MicroRNA-196a regulates bovine newborn ovary homeobox gene (NOBOX) expression during early embryogenesis

BackgroundOocyte-derived maternal RNAs drive early embryogenesis when the newly formed embryo is transcriptionally inactive. Recent studies in zebrafish have identified the role of microRNAs during the maternal-to-embryonic transition (MET). MicroRNAs are short RNAs that bind to the 3' UTR of target mRNAs to repress their translation and accelerate their decay. Newborn ovary homeobox gene (NOBOX) is a transcription factor that is preferentially expressed in oocytes and essential for folliculogenesis in mice. NOBOX knockout mice are infertile and lack of NOBOX disrupts expression of many germ-cell specific genes and microRNAs. We recently reported the cloning and expression of bovine NOBOX during early embryonic development and our gene knockdown studies indicate that NOBOX is a maternal effect gene essential for early embryonic development. As NOBOX is a maternal transcript critical for development and NOBOX is depleted during early embryogenesis, we hypothesized that NOBOX is targeted by microRNAs for silencing and/or degradation.ResultsUsing an algorithm "MicroInspector", a potential microRNA recognition element (MRE) for miR-196a was identified in the 3' UTR of the bovine NOBOX mRNA. Expression analysis of miR-196a in bovine oocytes and during early embryonic development indicated that it is expressed both in oocytes and embryos and tends to increase at the four-cell and eight-cell stages. Ectopic expression of NOBOX and miR-196a in HeLa cells inhibited the expression of NOBOX protein compared to the control cells without miR-196a. Similarly, the activity of a luciferase construct containing the entire 3' UTR of bovine NOBOX was suppressed, and the regulation was abolished by mutations in the miR-196a binding site indicating that the predicted MRE is critical for the direct and specific binding of miR-196a to the NOBOX mRNA. Furthermore, ectopic expression of miR-196a mimic in bovine early embryos significantly reduced the NOBOX expression at the both mRNA and protein levels.ConclusionCollectively, our results demonstrate that miR-196a is a bona fide negative regulator of NOBOX during bovine early embryogenesis.

[1]  G. Schultz,et al.  Transition from maternal to embryonic control in early mammalian development: A comparison of several species , 1990, Molecular reproduction and development.

[2]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[3]  G. Woude,et al.  The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs , 1989, Nature.

[4]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[5]  S. Elledge,et al.  Dicer is essential for mouse development , 2003, Nature Genetics.

[6]  P. Galle,et al.  High miR-196a levels promote the oncogenic phenotype of colorectal cancer cells. , 2009, World journal of gastroenterology.

[7]  Phillip D Zamore,et al.  Translational regulation in development , 1995, Cell.

[8]  D. Bartel,et al.  MicroRNA-Directed Cleavage of HOXB8 mRNA , 2004, Science.

[9]  G. Hannon,et al.  RNase III enzymes and the initiation of gene silencing , 2004, Nature Structural &Molecular Biology.

[10]  A. Pasquinelli,et al.  Regulation by let-7 and lin-4 miRNAs Results in Target mRNA Degradation , 2005, Cell.

[11]  N. Rajewsky.  microRNA target predictions in animals , 2006, Nature Genetics.

[12]  Isaac Bentwich.  Prediction and validation of microRNAs and their targets , 2005, FEBS letters.

[13]  A. Rajkovic,et al.  NOBOX homeobox mutation causes premature ovarian failure. , 2007, American journal of human genetics.

[14]  Jane Y. Wu,et al.  Misexpression of miR-196a induces eye anomaly in Xenopus laevis , 2009, Brain Research Bulletin.

[15]  Zi-jiang Chen,et al.  Mutation analysis of NOBOX homeodomain in Chinese women with premature ovarian failure. , 2009, Fertility and sterility.

[16]  M. Manzanares,et al.  An early developmental role for miRNAs in the maintenance of extraembryonic stem cells in the mouse embryo. , 2010, Developmental cell.

[17]  Lei Li,et al.  Maternal control of early mouse development , 2010, Development.

[18]  J. Steitz,et al.  Metazoan oocyte and early embryo development program: a progression through translation regulatory cascades. , 2006, Genes & development.

[19]  R. Rempel,et al.  Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. , 1988, Developmental biology.

[20]  Martin M Matzuk,et al.  Deletion of Dicer in somatic cells of the female reproductive tract causes sterility. , 2008, Molecular endocrinology.

[21]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[22]  Benedikt Brors,et al.  Microarray-based approach identifies microRNAs and their target functional patterns in polycystic kidney disease , 2008, BMC Genomics.

[23]  Jan Krützfeldt,et al.  Strategies to determine the biological function of microRNAs , 2006, Nature Genetics.

