Abstract
The mRNAs stored in oocytes undergo general decay during the maternal-zygotic transition (MZT), and their stability is tightly interconnected with meiotic cell-cycle progression. However, the factors that trigger decay of maternal mRNA and couple this event to oocyte meiotic maturation remain elusive. Here, we identified B-cell translocation gene-4 (BTG4) as an MZT licensing factor in mice. BTG4 bridged CNOT7, a catalytic subunit of the CCR4–NOT deadenylase, to eIF4E, a key translation initiation factor, and facilitated decay of maternal mRNA. Btg4-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. The intrinsic MAP kinase cascade in oocytes triggered translation of Btg4 mRNA stored in fully grown oocytes by targeting the 3′ untranslated region, thereby coupling CCR4–NOT deadenylase–mediated decay of maternal mRNA with oocyte maturation and fertilization. This is a key step in oocyte cytoplasmic maturation that determines the developmental potential of mammalian embryos.
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References
Schier, A.F. The maternal-zygotic transition: death and birth of RNAs. Science 316, 406–407 (2007).
Li, L., Lu, X. & Dean, J. The maternal to zygotic transition in mammals. Mol. Aspects Med. 34, 919–938 (2013).
Schultz, R.M. From egg to embryo: a peripatetic journey. Reproduction 130, 825–828 (2005).
Li, L., Baibakov, B. & Dean, J. A subcortical maternal complex essential for preimplantation mouse embryogenesis. Dev. Cell 15, 416–425 (2008).
Christians, E., Davis, A.A., Thomas, S.D. & Benjamin, I.J. Maternal effect of Hsf1 on reproductive success. Nature 407, 693–694 (2000).
Burns, K.H. et al. Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos. Science 300, 633–636 (2003).
Wu, X. et al. Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyte-to-embryo transition. Nat. Genet. 33, 187–191 (2003).
Walser, C.B. & Lipshitz, H.D. Transcript clearance during the maternal-to-zygotic transition. Curr. Opin. Genet. Dev. 21, 431–443 (2011).
Schellander, K., Hoelker, M. & Tesfaye, D. Selective degradation of transcripts in mammalian oocytes and embryos. Theriogenology 68 (Suppl. 1), S107–S115 (2007).
Hou, Y. et al. Genome analyses of single human oocytes. Cell 155, 1492–1506 (2013).
Gallardo, T.D. et al. Genomewide discovery and classification of candidate ovarian fertility genes in the mouse. Genetics 177, 179–194 (2007).
Qiu, Z. et al. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases. Nucleic Acids Res. 41, e120 (2013).
Huang, P. et al. Heritable gene targeting in zebrafish using customized TALENs. Nat. Biotechnol. 29, 699–700 (2011).
Winkler, G.S. The mammalian anti-proliferative BTG/Tob protein family. J. Cell. Physiol. 222, 66–72 (2010).
Tirone, F. The gene PC3(TIS21/BTG2), prototype member of the PC3/BTG/TOB family: regulator in control of cell growth, differentiation, and DNA repair? J. Cell. Physiol. 187, 155–165 (2001).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).
Charlesworth, A., Meijer, H.A. & de Moor, C.H. Specificity factors in cytoplasmic polyadenylation. Wiley Interdiscip. Rev. RNA 4, 437–461 (2013).
Buanne, P. et al. Cloning of PC3B, a novel member of the PC3/BTG/TOB family of growth inhibitory genes, highly expressed in the olfactory epithelium. Genomics 68, 253–263 (2000).
Mauxion, F., Chen, C.Y., Séraphin, B. & Shyu, A.B. BTG/TOB factors impact deadenylases. Trends Biochem. Sci. 34, 640–647 (2009).
Doidge, R., Mittal, S., Aslam, A. & Winkler, G.S. Deadenylation of cytoplasmic mRNA by the mammalian Ccr4-Not complex. Biochem. Soc. Trans. 40, 896–901 (2012).
Doidge, R., Mittal, S., Aslam, A. & Winkler, G.S. The anti-proliferative activity of BTG/TOB proteins is mediated via the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase subunits of the Ccr4-not complex. PLoS One 7, e51331 (2012).
Miller, J.E. & Reese, J.C. Ccr4-Not complex: the control freak of eukaryotic cells. Crit. Rev. Biochem. Mol. Biol. 47, 315–333 (2012).
