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• W4229042109 abstract "•Oocytes accumulate translationally repressed pou5f3 mRNA as granular structures•These RNA granules exhibit solid-like properties•After fertilization, pou5f3 RNA granules become to be liquid-like droplets•Changes in their physical properties promote translation and embryonic development Fertilized eggs begin to translate mRNAs at appropriate times and placements to control development, but how the translation is regulated remains unclear. Here, we found that pou5f3 mRNA encoding a transcriptional factor essential for development formed granules in a dormant state in zebrafish oocytes. Although the number of pou5f3 granules remained constant, Pou5f3 protein accumulated after fertilization. Intriguingly, signals of newly synthesized peptides and a ribosomal protein became colocalized with pou5f3 granules after fertilization and, moreover, nascent Pou5f3 was shown to be synthesized in the granules. This functional change was accompanied by changes in the state and internal structure of granules. Dissolution of the granules reduced the rate of protein synthesis. Similarly, nanog and sox19b mRNAs in zebrafish and Pou5f1/Oct4 mRNA in mouse assembled into granules. Our results reveal that subcellular compartments, termed embryonic RNA granules, function as activation sites of translation after changing physical properties for directing vertebrate development. Fertilized eggs begin to translate mRNAs at appropriate times and placements to control development, but how the translation is regulated remains unclear. Here, we found that pou5f3 mRNA encoding a transcriptional factor essential for development formed granules in a dormant state in zebrafish oocytes. Although the number of pou5f3 granules remained constant, Pou5f3 protein accumulated after fertilization. Intriguingly, signals of newly synthesized peptides and a ribosomal protein became colocalized with pou5f3 granules after fertilization and, moreover, nascent Pou5f3 was shown to be synthesized in the granules. This functional change was accompanied by changes in the state and internal structure of granules. Dissolution of the granules reduced the rate of protein synthesis. Similarly, nanog and sox19b mRNAs in zebrafish and Pou5f1/Oct4 mRNA in mouse assembled into granules. Our results reveal that subcellular compartments, termed embryonic RNA granules, function as activation sites of translation after changing physical properties for directing vertebrate development. Protein syntheses at appropriate times and placements direct various biological processes in almost all organisms including development of animals. Because transcription is silent shortly before fertilization until zygotic genome activation at around the 1000-cell stage in zebrafish and the 2-cell stage in mouse (Hamatani et al., 2004Hamatani T. Carter M.G. Sharov A.A. Ko M.S. Dynamics of global gene expression changes during mouse preimplantation development.Dev. Cell. 2004; 6: 117-131https://doi.org/10.1016/s1534-5807(03)00373-3Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Harvey et al., 2013Harvey S.A. Sealy I. Kettleborough R. Fenyes F. White R. Stemple D. Smith J.C. Identification of the zebrafish maternal and paternal transcriptomes.Development. 2013; 140: 2703-2710https://doi.org/10.1242/dev.095091Crossref PubMed Scopus (124) Google Scholar; Kane and Kimmel, 1993Kane D.A. Kimmel C.B. The zebrafish midblastula transition.Development. 