In the absence of transcription, the regulation of gene expression in oocytes is controlled almost exclusively at the level of transcriptome and proteome stabilization, and translation. A subset of maternal transcripts is stored in a translationally dormant state in the oocyte, and temporally driven translation of specific mRNAs propel meiotic progression, oocyte-to-embryo transition and early embryo development. We identified Ank2.3 as the only transcript variant present in the mouse oocyte and discovered that it is translated after nuclear envelope breakdown. Here we show that Ank2.3 mRNA is localized in higher concentration in the oocyte nucleoplasm and, after nuclear envelope breakdown, in the newly forming spindle where its translation occurs. Furthermore, we reveal that Ank2.3 mRNA contains an oligo-pyrimidine motif at 5'UTR that predetermines its translation through a cap-dependent pathway. Lastly, we show that prevention of ANK2 translation leads to abnormalities in oocyte cytokinesis.

Department of Cell Biology Faculty of Science Charles University Prague Czech Republic

Laboratory of Biochemistry and Molecular Biology of Germ Cells IAPG CAS Libechov Czech Republic

Erratum v

Sci Rep. 2020 Feb 18;10(1):3222. doi: 10.1038/s41598-020-60116-0. PubMed

Erratum v

Sci Rep. 2020 May 19;10(1):8572. doi: 10.1038/s41598-020-65541-9. PubMed

Zobrazit více v PubMed

De La Fuente R, et al. Major chromatin remodeling in the germinal vesicle (GV) of mammalian oocytes is dispensable for global transcriptional silencing but required for centromeric heterochromatin function. Dev. Biol. 2004;275:447–458. doi: 10.1016/j.ydbio.2004.08.028. PubMed DOI

Lasko P. mRNA Localization and Translational Control in Drosophila Oogenesis. Cold Spring Harbor Perspectives in Biology. 2012;4(10):a012294–a012294. doi: 10.1101/cshperspect.a012294. PubMed DOI PMC

Bachvarova R, De Leon V. Polyadenylated RNA of mouse ova and loss of maternal RNA in early development. Dev. Biol. 1980;74:1–8. doi: 10.1016/0012-1606(80)90048-2. PubMed DOI

Brower PT, Gizang E, Boreen SM, Schultz RM. Biochemical studies of mammalian oogenesis: synthesis and stability of various classes of RNA during growth of the mouse oocyte in vitro. Dev. Biol. 1981;86:373–383. doi: 10.1016/0012-1606(81)90195-0. PubMed DOI

Schultz RM. Regulation of zygotic gene activation in the mouse. BioEssays. 1993;15:531–538. doi: 10.1002/bies.950150806. PubMed DOI

Tadros W, Lipshitz HD. The maternal-to-zygotic transition: a play in two acts. Development. 2009;136:3033–3042. doi: 10.1242/dev.033183. PubMed DOI

Susor A, Kubelka M. Translational Regulation in the Mammalian Oocyte. Results Probl Cell Differ. 2017;63:257–295. doi: 10.1007/978-3-319-60855-6_12. PubMed DOI

Sonenberg N, Hinnebusch AG. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell. 2009;136:731–745. doi: 10.1016/j.cell.2009.01.042. PubMed DOI PMC

Terenzio M, et al. Science. 2018. Locally translated mTOR controls axonal local translation in nerve injury; pp. 1416–1421. PubMed PMC

King ML, Messitt TJ, Mowry KL. Putting RNAs in the right place at the right time: RNA localization in the frog oocyte. Biology of the Cell. 2005;97:19–33. doi: 10.1042/BC20040067. PubMed DOI

Holt CE, Bullock SL. Subcellular mRNA Localization in Animal Cells and Why It Matters. Science. 2009;326:1212–1216. doi: 10.1126/science.1176488. PubMed DOI PMC

Dubowy J, Macdonald PM. Localization of mRNAs to the oocyte is common in Drosophila ovaries. Mechanisms of Development. 1998;70:193–195. doi: 10.1016/S0925-4773(97)00185-8. PubMed DOI

Nieuwkoop PD. Inductive interactions in early amphibian development and their general nature. Development. 1985;89:333–347. PubMed

Susor, A. et al. Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR–eIF4F pathway. Nat Commun6 (2015). PubMed PMC

Jansova D, Tetkova A, Koncicka M, Kubelka M, Susor A. Localization of RNA and translation in the mammalian oocyte and embryo. Plos One. 2018;13:e0192544. doi: 10.1371/journal.pone.0192544. PubMed DOI PMC

