title: MicroRNA-196a regulates bovine newborn ovary homeobox gene (NOBOX) expression during early embryogenesis.
abstract: BACKGROUND
  Oocyte-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.
  
  
  RESULTS
  Using 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.
  
  
  CONCLUSIONS
  Collectively, our results demonstrate that miR-196a is a bona fide negative regulator of NOBOX during bovine early embryogenesis.
authors.affiliation: Laboratory of Animal Biotechnology and Genomics, Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506, USA.
authors.forename: Swamy K
authors.initials: SK
authors.lastname: Tripurani
authors.forename: Kyung-Bon
authors.initials: KB
authors.lastname: Lee
authors.forename: Gabbine
authors.initials: G
authors.lastname: Wee
authors.forename: George W
authors.initials: GW
authors.lastname: Smith
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yao
mesh_terms: D020413:3' Untranslated Regions; D000465:Algorithms; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002417:Cattle; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D010053:Ovary / Q000166:cytology / Q000196:embryology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D018398:Homeodomain Proteins; D035683:MicroRNAs; D014157:Transcription Factors