1993; 119: 447-456https://doi.org/10.1242/dev.119.2.447Crossref PubMed Google Scholar), timely and spatially controlled protein synthesis after fertilization is achieved by translational activation of the dormant mRNAs stored in eggs. Comprehensive analyses of gene expression demonstrated that zebrafish and mouse eggs accumulate more than eight thousand mRNAs during oogenesis (Aanes et al., 2011Aanes H. Winata C.L. Lin C.H. Chen J.P. Srinivasan K.G. Lee S.G. Lim A.Y. Hajan H.S. Collas P. Bourque G. et al.Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition.Genome Res. 2011; 21: 1328-1338https://doi.org/10.1101/gr.116012.110Crossref PubMed Scopus (208) Google Scholar; Chen et al., 2011Chen J. Melton C. Suh N. Oh J.S. Horner K. Xie F. Sette C. Blelloch R. Conti M. Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition.Genes Dev. 2011; 25: 755-766https://doi.org/10.1101/gad.2028911Crossref PubMed Scopus (161) Google Scholar; Harvey et al., 2013Harvey S.A. Sealy I. Kettleborough R. Fenyes F. White R. Stemple D. Smith J.C. Identification of the zebrafish maternal and paternal transcriptomes.Development. 2013; 140: 2703-2710https://doi.org/10.1242/dev.095091Crossref PubMed Scopus (124) Google Scholar). Of these mRNAs, more than two thousand mRNAs are stored as a translationally repressed form (Chen et al., 2011Chen J. Melton C. Suh N. Oh J.S. Horner K. Xie F. Sette C. Blelloch R. Conti M. Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition.Genes Dev. 2011; 25: 755-766https://doi.org/10.1101/gad.2028911Crossref PubMed Scopus (161) Google Scholar; Luong et al., 2020Luong X.G. Daldello E.M. Rajkovic G. Yang C.R. Conti M. Genome-wide analysis reveals a switch in the translational program upon oocyte meiotic resumption.Nucleic Acids Res. 2020; 48: 3257-3276https://doi.org/10.1093/nar/gkaa010Crossref PubMed Scopus (18) Google Scholar; Winata and Korzh, 2018Winata C.L. Korzh V. The translational regulation of maternal mRNAs in time and space.FEBS Lett. 2018; 592: 3007-3023https://doi.org/10.1002/1873-3468.13183Crossref PubMed Scopus (29) Google Scholar). Although when and where distinct mRNAs are translated remain largely unknown, the temporal and spatial control of global translation has been shown to be crucial for the proper promotion of developmental processes including zygotic genome activation, formation of embryonic axes, and cell differentiation (Aoki et al., 2003Aoki F. Hara K.T. Schultz R.M. Acquisition of transcriptional competence in the 1-cell mouse embryo: requirement for recruitment of maternal mRNAs.Mol. Reprod. Dev. 2003; 64: 270-274https://doi.org/10.1002/mrd.10227Crossref PubMed Scopus (44) Google Scholar; Kumari et al., 2013Kumari P. Gilligan P.C. Lim S. Tran L.D. Winkler S. Philp R. Sampath K. An essential role for maternal control of Nodal signaling.Elife. 2013; 2: e00683https://doi.org/10.7554/elife.00683Crossref PubMed Scopus (0) Google Scholar; Sun et al., 2018Sun J. Yan L. Shen W. Meng A. Maternal Ybx1 safeguards zebrafish oocyte maturation and maternal-to-zygotic transition by repressing global translation.Development. 2018; 145: dev166587Crossref PubMed Scopus (25) Google Scholar; Wang and Latham, 1997Wang Q. Latham K.E. Requirement for protein synthesis during embryonic genome activation in mice.Mol. Reprod. Dev. 1997; 47: 265-270https://doi.org/10.1002/(sici)1098-2795(199707)47:3<265::aid-mrd5>3.0.co;2-jCrossref PubMed Scopus (0) Google Scholar; Winata and Korzh, 2018Winata C.L. Korzh V. The translational regulation of maternal mRNAs in time and space.FEBS Lett. 2018; 592: 3007-3023https://doi.org/10.1002/1873-3468.13183Crossref PubMed Scopus (29) Google Scholar; Winata et al., 2018Winata C.L. Lapinski M. Pryszcz L. Vaz C. Bin Ismail M.H. Nama S. Hajan H.S. Lee S.G.P. Korzh V. Sampath P. et al.Cytoplasmic polyadenylation-mediated translational control of maternal mRNAs directs maternal-to-zygotic transition.Development. 