Kopecný V, Landa V, Pavlok A. Localization of nucleic acids in the nucleoli of oocytes and early embryos of mouse and hamster: an autoradiographic study. Mol. Reprod. Dev. 1995;41:449–458. doi: 10.1002/mrd.1080410407. PubMed DOI

Xie F, Timme KA, Wood JR. Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos. Scientific Reports. 2018;8:7930. doi: 10.1038/s41598-018-26345-0. PubMed DOI PMC

Bennett V, Baines AJ, Davis JQ. Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes. J. Cell. Biochem. 1985;29:157–169. doi: 10.1002/jcb.240290210. PubMed DOI

Srinivasan Y, Elmer L, Davis J, Bennett V, Angelides K. Ankyrin and spectrin associate with voltage-dependent sodium channels in brain. Nature. 1988;333:177–180. doi: 10.1038/333177a0. PubMed DOI

Bouvier J, Richaud C, Richaud F, Patte JC, Stragier P. Nucleotide sequence and expression of the Escherichia coli dapB gene. J. Biol. Chem. 1984;259:14829–14834. PubMed

Gross-Thebing, T., Paksa, A. & Raz, E. Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos. BMC Biol12 (2014). PubMed PMC

Hutchinson JN, et al. A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics. 2007;8:39. doi: 10.1186/1471-2164-8-39. PubMed DOI PMC

Hausnerová VV, Lanctôt C. Transcriptional Output Transiently Spikes Upon Mitotic Exit. Scientific Reports. 2017;7:12607. doi: 10.1038/s41598-017-12723-7. PubMed DOI PMC

Pichon X, et al. Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells. J. Cell Biol. 2016;214:769–781. doi: 10.1083/jcb.201605024. PubMed DOI PMC

Decker CJ, Parker R. Diversity of cytoplasmic functions for the 3′ untranslated region of eukaryotic transcripts. Current Opinion in Cell Biology. 1995;7:386–392. doi: 10.1016/0955-0674(95)80094-8. PubMed DOI

Hogan DJ, Riordan DP, Gerber AP, Herschlag D, Brown PO. Diverse RNA-Binding Proteins Interact with Functionally Related Sets of RNAs, Suggesting an Extensive Regulatory System. PLOS Biology. 2008;6:e255. doi: 10.1371/journal.pbio.0060255. PubMed DOI PMC

Afonina E, Stauber R, Pavlakis GN. The human poly(A)-binding protein 1 shuttles between the nucleus and the cytoplasm. J. Biol. Chem. 1998;273:13015–13021. doi: 10.1074/jbc.273.21.13015. PubMed DOI

Gray NK, Hrabálková L, Scanlon JP, Smith RWP. Poly(A)-binding proteins and mRNA localization: who rules the roost? Biochem. Soc. Trans. 2015;43:1277–1284. doi: 10.1042/BST20150171. PubMed DOI

Dai X-X, et al. A combinatorial code for mRNA 3′-UTR-mediated translational control in the mouse oocyte. Nucleic Acids Res. 2019;47:328–340. doi: 10.1093/nar/gky971. PubMed DOI PMC

Martin KC, Ephrussi A. mRNA Localization: Gene Expression in the Spatial Dimension. Cell. 2009;136:719. doi: 10.1016/j.cell.2009.01.044. PubMed DOI PMC

Ben-Shem A, et al. The structure of the eukaryotic ribosome at 3.0 Å resolution. Science. 2011;334:1524–1529. doi: 10.1126/science.1212642. PubMed DOI

Dieck Stom, et al. Direct visualization of identified and newly synthesized proteins in situ. Nat Methods. 2015;12:411–414. doi: 10.1038/nmeth.3319. PubMed DOI PMC

Levy S, Avni D, Hariharan N, Perry RP, Meyuhas O. Oligopyrimidine tract at the 5′ end of mammalian ribosomal protein mRNAs is required for their translational control. Proc. Natl. Acad. Sci. USA. 1991;88:3319–3323. doi: 10.1073/pnas.88.8.3319. PubMed DOI PMC

Avni D, Biberman Y, Meyuhas O. The 5′ terminal oligopyrimidine tract confers translational control on TOP mRNAs in a cell type- and sequence context-dependent manner. Nucleic Acids Res. 1997;25:995–1001. doi: 10.1093/nar/25.5.995. PubMed DOI PMC

