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

Swamy K Tripurani1, Kyung-Bon Lee2, Gabbine Wee2, George W Smith23 and Jianbo Yao1*

Author Affiliations

1 Laboratory of Animal Biotechnology and Genomics, Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506, USA

2 Laboratory of Mammalian Reproductive Biology and Genomics, Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA

3 Department of Physiology, Michigan State University, East Lansing, MI 48824, USA

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BMC Developmental Biology 2011, 11:25  doi:10.1186/1471-213X-11-25

Published: 6 May 2011



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.


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.


Collectively, our results demonstrate that miR-196a is a bona fide negative regulator of NOBOX during bovine early embryogenesis.

NOBOX; bovine; oocyte; early embryogenesis; microRNA; maternal to zygotic transition