2018; 145: dev159566https://doi.org/10.1242/dev.159566Crossref PubMed Scopus (26) Google Scholar; Zaucker et al., 2020Zaucker A. Kumari P. Sampath K. Zebrafish embryogenesis - a framework to study regulatory RNA elements in development and disease.Dev. Biol. 2020; 457: 172-180https://doi.org/10.1016/j.ydbio.2019.01.008Crossref PubMed Scopus (4) Google Scholar). However, the mechanisms by which translation of the dormant mRNAs is temporally and spatially controlled after fertilization remain largely unresolved. pou5f3 mRNA encodes the Pou-domain transcription factor Pou5f3 (also known as Pou2/Pou5f1), a homolog of mammalian Oct4/Pou5f1 (Takeda et al., 1994Takeda H. Matsuzaki T. Oki T. Miyagawa T. Amanuma H. A novel POU domain gene, zebrafish pou2: expression and roles of two alternatively spliced twin products in early development.Genes Dev. 1994; 8: 45-59https://doi.org/10.1101/gad.8.1.45Crossref PubMed Scopus (102) Google Scholar), and was identified as one of the mRNAs extensively translated in polysomes of zebrafish embryos at the cleavage stage (Lee et al., 2013Lee M.T. Bonneau A.R. Takacs C.M. Bazzini A.A. DiVito K.R. Fleming E.S. Giraldez A.J. Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition.Nature. 2013; 503: 360-364https://doi.org/10.1038/nature12632Crossref PubMed Scopus (274) Google Scholar). Identification of pou5f3 mutants in zebrafish that possess the maternally expressed pou5f3 transcript but are deficient in zygotically expressed pou5f3 demonstrated that Pou5f3 is essential for brain morphogenesis (Belting et al., 2001Belting H.G. Hauptmann G. Meyer D. Abdelilah-Seyfried S. Chitnis A. Eschbach C. Soll I. Thisse C. Thisse B. Artinger K.B. et al.Spiel ohne grenzen/pou2 is required during establishment of the zebrafish midbrain-hindbrain boundary organizer.Development. 2001; 128: 4165-4176https://doi.org/10.1242/dev.128.21.4165Crossref PubMed Google Scholar). Moreover, generation and analyses of maternal and zygotic pou5f3 mutants demonstrated that Pou5f3 is essential in early embryogenesis for differentiation of endoderm cells and specification of dorsal-ventral regions (Belting et al., 2011Belting H.G. Wendik B. Lunde K. Leichsenring M. Mossner R. Driever W. Onichtchouk D. Pou5f1 contributes to dorsoventral patterning by positive regulation of vox and modulation of fgf8a expression.Dev. Bio. 2011; 356: 323-336https://doi.org/10.1016/j.ydbio.2011.05.660Crossref PubMed Scopus (38) Google Scholar; Lunde et al., 2004Lunde K. Belting H.G. Driever W. Zebrafish pou5f1/pou2, homolog of mammalian Oct4, functions in the endoderm specification cascade.Curr. Biol. 2004; 14: 48-55https://doi.org/10.1016/j.cub.2003.11.022Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar; Reim and Brand, 2006Reim G. Brand M. Maternal control of vertebrate dorsoventral axis formation and epiboly by the POU domain protein Spg/Pou2/Oct4.Development. 2006; 133: 2757-2770https://doi.org/10.1242/dev.02391Crossref PubMed Scopus (78) Google Scholar; Reim et al., 2004Reim G. Mizoguchi T. Stainier D.Y. Kikuchi Y. Brand M. The POU domain protein spg (pou2/Oct4) is essential for endoderm formation in cooperation with the HMG domain protein casanova.Dev. Cell. 2004; 6: 91-101https://doi.org/10.1016/s1534-5807(03)00396-4Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In addition, Pou5f3 has been shown to trigger zygotic genome activation cooperatively with Nanog and Sox19b, which are also extensively translated in cleavage-stage embryos (Lee et al., 2013Lee M.T. Bonneau A.R. Takacs C.M. Bazzini A.A. DiVito K.R. Fleming E.S. Giraldez A.J. Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition.Nature. 