Thoreen CC, et al. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature. 2012;485:109–113. doi: 10.1038/nature11083. PubMed DOI PMC

Sekiyama N, et al. Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1. PNAS. 2015;112:E4036–E4045. doi: 10.1073/pnas.1512118112. PubMed DOI PMC

Heesom KJ, Gampel A, Mellor H, Denton RM. Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1) Curr. Biol. 2001;11:1374–1379. doi: 10.1016/S0960-9822(01)00422-5. PubMed DOI

Corey, D. R. & Abrams, J. M. Morpholino antisense oligonucleotides: tools for investigating vertebrate development. Genome Biol2, reviews1015.1-reviews1015.3 (2001). PubMed PMC

Hashimoto N, Kishimoto T. Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation. Dev. Biol. 1988;126:242–252. doi: 10.1016/0012-1606(88)90135-2. PubMed DOI

Mayer S, Wrenzycki C, Tomek W. Inactivation of mTor arrests bovine oocytes in the metaphase-I stage, despite reversible inhibition of 4E-BP1 phosphorylation. Mol. Reprod. Dev. 2014;81:363–375. doi: 10.1002/mrd.22305. PubMed DOI

Boothby TC, Zipper RS, van der Weele CM, Wolniak SM. Removal of retained introns regulates translation in the rapidly developing gametophyte of Marsilea vestita. Dev. Cell. 2013;24:517–529. doi: 10.1016/j.devcel.2013.01.015. PubMed DOI

Bahar Halpern K, et al. Nuclear Retention of mRNA in Mammalian Tissues. Cell Rep. 2015;13:2653–2662. doi: 10.1016/j.celrep.2015.11.036. PubMed DOI PMC

Jambor, H. et al. Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. Elife4 (2015). PubMed PMC

Lécuyer E, et al. Global Analysis of mRNA Localization Reveals a Prominent Role in Organizing Cellular Architecture and Function. Cell. 2007;131:174–187. doi: 10.1016/j.cell.2007.08.003. PubMed DOI

Jefferies HB, et al. Rapamycin suppresses 5′TOP mRNA translation through inhibition of p70s6k. EMBO J. 1997;16:3693–3704. doi: 10.1093/emboj/16.12.3693. PubMed DOI PMC

Severance AL, Latham KE. PLK1 regulates spindle association of phosphorylated eukaryotic translation initiation factor 4E-binding protein and spindle function in mouse oocytes. Am. J. Physiol., Cell Physiol. 2017;313:C501–C515. doi: 10.1152/ajpcell.00075.2017. PubMed DOI PMC

Jansova D, et al. Regulation of 4E-BP1 activity in the mammalian oocyte. Cell Cycle. 2017;16:927–939. doi: 10.1080/15384101.2017.1295178. PubMed DOI PMC

Yu J, Yaba A, Kasiman C, Thomson T, Johnson J. mTOR controls ovarian follicle growth by regulating granulosa cell proliferation. PLoS ONE. 2011;6:e21415. doi: 10.1371/journal.pone.0021415. PubMed DOI PMC

Kogasaka Y, Hoshino Y, Hiradate Y, Tanemura K, Sato E. Distribution and association of mTOR with its cofactors, raptor and rictor, in cumulus cells and oocytes during meiotic maturation in mice. Mol. Reprod. Dev. 2013;80:334–348. doi: 10.1002/mrd.22166. PubMed DOI

Lopez-Bonet E, et al. Serine 2481-autophosphorylation of mammalian target of rapamycin (mTOR) couples with chromosome condensation and segregation during mitosis: confocal microscopy characterization and immunohistochemical validation of PP-mTOR(Ser2481) as a novel high-contrast mitosis marker in breast cancer core biopsies. Int. J. Oncol. 2010;36:107–115. PubMed

Rong L, et al. Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs. RNA. 2008;14:1318–1327. doi: 10.1261/rna.950608. PubMed DOI PMC

Chan CC, et al. eIF4A3 is a novel component of the exon junction complex. RNA. 2004;10:200–209. doi: 10.1261/rna.5230104. PubMed DOI PMC

Shibuya T, Tange TØ, Sonenberg N, Moore MJ. eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. Mol. Biol. 2004;11:346–351. doi: 10.1038/nsmb750. PubMed DOI

Ellederová Z, et al. ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes. Mol. Reprod. Dev. 2008;75:309–317. doi: 10.1002/mrd.20690. PubMed DOI

