Mammalian Emi2 mediates cytostatic arrest and
transduces the signal for meiotic exit via Cdc20
Shisako Shoji1 , Naoko Yoshida1, Manami Amanai1, Maki Ohgishi1 ,
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Tomoyuki Fukui1, Satoko Fujimoto1, Yoshikazu Nakano1, Eriko Kajikawa1
and Anthony C. F. Perry1*
1Laboratory
of Mammalian Molecular Embryology, RIKEN Center for Developmental Biology, 2-2-3
Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan
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Supplementary Material
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Supplementary materials and methods
Plasmids
To produce EGFP fusions for evaluation of RNAi in cell lines, Emi1 cDNA (amplified by RT-PCR of
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mouse mII oocytes with primers [5'  3'] CCGAATTCATGAGCCGGCGCACCT and
CGCTCGAGCACAATCTTTGTAAGTTCTTT) was subcloned into the EcoRI and XhoI sites of
plasmid pBT (Stratagene), and from there into the NotI and XhoI sites of a modified pCMV-tag vector
(Stratagene). EGFP cDNA (amplified by PCR with primers
CCGCTCGAGAATGGTGAGCAAGGGCGA and CGGCTCGAGTACTTGTACAGCTCGTCC from
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pCX-EGFP) was inserted into the XhoI site of the resultant construct. Emi2 cDNA (amplified by RTPCR of mouse mII oocyte total RNA with primers GGCATATGATGGACTCCTCTGCTGTC and
CGCTCGAGCAGAGGCGTTTTAAGTTCC) was subcloned into the SmaI and XhoI sites of pBT and
Shoji et al., 6:36 PM 6/3/16, page 1
from there into NotI/ XhoI-digested pCMV-tag, and EGFP cDNA inserted into the XhoI site as described
above. Over-expression was in the E. coli host, BL21-CodonPlus(DE3)-RIL (Stratagene) from pET19b
(Novagen) constructs containing cDNA encoding an N-terminal portion of Emi1 (residues 1-219;
amplified with primers CCACTAGTATGAGCCGGCGCACCT and
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GCGGATCCTCATAGGTGCTCCAGGCC) in pEmi1-NT, or a C-terminal portion of Emi2 (residues
485-641; amplified with primers GGACTAGTATACAGCTGAGAGCAAGT and
GCGGATCCTTCAGAGGCGTTTTAAGT) in pEmi2-CT.
For the mammalian cell culture expression experiments presented in Figure 3, the coding sequence of
mCherry was amplified by PCR from pRSET-B-mCherry (Shaner et al., 2004) and cloned into the large
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BamHI-BsrGI fragment of pECFP-N1 (Clontech). Emi2 cDNA amplified by PCR from mII oocytes was
cloned into the large KpnI-BamHI fragment of this construct to generate pCMV-IE/Emi2-mCherry; the
Emi2-mCherry-encoding XhoI-NotI fragment of this construct was sublconed into pCI-Neo (Promega) to
generate pCI-Neo/Emi2-mCherry, which was used for transfection. The Venus--tubulin construct was
produced by inserting the Venus coding sequence into the large, blunted NheI-BsrGI fragment of
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pEGFP-Tub (Clontech).
Constructs expressing Emi2 mutants were generated essentially by the method of Imai et al. (1991)
using pCI-Neo/Emi2-mCherry as input DNA.
For baculovirus over-expression of FLAG-tagged Emi2, we modified pFastBac1 (InVitrogen) to
encode 3 FLAG epitopes. Mouse Emi2 cDNA was cloned into the large SpeI-NotI fragment of this
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construct to generate pFastBac-3XFLAG-Emi2.
Preparation of antigen for antibody characterization
Anti-Emi1 and -Emi2 antibodies were characterized using Emi1-Myc-His and Emi2-Myc-His proteins
translated in vitro and purified with the MagZ Protein Purification System (Promega). Protein quantities
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were estimated by comparative silver staining using known amounts of bovine serum albumin as a
standard.