2013; 503: 360-364https://doi.org/10.1038/nature12632Crossref PubMed Scopus (274) Google Scholar; Leichsenring et al., 2013Leichsenring M. Maes J. Mossner R. Driever W. Onichtchouk D. Pou5f1 transcription factor controls zygotic gene activation in vertebrates.Science. 2013; 341: 1005-1009https://doi.org/10.1126/science.1242527Crossref PubMed Scopus (141) Google Scholar). These studies demonstrated the importance of Pou5f3 synthesis from stored mRNAs after fertilization for directing diverse developmental processes. However, how the translation of pou5f3 mRNA is controlled during embryogenesis remains unknown. mRNA localization at particular regions within cells has been found in various types of cells including oocytes, neurons, and cultured fibroblasts (Buxbaum et al., 2015Buxbaum A.R. Haimovich G. Singer R.H. In the right place at the right time: visualizing and understanding mRNA localization.Nat. Rev. Mol. Cell Biol. 2015; 16: 95-109https://doi.org/10.1038/nrm3918Crossref PubMed Scopus (331) Google Scholar; Kloc et al., 2002Kloc M. Zearfoss N.R. Etkin L.D. Mechanisms of subcellular mRNA localization.Cell. 2002; 108: 533-544https://doi.org/10.1016/s0092-8674(02)00651-7Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Martin and Ephrussi, 2009Martin K.C. Ephrussi A. mRNA localization: gene expression in the spatial dimension.Cell. 2009; 136: 719-730https://doi.org/10.1016/j.cell.2009.01.044Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). Furthermore, studies in which gene expression patterns in Drosophila embryos, larvae, and ovaries were comprehensively analyzed revealed that thousands of mRNAs exhibit subcellular localizations within cells (Jambor et al., 2015Jambor H. Surendranath V. Kalinka A.T. Mejstrik P. Saalfeld S. Tomancak P. Systematic imaging reveals features and changing localization of mRNAs in Drosophila development.Elife. 2015; 4: e05003https://doi.org/10.7554/elife.05003Crossref Scopus (0) Google Scholar; Lecuyer et al., 2007Lecuyer E. Yoshida H. Parthasarathy N. Alm C. Babak T. Cerovina T. Hughes T.R. Tomancak P. Krause H.M. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.Cell. 2007; 131: 174-187https://doi.org/10.1016/j.cell.2007.08.003Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar; Wilk et al., 2016Wilk R. Hu J. Blotsky D. Krause H.M. Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs.Genes Dev. 2016; 30: 594-609https://doi.org/10.1101/gad.276931.115Crossref PubMed Scopus (83) Google Scholar). These findings suggest that widespread mRNAs are post-transcriptionally regulated in spatially controlled manners, although the biological significance of most of the mRNA localizations remains unknown. Cytoplasmic RNA granules such as stress granules and processing bodies (P-bodies) are assembled in many types of cells in organisms ranging from yeast to mammals and are thought to function as sites of storage and/or degradation of translationally repressed mRNAs (Buchan and Parker, 2009Buchan J.R. Parker R. Eukaryotic stress granules: the ins and outs of translation.Mol. Cell. 2009; 36: 932-941https://doi.org/10.1016/j.molcel.2009.11.020Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar; Ivanov et al., 2019Ivanov P. Kedersha N. Anderson P. Stress granules and processing bodies in translational control.Cold Spring Harb. Perspect. Biol. 2019; 11: a032813https://doi.org/10.1101/cshperspect.a032813Crossref PubMed Scopus (141) Google Scholar). Neuronal granules, another type of RNA granules found in neurons, transport mRNAs in a translationally repressed form to dendritic and/or axonal regions (Kiebler and Bassell, 2006Kiebler M.A. Bassell G.J. Neuronal RNA granules: movers and makers.Neuron. 