Horiuchi, M. et al. Structural basis for the antiproliferative activity of the Tob-hCaf1 complex. J. Biol. Chem. 284, 13244–13255 (2009).
Winkler, G.S. & Balacco, D.L. Heterogeneity and complexity within the nuclease module of the Ccr4-Not complex. Front. Genet. 4, 296 (2013).
Gosselin, P. et al. Tracking a refined eIF4E-binding motif reveals Angel1 as a new partner of eIF4E. Nucleic Acids Res. 41, 7783–7792 (2013).
Matsuo, H. et al. Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein. Nat. Struct. Biol. 4, 717–724 (1997).
Marcotrigiano, J., Gingras, A.C., Sonenberg, N. & Burley, S.K. Cocrystal structure of the messenger RNA 5′ cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89, 951–961 (1997).
Ptushkina, M. et al. Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5′ cap in yeast involves a site partially shared by p20. EMBO J. 17, 4798–4808 (1998).
Sarkissian, M., Mendez, R. & Richter, J.D. Progesterone and insulin stimulation of CPEB-dependent polyadenylation is regulated by Aurora A and glycogen synthase kinase-3. Genes Dev. 18, 48–61 (2004).
Richter, J.D. Breaking the code of polyadenylation-induced translation. Cell 132, 335–337 (2008).
Mendez, R. et al. Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 404, 302–307 (2000).
Groisman, I. et al. CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division. Cell 103, 435–447 (2000).
Eliscovich, C., Peset, I., Vernos, I. & Méndez, R. Spindle-localized CPE-mediated translation controls meiotic chromosome segregation. Nat. Cell Biol. 10, 858–865 (2008).
Ellis, R.E. & Kimble, J. The fog-3 gene and regulation of cell fate in the germ line of Caenorhabditis elegans. Genetics 139, 561–577 (1995).
Chen, P.J., Cho, S., Jin, S.W. & Ellis, R.E. Specification of germ cell fates by FOG-3 has been conserved during nematode evolution. Genetics 158, 1513–1525 (2001).
Ezzeddine, N. et al. Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation. Mol. Cell. Biol. 27, 7791–7801 (2007).
Richter, J.D. & Lasko, P. Translational control in oocyte development. Cold Spring Harb. Perspect. Biol. 3, a002758 (2011).
Piqué, M., López, J.M., Foissac, S., Guigó, R. & Méndez, R. A combinatorial code for CPE-mediated translational control. Cell 132, 434–448 (2008).
Chen, J. et al. Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition. Genes Dev. 25, 755–766 (2011).
Wolf, J. et al. Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation. EMBO J. 33, 1514–1526 (2014).
Zheng, D. et al. Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells. J. Cell Biol. 182, 89–101 (2008).
Fan, H.Y. et al. MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science 324, 938–941 (2009).
Lan, Z.J., Xu, X. & Cooney, A.J. Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice. Biol. Reprod. 71, 1469–1474 (2004).
Shen, B. et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013).
Zhou, J. et al. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int. J. Biochem. Cell Biol. 46, 49–55 (2014).
Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Acknowledgements
This study was funded by the National Basic Research Program of China (2012CB944403) and the National Natural Science Foundation of China (31528016, 91519313, and 31371449) to H.-Y.F. We thank B. Zhang (Peking University) for vectors.
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H.-Y.F. and C.Y. conceived the project. H.-Y.F., C.Y., S.-Y.J., Q.-Q.S., Y.D., Y.-L.Z., and F.T. designed and analyzed experiments. C.Y., S.-Y.J., Q.-Q.S., Y.D., Y.-L.Z., J.-J.Z., Y.L., B.H., and S.-C.S. performed experiments. Z.-W.W. and Q.-Y.S. assisted in microinjection of mouse embryos. C.Y., Y.-L.Z., and H.-Y.F. wrote the paper. C.Y., S.-Y.J., Q.-Q.S., Y.D., J.-J.Z., and Y.-L.Z. contributed equally to this work.
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Supplementary Figure 1 Phenotypic analyses of Btg4-knockout mice.
