubulin is remark-
ably high in stage I oocytes compared to stage VI oocytes.
Maternal mRNAs are repressed by multiple mechanisms through-
out development (Colegrove-Otero et al., 2005), and during
oogenesis it is known that the mechanism of CPEB-mediated
repression changes as different partner proteins are expressed at
different stages (Radford et al., 2008). Therefore, it is conceivable
that Zar2 could provide yet another mechanism of translational
repression early in oogenesis. Identification of additional Zar2
target mRNAs and their regulation during oogenesis and embry-
ogenesis will help elucidate the physiological role of Zar2. 147
maternal Xenopus 30UTRs were identified as being candidates for
containing TCSs (Wang et al., 2008). Testing whether those
mRNAs can interact with Zar2 will advance this goal.
Zygote arrest proteins and pathological conditions
In this report, we have shown that Xenopus Zar2 binds to the
TCS in maternal mRNAs and represses translation in immature
oocytes. Because of the areas of conservation in the C-terminal,
we expect Zar1 to also bind RNA. There is also homology between
Zar1 and Zar2 in the N-terminal domain and so we expect Zar1 to
also regulate translation. Expression of Zar1 and Zar2 is normally
confined to oocytes and early embryos. However, recently, it has
been reported that Zar transcripts and Zar proteins are expressed
in a variety of cancers. Zar2 is expressed in human breast cancer
cells where it binds to the BRCA2 gene promoter and represses
expression of BRCA2 during G0/G1 (Misra et al., 2010). In addition,
the Zar1 gene is aberrantly methylated and over-expressed in
malignant melanoma cells (Shinojima et al., 2010). The Zar1 gene
is also hypermethylated in glioma cell lines and brain tumors
(Watanabe et al., 2010). Understanding the molecular function of
Zar proteins will help us understand their roles in early develop-
ment and in pathological conditions.
We would like to thank Dr. Jeff Coller, Case Western Reserve
University, OH; Dr. Nancy Standart, University of Cambridge, UK;
and Dr. Marvin Wickens, University of Wisconsin, Madison, for
reagents. We also thank Lori Sherman, Protein Production/Mab/
Tissue Culture Core Manger, University of Colorado Cancer Center
for bacluovirus infection of Sf9 cells, for protein purification. We
would like to thank Dr. Mike Wunder for statistical advice, and
Drs. Brad Stith and Aimee Bernard for critical reading of this
manuscript (Dept. Integrative Biology, University of Colorado
Denver). We would like to thank RNA Club at University of
Colorado Denver Anschutz Medical Campus for intellectual and
technical support. This work was funded in part by ACS RSG
0804401, NIH/NCRR RR20146, UAMS pilot study awards and
University of Colorado Denver start-up funds (to AC); and NIH
grant R01 HD35688, ACS RPG 101279 and the Arkansas BioS-
ciences Institute (to AMM). The DNA samples were sequenced by
the University of Colorado Cancer Center DNA Sequencing and
Analysis Core (http://DNASequencingCore.ucdenver.edu), which
is supported by a NIH/NCI Cancer Center Core Support Grant (P30
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