2006; 51: 685-690https://doi.org/10.1016/j.neuron.2006.08.021Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar; Thomas et al., 2014Thomas M.G. Pascual M.L. Maschi D. Luchelli L. Boccaccio G.L. Synaptic control of local translation: the plot thickens with new characters.Cell Mol. Life Sci. 2014; 71: 2219-2239https://doi.org/10.1007/s00018-013-1506-yCrossref PubMed Scopus (25) Google Scholar). We previously showed that zebrafish and mouse oocytes possess cytoplasmic RNA granules consisting of translationally repressed cyclin B1, mos, mad2, or emi2 mRNA (Horie and Kotani, 2016Horie M. Kotani T. Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation.Eur. J. Cell Biol. 2016; 95: 563-573https://doi.org/10.1016/j.ejcb.2016.10.001Crossref PubMed Scopus (9) Google Scholar; Kotani et al., 2013Kotani T. Yasuda K. Ota R. Yamashita M. Cyclin B1 mRNA translation is temporally controlled through formation and disassembly of RNA granules.J. Cell Biol. 2013; 202: 1041-1055https://doi.org/10.1083/jcb.201302139Crossref PubMed Scopus (56) Google Scholar, Kotani et al., 2017Kotani T. Maehata K. Takei N. Regulation of translationally repressed mRNAs in zebrafish and mouse oocytes.Results Probl. Cell Differ. 2017; 63: 297-324https://doi.org/10.1007/978-3-319-60855-6_13Crossref PubMed Scopus (7) Google Scholar; Takei et al., 2020Takei N. Takada Y. Kawamura S. Sato K. Saitoh A. Bormann J. Yuen W.S. Carroll J. Kotani T. Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.J. Cell Sci. 2020; 133: jcs249128https://doi.org/10.1242/jcs.249128Crossref PubMed Scopus (5) Google Scholar, Takei et al., 2021Takei N. Sato K. Takada Y. Iyyappan R. Susor A. Yamamoto T. Kotani T. Tdrd3 regulates the progression of meiosis II through translational control of Emi2 mRNA in mouse oocytes.Curr. Res. Cell Biol. 2021; 2: 100009https://doi.org/10.1016/j.crcbio.2021.100009Crossref Google Scholar). These granules disassembled at different timings after initiation of meiosis, coinciding with the translational activation of assembled mRNA. Collectively, the results of most studies have suggested that cytoplasmic RNA granules function as sites of storage and/or cargo of dormant mRNAs within cells. In this study, we found that pou5f3 mRNA assembled into cytoplasmic RNA granules in a translationally repressed form in zebrafish oocytes. The granular structure of pou5f3 mRNA persisted after fertilization until, at least, the gastrulation stage, despite an increase in Pou5f3 protein. Staining of translating proteins and a ribosome protein indicated that newly synthesized peptides and the ribosomal protein were not colocalized with pou5f3 RNA granules in fertilized eggs but became colocalized in later stages. Moreover, nascent Pou5f3 polypeptides translated in polysomes were detected within pou5f3 RNA granules. The state and internal structure of granules were found to be changed after fertilization, and their change into liquid droplets was shown to be important for effective translation. These findings provide a model in which RNA granules, termed embryonic RNA granules, function as both repression and activation sites of translation for directing developmental processes. In zebrafish oocytes, pou5f3 mRNA was shown to be localized at the animal polar cytoplasm (Howley and Ho, 2000Howley C. Ho R.K. mRNA localization patterns in zebrafish oocytes.Mech. Dev. 2000; 92: 305-309https://doi.org/10.1016/s0925-4773(00)00247-1Crossref PubMed Scopus (0) Google Scholar). We first confirmed the localization of pou5f3 mRNA in growing and fully grown oocytes by in situ hybridization of zebrafish ovaries (Figures 1A and 1B ). The micropyle is a structure located at the top of the animal pole, through