(a) Quantitative RT-PCR results showing relative expression levels of mouse Btg4 in somatic tissues, oocytes (GV and MII), and early embryos (1-cell, 2-cell, 4-cell and blastula). Error bars indicate S.E.M. (b) PCR results of tail genomic DNAs. The WT allele (“+”) was amplified with WT-F and WT-R primers, and a 7-nucleotide-deleted null allele (“−”) was amplified with the primer pairs GT-F and GT-R. “+/–” and “−/−” refer to mice heterozygous and homozygous for the Btg4 mutant allele, respectively. (c) Average body weights of 3-week-old WT, Btg4+/−, and Btg4−/− females. n=9 for each genotype. Error bars indicate S.E.M. (d) Ovarian histology of WT and Btg4−/− females (3-week and 18-week old). Scale bar: 200 μm. (e) The average numbers of ovulated oocytes by 3-week-old WT (n=4) and Btg4−/− (n=5) females, as determined by a superovulation assay. Error bars indicate S.E.M. (f) Average in vitro GVBD and PB1 emission (PBE) rates of GV oocytes collected from WT and Btg4−/− females. The numbers of WT and Btg4−/− oocytes analyzed were 87 and 105, respectively. Error bars indicate S.E.M. (g) Representative images of early embryos collected from the oviducts of WT, Btg4−/− and Btg4 C-terminal truncated (Btg4ΔC/ΔC) females. n = 5 mice for each genotype at different developmental stages. Scale bar: 100 μm. (h) Pups born from Btg4−/− and WT foster mothers after embryo transfer. The genotypes of foster mothers and transferred embryos are indicated. (i) Genotyping results showing that the pups born by Btg4−/− foster mothers are Btg4 wild types.
Supplementary Figure 2 Immunofluorescence staining for markers of DNA damage and genome reprogramming in embryos derived from WT and Btg4−/− females.
(a) Immunofluorescent staining for DNA damage marker (γH2AX) in embryos derived from WT and Btg4−/− females. Scale bar; 10 μm. (b) BrdU incorporation assay showing DNA replication in zygotes derived from WT and Btg4−/− females mated with WT males. More than 20 embryos were analyzed for each genotype with different markers. Scale bar: 10 μm. (c) Immunofluorescent staining of 5‑methylcytosine (5mC) and 5‑hydroxymethylcytosine (5hmC) in zygotes derived from WT and Btg4−/− females mated with WT males. Scale bar: 10 μm. (d,e) Immunofluorescent staining of trimethylated histone H3 at lysine-4 (H3K4me3) (d) and lysine-9 (H3K9me3) (e) in zygotes derived from WT and Btg4−/− females mated with WT males. Scale bar: 10 μm.
Supplementary Figure 3 Immunofluorescence staining of genome transcriptional-activation markers in embryos derived from WT and Btg4−/− females.
(a) Immunofluorescent staining of phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2) in 1- or 2-cell embryos derived from WT and Btg4−/− females mated with WT males. Scale bar: 10 μm. (b-d) Immunofluorescent staining of acetylated histone H2B at lysine-5 (H2BK5-ace) (b), H3 at lysine-9 (H2BK9-ace) (c), and H3 at lysine-18 (H2BK18-ace) (d), in 1- or 2-cell embryos derived from WT and Btg4−/− females mated with WT males. Scale bar: 10 μm.
Supplementary Figure 4 Degradation of maternal mRNA during meiotic maturation and fertilization in WT and Btg4−/− oocytes.
(a) Numbers of gene transcripts detected by RNA-seq. (b) Down-regulated and up-regulated genes in WT and Btg4−/− oocytes/embryos at different stages. When comparing two samples, the up-regulation and down-regulation means the upper right one compared to the lower left one. (c) Heatmap illustration showing differentially expressed transcripts in WT oocytes/embryos. Cluster I, transcripts that are significantly degraded at GV to MII transition and not re-accumulate after that in WT oocytes. Cluster II, transcripts that are significantly degraded upon fertilization in WT oocytes. Cluster III, transcripts that are significantly up-regulated at zygote to 2-cell transition (or, zygotic genes). Cluster IV, transcripts that are significantly degraded at GV to MII transition but re-accumulated at zygote to 2-cell transition. (d) Heatmap illustration showing gene transcripts that are degraded during meiotic maturation in WT oocytes but failed to be degraded in Btg4−/− oocytes. (e) Heatmap illustration showing gene transcripts that are up-regulated during zygote to 2-cell transition in WT embryos but are not up-regulated in Btg4−/− 2-cell embryos.
Supplementary Figure 5 Btg4 knockout impairs zygotic genome activation.