Antibody purification and immunoblotting
Anti-Emi1 antibodies were affinity purified from immune serum using recombinant Emi1-blotted
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PROTRAN nitrocellulose (Schleicher & Schuell Bioscience, Germany). Anti-Emi2 antibodies were
Shoji et al., 6:36 PM 6/3/16, page 2
enriched from immune serum using IgG by Protein G-Sepharose (Amersham Bioscience). Following
standard separation, blocking (Block Ace; Yukijirusi Nyugyo, Japan) and blotting, immunodetection was
typically performed using (Toyobo Co., Ltd., Japan) enhancer with a LumiGLO Reserve
Chemiluminescent Substrate Kit (Kirkegard & Perry Laboratories, Inc.).
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Blots were stripped with
Western blot stripping solution (Nakalai Tesque Inc., Japan) and re-probed with anti--tubulin
antibodies to confirm equal loadings. Where appropriate, comparative immunoblotting was with the
same number of oocytes in the range 100-135 for a given filter.
siRNA design
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Double stranded siRNAs (iGENE Therapeutics Inc., Japan) were designed using top-scoring sequences
identified by Target Finder (Gene Script Corp.; https://www.genscript.com/ssl-bin/app/rnai) and the RNAi
software tools of the Hannon laboratory (http://katahdin.cshl.org:9331/portal/scripts/main2.pl), and
corresponded to cDNAs for mouse Emi1 (NM_025995: #2, nt 1090-1114; #3, nt 337-361), Emi2
(BC_098484: #1, nt 613-637; #2, nt 1042-1066), Cdc20 (NM_023223: #1, nt 892-916; #2, nt 1037-
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1061), Mos (J00372: nt 2708-2732) and EGFP (pCX-EGFP: nt 2322-2346).
Tissue culture
NIH3T3 and HEK293T cells were maintained in Dulbecco’s medium supplemented with 10% (v/v) FBS
in a 100 mm dish. siRNAs were evaluated by co-transfection of cells in 6 well plates with 100 pmol of
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siRNA and 1 g of plasmid DNA, using X-tremeGENE siRNA Transfection Reagent (Roche Applied
Science). 48 h after transfection, cells were examined by epifluoresence microscopy and/or harvested in
PBS for analysis by RT-PCR.
RT-PCR
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For analysis of siRNA-mediated RNAi, 1-3 cumulus-denuded oocytes or embryos were collected in 2 l
0.1% (w/v) sarkosyl, heated at 65ºC for 5 min and subjected to oligo (d)T-primed first-strand cDNA
synthesis in 25 l with SuperScript III RT (Invitrogen). 0.05x or 0.1x cDNA was used to program PCR
with Pwo DNA polymerase (Roche). All test reactions were accompanied by negative, RT-minus
controls and were performed on at least two independent preparations.
Shoji et al., 6:36 PM 6/3/16, page 3
Ratiometric quantitation of mRNAs with QuantiTect SYBR Green PCR Master Mix (Qiagen)
utilized approximately 10% of first-strand reverse-transcription reaction (0.2 cell equivalents) in 25 l.
PCR primers yielded negligible dimers and were typically intron-flanking. Product quantification was
on a Prism 7000 Sequence Detection System (Applied Biosystems). To determine the number of cycles
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required to reach threshold values for each primer set, standard curves were constructed using serial
dilutions of mouse testis cDNA. Normalized ratiometric values were obtained by dividing the raw value
for the query sequence by that concurrently obtained from the same cDNA preparation for -actin or
H2A.Z (Jeong et al., 2005).
For tissue RT-PCR, total RNA was extracted using Isogen (Nippon Gene) and 200 ng used for first-
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strand cDNA synthesis with SuperScript III RT (Invitrogen). RT-PCR for embryos was programmed
with RNA from respectively 58, 33 and 23 embryos of 2- and 8-cells and blastocysts. PCR was with
GeneTaq NT (Nippon Gene).
PCR primers
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Standard RT-PCR was with the following primers (5'  3') for mouse:
-actin: GGCATTGTTACCAACTGGGACGAC and CCAGAGGCATACAGGGACAGCACAG.
Emi1: CTGAGCTCTCCCGCAG and GCGGATCCATCACAATCTTTGTAAGTTCT.
Emi2: AGGAATTCTTTGAGGACAG and GCGGATCCTTCAGAGGCGTTTTAAGT.
Cdc20: GCATTCTGAGGTTTGCCG and GCAGTTCGTGTTCGAGAG.
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Cdh1: GGACTCAGTGACTTCCGTTGGC and TTCTTCCCAGCAGCAGCGTC.
Cdc2: GTACTTACGGTGTGGTGTAT and TAATCTGATTGTCCAAGTC.
Mos: CAGCCTAGGTACCATAATC and CGGGATCCCTCAGCCTAGTGCCCCT.
RT-PCR for Xenopus laevis was with the following:
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GAPDH: CTCCTCTCGCAAAGGTCATC and GGAAAGCCATTCCGGTTATT.
Emi1: GCAAGTAACCAGTCTCCA and CGACATCAACAGCATTCC.
Emi2: GTCGATCAGCTCTAAGATC and CTAGCTTCAAAGTCTCTTC.
Quantitative mouse RT-PCR was with the following primers:
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-actin: TGACAGGATGCAGAAGGAGA and GCTGGAAGGTGGACAGTGAG.
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H2A.Z (NM_016750): GCGTATCACCCCTCGTCACTTG and TCTTCTGTTGTCCTTTCTTCCCG.
Emi1: AAATCAAGTCCTCCAGTCAGCGTG and TTCGTTGTTCTTCAATGTCTTTGCC.
Emi2: AGTGGTGAGCAGGTTCCAACTCTG and TGTTTACTCCGTAGGTGGGTGAGG.
Cdc20: GGCACATTCGCATTTGGAACG and TAGTGGGGAGACCAGAGGATGGAG.
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Cdh1: GGACTCAGTGACTTCCGTTGGC and TTCTTCCCAGCAGCAGCGTC.
Mos: CAGTGGTTGCCTACAATCTGCG and AGCCTTGAGGTCCCTTTGGAG.
MBP and H1 kinase assays
MBP and H1 kinases were assayed respectively to determine MAPK and MPF activities in 1-3 oocytes
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or embryos essentially as described (Svoboda et al., 2000).
cRNA preparation
T7-directed transcription was programmed in vitro by pCI-Neo/Emi2-mCherry or derivatives using an
AmpliCap-Max T7 High Yield Message Maker Kit (Epicentre Biotechnologies). cRNA purification was
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with the MEGA clear system (Ambion) according to the manufacturer's instructions.
Immunofluorescence microscopy
For immunofluorescence imaging, oocytes were fixed in 4% (w/v) paraformaldehyde and labeled with
mouse anti--tubulin antibodies (Sigma) followed by anti-mouse IgG Alexa488 conjugate (Molecular
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Probes) and propidium iodide (Sigma).
Fluorescence was visualized on a Nikon Eclipse E600
microscope equipped with a Radiance 2100 laser scanning confocal system (BioRad).
Movie
GV oocytes were injected with siEmi2#2 as described above and transferred to the stage of a Zeiss
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Axiovert 200 microscope in a 37ºC incubation chamber containing 5% (v/v in air) CO 2. Bright-field
images (10x objective) were captured every 10 min by an Olympus DP-70 digital camera driven by
MetaMorph (Molecular Devices) imaging software. The movie plays at 30 frames/sec and corresponds
to 33 h 14 min.
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In vitro binding assays
Shoji et al., 6:36 PM 6/3/16, page 5
Emi1, Emi2, and Cdc20 cDNAs were subcloned into the T7 expression vector, pcDNA3.1/myc-His
(Invitrogen) to generate Myc-tagged fusions. In vitro reactions were performed with the TNT Coupled
Transcription/Translation System (Promega) in the presence of L-[35S] methionine (30 Ci, Amersham
Biosciences) in a final volume of 50 l, according to the manufacturer's recommendations. Protein
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products were immunoprecipitated with anti-Cdc20 or -Myc monoclonal antibodies followed by
incubation with Protein G-Sepharose 4FF (Amersham).
Immunocomplexes were collected by
centrifugation (20,400 g at 4ºC for 5 min), washed, separated by 10% SDS-PAGE and labeled proteins
visualized with a BioImage Analyzer (Fuji Film) following X-radiography at room temperature for 12 to
16 h.