which a sperm enters into the egg cytoplasm (Figures 1B–1L). The region of localization of pou5f3 mRNA was similar to that of cyclin B1 mRNA (Figure 1C) (Kondo et al., 2001Kondo T. Kotani T. Yamashita M. Dispersion of cyclin B mRNA aggregation is coupled with translational activation of the mRNA during zebrafish oocyte maturation.Dev. Biol. 2001; 229: 421-431https://doi.org/10.1006/dbio.2000.9990Crossref PubMed Scopus (38) Google Scholar), which encodes Cyclin B1, a regulatory subunit of maturation-promoting factor (MPF). To examine the distribution pattern of pou5f3 mRNA, we performed fluorescence in situ hybridization (FISH) of zebrafish ovaries. pou5f3 mRNA was found to be distributed as granular structures in the animal polar cytoplasm (Figure 1D). No signal was detected with the pou5f3 sense probe (Figures S1A and S1B), confirming the specificity of signals. Interestingly, pou5f3 mRNA and cyclin B1 mRNA appeared to be assembled into different granules in the same region (Figures 1E–1H). The assembly of pou5f3 and cyclin B1 mRNAs into distinct granules in the same region was also observed in growing oocytes (Figures S1C and S1D). In our previous study, mos and cyclin B1 mRNAs were shown to be assembled into different granules at the animal polar cytoplasm of fully grown oocytes (Horie and Kotani, 2016Horie M. Kotani T. Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation.Eur. J. Cell Biol. 2016; 95: 563-573https://doi.org/10.1016/j.ejcb.2016.10.001Crossref PubMed Scopus (9) Google Scholar). pou5f3 RNA granules were also different from mos RNA granules (Figure 1O). In contrast to these mRNAs assembling into granules, α-tubulin and β-actin mRNAs were diffusely distributed in the oocyte cytoplasm (Figures S1E–S1G). The meiosis of fully grown oocytes is arrested at the prophase of meiosis I. These immature oocytes resume meiosis in response to hormonal stimuli and are arrested again at the metaphase of meiosis II. This process is called oocyte maturation, through which oocytes become matured and acquire fertility. We have shown that translationally repressed mRNAs such as cyclin B1, mos, mad2, and emi2 mRNAs form RNA granules in immature oocytes and that these granules disassemble during oocyte maturation at the times of translational activation (Horie and Kotani, 2016Horie M. Kotani T. Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation.Eur. J. Cell Biol. 2016; 95: 563-573https://doi.org/10.1016/j.ejcb.2016.10.001Crossref PubMed Scopus (9) Google Scholar; Kotani et al., 2013Kotani T. Yasuda K. Ota R. Yamashita M. Cyclin B1 mRNA translation is temporally controlled through formation and disassembly of RNA granules.J. Cell Biol. 2013; 202: 1041-1055https://doi.org/10.1083/jcb.201302139Crossref PubMed Scopus (56) Google Scholar; Takei et al., 2020Takei N. Takada Y. Kawamura S. Sato K. Saitoh A. Bormann J. Yuen W.S. Carroll J. Kotani T. Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.J. Cell Sci. 2020; 133: jcs249128https://doi.org/10.1242/jcs.249128Crossref PubMed Scopus (5) Google Scholar, Takei et al., 2021Takei N. Sato K. Takada Y. Iyyappan R. Susor A. Yamamoto T. Kotani T. Tdrd3 regulates the progression of meiosis II through translational control of Emi2 mRNA in mouse oocytes.Curr. Res. Cell Biol. 2021; 2: 100009https://doi.org/10.1016/j.crcbio.2021.100009Crossref Google Scholar). Consistent with the results of previous studies, cyclin B1 RNA granules had almost completely disassembled in mature oocytes (Figures 1J and 1M). In contrast, pou5f3 RNA granules remained in mature oocytes (Figures 1I–1M). We then analyzed polyadenylation of pou5f3 mRNA by using a poly(A) test (PAT) assay. Polyadenylation of dormant mRNAs has been shown to direct the translational activation of the mRNAs (Richter and Sonenberg, 2005Richter J.D. Sonenberg N. Regulation of cap-dependent translation by eIF4E inhibitory proteins.Nature. 