[24]  Geraldine Seydoux,et al.  A clean start: degradation of maternal proteins at the oocyte-to-embryo transition. , 2004, Trends in cell biology.

[25]  F. Tang,et al.  Maternal microRNAs are essential for mouse zygotic development. , 2007, Genes & development.

[26]  Anton J. Enright,et al.  Materials and Methods Figs. S1 to S4 Tables S1 to S5 References and Notes Micrornas Regulate Brain Morphogenesis in Zebrafish , 2022 .

[27]  M. Sheets,et al.  Deadenylation of maternal mRNAs mediated by miR-427 in Xenopus laevis embryos. , 2009, RNA.

[28]  Vesselin Baev,et al.  MicroInspector: a web tool for detection of miRNA binding sites in an RNA sequence , 2005, Nucleic Acids Res..

[29]  Martha L Bulyk,et al.  Microarray Analyses of Newborn Mouse Ovaries Lacking Nobox1 , 2007, Biology of reproduction.

[30]  A. Bettegowda,et al.  Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development. , 2007, Frontiers in bioscience : a journal and virtual library.

[31]  Anton J. Enright,et al.  Zebrafish MiR-430 Promotes Deadenylation and Clearance of Maternal mRNAs , 2006, Science.

[32]  Changning Yan,et al.  Nobox is a homeobox-encoding gene preferentially expressed in primordial and growing oocytes , 2002, Mechanisms of Development.

[33]  Yitzhak Pilpel,et al.  Composition and regulation of maternal and zygotic transcriptomes reflects species-specific reproductive mode , 2010, Genome Biology.

[34]  C. Sander,et al.  A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing , 2007, Cell.

[35]  R. Schultz,et al.  The molecular foundations of the maternal to zygotic transition in the preimplantation embryo. , 2002, Human reproduction update.

[36]  K. Echeverri,et al.  miR-196 is an essential early-stage regulator of tail regeneration, upstream of key spinal cord patterning events. , 2009, Developmental biology.

[37]  W. Filipowicz,et al.  Role of Dicer in posttranscriptional RNA silencing. , 2008, Current topics in microbiology and immunology.

[38]  Martin M Matzuk,et al.  NOBOX Deficiency Disrupts Early Folliculogenesis and Oocyte-Specific Gene Expression , 2004, Science.

[39]  A. Bosserhoff,et al.  MicroRNA miR-196a is a central regulator of HOX-B7 and BMP4 expression in malignant melanoma , 2010, Cellular and Molecular Life Sciences.

[40]  R. Russell,et al.  Principles of MicroRNA–Target Recognition , 2005, PLoS biology.

[41]  Fugaku Aoki,et al.  Degradation of maternal mRNA in mouse embryos: Selective degradation of specific mRNAs after fertilization , 2005, Molecular reproduction and development.

[42]  J. Ireland,et al.  Quantitative analysis of messenger RNA abundance for ribosomal protein L‐15, cyclophilin‐A, phosphoglycerokinase, β‐glucuronidase, glyceraldehyde 3‐phosphate dehydrogenase, β‐actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro , 2006, Molecular reproduction and development.

[43]  P. Coussens,et al.  JY-1, an oocyte-specific gene, regulates granulosa cell function and early embryonic development in cattle , 2007, Proceedings of the National Academy of Sciences.

[44]  J. Yao,et al.  Role of Importin Alpha8, a New Member of the Importin Alpha Family of Nuclear Transport Proteins, in Early Embryonic Development in Cattle1 , 2009, Biology of reproduction.

[45]  Andrew D. Johnson,et al.  Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes. , 1985, Developmental biology.

[46]  G. Hannon,et al.  The miRNA-Processing Enzyme Dicer Is Essential for the Morphogenesis and Maintenance of Hair Follicles , 2006, Current Biology.

[47]  A. Iritani,et al.  Mechanism of Translation in the Period of Oocyte to Zygote Transition in Mammals , 2005 .

[48]  T. Hennet,et al.  Differential regulation of the zebrafish orthopedia1 gene during fate determination of diencephalic neurons , 2006, BMC Developmental Biology.

[49]  J. Yao,et al.  Cloning and analysis of fetal ovary microRNAs in cattle. , 2010, Animal reproduction science.

[50]  Lei Wang,et al.  A novel functional role for the oocyte-specific transcription factor newborn ovary homeobox (NOBOX) during early embryonic development in cattle. , 2011, Endocrinology.

[51]  G. Begemann.  MicroRNAs and RNA interference in zebrafish development. , 2008, Zebrafish.

[52]  Kunio Inoue.  [MicroRNA function in animal development]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[53]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[54]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature Reviews Genetics.

[55]  J. Castle,et al.  Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs , 2005, Nature.

[56]  Shridar Ganesan,et al.  Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. , 2005, Genes & development.