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Baculovirus protein expression and purification
Baculovirus cells (sf21) were transfected with pFastBac-3xFLAG-Emi2 for Emi2-FLAG expression
essentially according to the recommendations of the Bac-to-Bac Expression System (Invitrogen). 100200 ml of culture optimized for protein expression was washed in PBS and resuspended in 5-10 ml of
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lysis buffer containing 0.5% (v/v) triton X-100, 1 mM PMSF, 10% (v/v) glycerol and protease inhibitor
cocktail (Sigma) in PBS. Following sonication by an ultrasonic disruptor (UD-201; Tomy, Japan) at
70% power for 2 x 10 sec, the mixture was centrifuged at 20,400 g for 10 min at 4ºC. Supernatant was
then mixed on a rotating platform for 3 h at 4ºC with 300 l anti-FLAG M2 agarose beads (Sigma) that
had been pre-washed 3 times in lysis buffer. Following supernatant incubation, beads were washed 5-6
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times in a buffer containing 150 mM NaCl, 0.01% (v/v) NP-40 and 10% (v/v) glycerol in PBS, and
finally resuspended in an elution buffer containing 150 g/ml 3x FLAG peptide (Sigma),
MDYKDHDGDYKDHDIDYKDDDDK, in PBS.
After incubation for 10 min on ice, beads were
pelleted, and eluate collected and stored at 4ºC and the residual beads subjected to 3 further rounds of
elution.
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Pooled eluate was dialyzed for 3 h at 4ºC against PBS and concentrated with Amicon
ultracentrifugal filter devices (Millipore, USA) to a final protein concentration of 0.6-1.8 mg/ml as
determined by Advanced protein assay reagent (Cytoskeleton, USA). The integrity of recombinant Emi2
protein was determined by SDS-PAGE followed by silver staining (Wako, Japan) or Western blotting
(Figure 4G).
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Shoji et al., 6:36 PM 6/3/16, page 6
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RT-PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos.
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Supplementary figure legends
Supplementary Figure S1 RNAi-induced reduction of recombinant Emi1 and Emi2 proteins in cell
culture. (A) NIH3T3 or HEK293T cells were transfected with a plasmid directing Emi1-eGFP-Myc
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fusion protein expression from a CMV promoter either with or without siRNAs siEmi1#2 (#2) or
siEmi1#3 (#3), showing bright field (upper) and 480 nm epifluorescence (lower). Emi1 and -actin
transcripts were analyzed (B) by RT-PCR and (C) recombinant Emi1 protein by Western blotting with
anti-His-Emi1-NT antibody. (D) NIH3T3 or HEK293T cells were transfected with a plasmid directing
Emi2-eGFP-Myc fusion protein expression from a CMV promoter either with or without siRNAs
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siEmi2#1 (#1) or siEmi2#2 (#2), showing bright field (upper) and 480 nm epifluorescence (lower). (E)
Emi2 and -actin transcripts analyzed by RT-PCR. Bar = 50 m throughout.
Supplementary Figure S2
Ratiometric semi-quantitative RT-PCR of oocytes for siRNA targets.
Ratiometric real-time RT-PCR analysis of (A) GV and (B) mII oocytes 24 h after injection with siRNAs
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to determine the resultant relative abundance of indicated transcripts. Raw values were first normalized
with respect to -actin mRNA levels determined for the same sample and plotted as a fraction of the value
obtained for control oocytes not injected with siRNA; corresponding average values are represented
beneath as percentages. Note that Y-axes are logarithmic.
Shoji et al., 6:36 PM 6/3/16, page 7
Supplementary Figure S3 Ratiometric semi-quantitative RT-PCR of uninjected mII oocytes following
culture in vitro.
Ratiometric real-time RT-PCR analysis of mII oocytes 24 h (A) after collection
(corresponding to ~36 h post-hCG). Non-detected Mos mRNA is indicated by an asterisk. These data are
included in (B), which additionally shows mRNA levels similarly detected in oocytes cultured for shorter
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time periods as shown. Raw values are expressed ratiometrically with respect to -actin levels determined
for the same sample in each case. Values for each mRNA are normalized with respect to 4 h, which is set
at 1.0. The ratios of H2A.Z:-actin mRNA levels at 24 h and 8 h when expressed as a ratio (24 h:8 h) =
1.28 (±0.12).
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Supplementary movie S1 Maturing oocytes following injection with siEmi2#2. Images were captured
every 10 min and normally plays at 30 frames/sec.
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