2005; 433: 477-480https://doi.org/10.1038/nature03205Crossref PubMed Scopus (745) Google Scholar), and previous studies showed that the poly(A) tails of cyclin B1, mos, mad2, and emi2 are elongated at timings consistent with those of granule disassembly (Horie and Kotani, 2016Horie M. Kotani T. Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation.Eur. J. Cell Biol. 2016; 95: 563-573https://doi.org/10.1016/j.ejcb.2016.10.001Crossref PubMed Scopus (9) Google Scholar; Kotani et al., 2013Kotani T. Yasuda K. Ota R. Yamashita M. Cyclin B1 mRNA translation is temporally controlled through formation and disassembly of RNA granules.J. Cell Biol. 2013; 202: 1041-1055https://doi.org/10.1083/jcb.201302139Crossref PubMed Scopus (56) Google Scholar; Takei et al., 2020Takei N. Takada Y. Kawamura S. Sato K. Saitoh A. Bormann J. Yuen W.S. Carroll J. Kotani T. Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.J. Cell Sci. 2020; 133: jcs249128https://doi.org/10.1242/jcs.249128Crossref PubMed Scopus (5) Google Scholar, Takei et al., 2021Takei N. Sato K. Takada Y. Iyyappan R. Susor A. Yamamoto T. Kotani T. Tdrd3 regulates the progression of meiosis II through translational control of Emi2 mRNA in mouse oocytes.Curr. Res. Cell Biol. 2021; 2: 100009https://doi.org/10.1016/j.crcbio.2021.100009Crossref Google Scholar). We confirmed that cyclin B1 mRNA was polyadenylated in mature oocytes (Figure 1N). In contrast, the poly(A) tails of pou5f3 mRNA were not elongated (Figure 1N). Taken together, these results demonstrate that pou5f3 RNA granules are assembled and regulated differently from RNA granules consisting of dormant mRNAs that are translated during meiosis. To confirm the synthesis of Pou5f3 protein during zebrafish oogenesis and embryogenesis, we produced antibodies against the N-terminus region of zebrafish Pou5f3 (amino acids 1–122). All three affinity-purified antibodies recognized mainly a protein with a molecular mass of ∼55 kDa and also slightly recognized a protein with a molecular mass of ∼62 kDa in immunoblots of embryos at 3 and 6 h post fertilization (hpf) (Figure S2A, see also Figure 2A ). The intensities of both signals were significantly reduced when the antibody was preincubated with recombinant Pou5f3 (Figure S2B) and when the translation of pou5f3 mRNA was inhibited with the antisense MO (Figures S2C and S2D), confirming that the antibodies specifically recognize Pou5f3. The protein with a higher molecular mass may be a phosphorylated form of Pou5f3 as shown in a previous study (Lippok et al., 2014Lippok B. Song S. Driever W. Pou5f1 protein expression and posttranslational modification during early zebrafish development.Dev. Dyn. 2014; 243: 468-477https://doi.org/10.1002/dvdy.24079Crossref PubMed Scopus (22) Google Scholar). Time course analysis of oocytes and embryos showed that the amount of Pou5f3 was significantly increased from 3 hpf until 9 hpf (Figures 2A and 2B). No statistical significance was shown between immature and mature oocytes (Figure 2B), although the amount of Pou5f3 was slightly increased between immature and fertilized eggs (0 hpf). Given that poly(A) tails of pou5f3 mRNA were not changed (Figure 1N) and Pou5f3 protein was not accumulated in mature oocytes (Figure 2B), pou5f3 RNA granules observed in immature and mature oocytes exhibit the assembly of mRNAs in a translationally repressed form. We then analyzed the distribution patterns of pou5f3 mRNA after fertilization by whole mount in situ hybridization. The cortical cytoplasm of zebrafish eggs accumulates at the animal pole, resulting in blastodisc formation and progression of mitotic cleavages in this region (Figure 2C, insets). As reported previously (Takeda et al., 1994Takeda H. Matsuzaki T. Oki T. Miyagawa T. Amanuma H. A novel POU domain gene, zebrafish pou2: expression and roles of two alternatively spliced twin products in early development.Genes Dev. 1994; 8: 45-59https://doi.org/10.1101/gad.8.1.45Crossref PubMed Scopus (102) Google Scholar), pou5f3 mRNA was ubiquitously detected in the blastodisc and whole cells during early development (Figure 2C). High-resolution images showed that pou5f3 mRNA was present as granular structures in fertilized eggs (0 hpf) (Figure 2D). Intriguingly, the granular structures were maintained in embryos at 1.5, 3, and 6 hpf (Figures 2D and 2E), at which times Pou5f3 protein was significantly accumulated (Figures 2A and 2B). pou5f3 RNA granules were dominantly distributed in the cytoplasm of cells in cleavage-stage embryos (Figure 2F). A PAT assay showed that the poly(A) tails of pou5f3 mRNA were significantly elongated in embryos at 1.5 and 3 hpf (Figure 2G). These results suggest that pou5f3 mRNA starts to be translated after fertilization and that the translational activation is not coupled with apparent granule disassembly. To assess whether the pou5f3 mRNA is translated within granular structures, we used the ribopuromycylation method (David et al., 2012David A. Dolan B.P. Hickman H.D. Knowlton J.J. Clavarino G. Pierre P. Bennink J.R. Yewdell J.W. Nuclear translation visualized by ribosome-bound nascent chain puromycylation.J. Cell Biol. 2012; 197: 45-57https://doi.org/10.1083/jcb.201112145Crossref PubMed Scopus (176) Google Scholar) in embryos. This method is based on properties of the translational inhibitor puromycin, which enters into the ribosome A-site and is subsequently incorporated into the nascent chain C terminus by the peptidyl transferase activity of ribosomes (Figure 3A ) (David et al., 2012David A. Dolan B.P. Hickman H.D. Knowlton J.J. Clavarino G. Pierre P. Bennink J.R. Yewdell J.W. Nuclear translation visualized by ribosome-bound nascent chain puromycylation.J. Cell Biol. 2012; 197: 45-57https://doi.org/10.1083/jcb.201112145Crossref PubMed Scopus (176) Google Scholar). Incubation with cycloheximide (CHX) before and during puromycin treatment stabilizes translating ribosomes on mRNAs and detection of puromycin with an anti-puromycin antibody visualizes the newly synthesized peptides (Figure 3A) (David et al., 2012David A. Dolan B.P. Hickman H.D. Knowlton J.J. Clavarino G. Pierre P. Bennink J.R. Yewdell J.W. Nuclear translation visualized by ribosome-bound nascent chain puromycylation.J. Cell Biol. 2012; 197: 45-57https://doi.org/10.1083/jcb.201112145Crossref PubMed Scopus (176) Google Scholar; Moissoglu et al., 2019Moissoglu K. Yasuda K. Wang T. Chrisafis G. Mili S. Translational regulation of protrusion-localized RNAs involves silencing and clustering after transport.Elife. 2019; 8: e44752https://doi.org/10.7554/elife.44752Crossref PubMed Scopus (0) Google Scholar). This method has been developed using cultured cells but has not b" @default.
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• W4229042109 date "2022-06-01" @default.
• W4229042109 modified "2023-10-14" @default.
• W4229042109 title "Identification of embryonic RNA granules that act as sites of mRNA translation after changing their physical properties" @default.
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• W4229042109 doi "https://doi.org/10.1016/j.isci.2022.104344" @default.
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