(a) Overlapping results showing the numbers and percentages of genes up-regulated in Btg4-deleted oocytes at MII oocytes and zygotes. (b) Overlapping results showing the numbers and percentages of genes up-regulated and down-regulated in Btg4−/− at 2-cell stage when compared to WT 2-cell embryos. (c) RNA-seq results showing the relative expression levels of representative genes. The error bars indicate S.E.M. (d) qRT-PCR showing the relative levels of indicated transcripts in oocytes/zygotes of WT and Btg4−/− females. Error bars indicates S.E.M. (e) RNA-seq results showing the expression levels of Btg and Tob family genes in human oocytes and early embryos. FPKM numbers were extracted from previously published data1. (f) RNA-seq results showing mRNA expression levels of Btg/Tob family genes in mouse oocytes and early embryos. (g) RNA-seq results showing mRNA expression levels of Cnot7/8 in human oocytes and early embryos. FPKM numbers were extracted from previously published data. (h) Quantitative RT-PCR results showing the relative expression levels of mouse Cnot7/8 in somatic tissues, oocytes, and early embryos. Error bars indicate S.E.M.
Supplementary Figure 6 BTG proteins interact with eIF4E.
(a) Deadenylation assay demonstrating that BTG4 binding did not affect CNOT7 activity in vitro. (b) Co-IP results showing the interactions of BTG2, BTG3, and BTG4 with eIF4E. (c) Co-IP results showing the interactions of BTG2 and BTG3 mutant forms with eIF4E. (d-e) Co-IP results showing the interactions of eIF4E4 mutant forms with BTG4.
Supplementary Figure 7 Regulation of Btg4-mRNA translation by its 3′ UTR.
(a-b) Epifluorescence (a) and Western blot (b) results showing that injected mRNAs encoded for GFP-BTG4 were expressed in both GV and MII oocytes, but endogenous BTG4 was only detected in MII oocytes. Scale bar: 100 μm. (c) Fluorescent microscopy results showing meiotic maturation coupled activation of Btg4 3′-UTR. Scale bar: 100 μm. In vitro transcribed FLAG-GFP-(Btg4)3′-UTR mRNA and mCherry mRNA were co-injected into GV oocytes. The injected oocytes were then further cultured in M2 medium with or without milrinone for 12 h. (d) Polyadenylation enabled the expression of FLAG-GFP-(Btg4)3′-UTR mRNA in GV oocytes, even in the absence of CPE elements. Scale bar: 100 μm. In vitro transcribed WT and CPE-deleted FLAG-GFP-(Btg4)3′-UTR mRNAs were polyadenylated in vitro and injected into GV oocytes. The injected oocytes were than further cultured in M2 medium with or without milrinone for 12 h.
Supplementary Figure 8 Effects of Cpeb1 depletion and aurora A inhibition on mouse oocyte maturation.
(a) qRT-PCR results showing the knockdown efficiency of Cpeb1 in GV oocytes. Error bars indicate S.E.M. (b-c) Injection of Cpeb1 targeted siRNA (siCpeb1) impaired PB1 emission during in vitro mouse oocyte maturation. Error bars indicate S.E.M. Scale bar: 100 μm. (d) Injection of siCpeb1 impaired PB1 emission and disrupted chromosome alignment in the middle of the spindle. Scale bar: 10 μm. (e) MLN8273 did not block BTG4 expression and ERK1/2 phosphorylation in mature oocytes. (f-g) The aurora A inhibitor, MLN8273 blocked PB1 emission, and disrupted chromosome alignment in the middle of the spindle during in vitro mouse oocyte maturation. Scale bar: 10 μm. Error bars indicate S.E.M. (h) The morphology of MLN8237 treated oocytes showed that MLN8273 blocked PB1 emission during in vitro mouse oocyte maturation. Scale bar: 100 μm.
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Supplementary Figures 1–8 and Supplementary Tables 2–4 (PDF 8657 kb)
Supplementary Table 1
FPKM of RNA-seq results (XLS 8170 kb)
Supplementary Data Set 1
Original images of gels and blots used in this study (PDF 5498 kb)
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Yu, C., Ji, SY., Sha, QQ. et al. BTG4 is a meiotic cell cycle–coupled maternal-zygotic-transition licensing factor in oocytes. Nat Struct Mol Biol 23, 387–394 (2016). https://doi.org/10.1038/nsmb.3204
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DOI: https://doi.org/10.1038/nsmb.3204
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