Romasko EJ, Amarnath D, Midic U, Latham KE. Association of maternal mRNA and phosphorylated EIF4EBP1 variants with the spindle in mouse oocytes: localized translational control supporting female meiosis in mammals. Genetics. 2013;195:349–358. doi: 10.1534/genetics.113.154005. PubMed DOI PMC

Tomek W, et al. Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1. Biol. Reprod. 2002;66:1274–1282. doi: 10.1095/biolreprod66.5.1274. PubMed DOI

Susor A, et al. Regulation of cap-dependent translation initiation in the early stage porcine parthenotes. Mol. Reprod. Dev. 2008;75:1716–1725. doi: 10.1002/mrd.20913. PubMed DOI

Shuda M, et al. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation. Proc. Natl. Acad. Sci. USA. 2015;112:5875–5882. doi: 10.1073/pnas.1505787112. PubMed DOI PMC

Bischof J, et al. A cdk1 gradient guides surface contraction waves in oocytes. Nature Communications. 2017;8:849. doi: 10.1038/s41467-017-00979-6. PubMed DOI PMC

Blower MD, Feric E, Weis K, Heald R. Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules. J. Cell Biol. 2007;179:1365–1373. doi: 10.1083/jcb.200705163. PubMed DOI PMC

VerMilyea MD, et al. Transcriptome asymmetry within mouse zygotes but not between early embryonic sister blastomeres. The EMBO Journal. 2011;30:1841–1851. doi: 10.1038/emboj.2011.92. PubMed DOI PMC

Jang C-Y, Kim HD, Zhang X, Chang J-S, Kim J. Ribosomal protein S3 localizes on the mitotic spindle and functions as a microtubule associated protein in mitosis. Biochem. Biophys. Res. Commun. 2012;429:57–62. doi: 10.1016/j.bbrc.2012.10.093. PubMed DOI

Schweizer N, Pawar N, Weiss M, Maiato H. An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region. J Cell Biol. 2015;210:695–704. doi: 10.1083/jcb.201506107. PubMed DOI PMC

Yi K, et al. Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes. Nat. Cell Biol. 2011;13:1252–1258. doi: 10.1038/ncb2320. PubMed DOI PMC

Verlhac M-H, Lefebvre C, Guillaud P, Rassinier P, Maro B. Asymmetric division in mouse oocytes: with or without Mos. Current Biology. 2000;10:1303–1306. doi: 10.1016/S0960-9822(00)00753-3. PubMed DOI

Azoury J, Verlhac M-H, Dumont J. Actin filaments: key players in the control of asymmetric divisions in mouse oocytes. Biol. Cell. 2009;101:69–76. doi: 10.1042/BC20080003. PubMed DOI

Uraji, J., Scheffler, K. & Schuh, M. Functions of actin in mouse oocytes at a glance. J. Cell. Sci. 131 (2018). PubMed

Chaigne A, et al. F-actin mechanics control spindle centring in the mouse zygote. Nat Commun. 2016;7:10253. doi: 10.1038/ncomms10253. PubMed DOI PMC

van Oort RJ, Altamirano J, Lederer WJ, Wehrens XHT. Alternative splicing: a key mechanism for ankyrin-B functional diversity? J Mol Cell Cardiol. 2008;45:709–711. doi: 10.1016/j.yjmcc.2008.08.016. PubMed DOI PMC

Sobel JS, Pinto-Correia C, Goldstein EG. Identification of an M(r) 60,000 polypeptide unique to the meiotic spindle of the mouse oocyte. Mol. Reprod. Dev. 1995;40:467–480. doi: 10.1002/mrd.1080400411. PubMed DOI

Skop AR, Liu H, Yates J, Meyer BJ, Heald R. Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms. Science. 2004;305:61–66. doi: 10.1126/science.1097931. PubMed DOI PMC

Tetkova, A. & Hancova, M. Mouse Oocyte Isolation, Cultivation and RNA Microinjection. BIO-PROTOCOL6 (2016).

Tal M. Metal ions and ribosomal conformation. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis. 1969;195:76–86. doi: 10.1016/0005-2787(69)90604-2. PubMed DOI

Spatial positioning of preimplantation mouse embryo cells is regulated by mTORC1 and m7G-cap-dependent translation at the 8- to 16-cell transition

Multiple Roles of PLK1 in Mitosis and Meiosis

ncRNA BC1 influences translation in the oocyte

Oocyte specific lncRNA variant Rose influences oocyte and embryo development

The neglected part of early embryonic development: maternal protein degradation