Exploration Of The Role And Molecular Mechanism Of RNF114Protein In Mouse Early Embryo Development

In mammals，the genome of germ cells is transcription silencing during meiosis.In mice, a wide range of embryonic genome activation starts at the late stage of2-cellembryo; while in human it occurs at the4-8cell stage. Before the embryonic genomeactivation,embryonic development is under the regulation of maternal factors(including mRNA and protein)，which are accumulated and stored in cytoplasm ofmature oocytes. In the fertilized or nuclear transfer embryos，the maternal factorssupport initial embryonic development and activate the transcription of embryonicgenome，ensure the expression of new genes and biosynthesis of new proteins，anda cascade reaction to produce more and more factors，which maintains the embryodevelopment.Even after the activation of the embryonic genome，the persistence ofmaternal factors can promote the early embryonic development by still being togetherwith other maternal factors or interacting with the new genes expressed in theembyo.So the study of maternal factors will be helpful to in-depth understanding ofearly embryonic developmental events and regulatory mechanisms as well as to findthe reason of the failure of early embyo culture in vitro and provide a theoretical basisto improve the success rate.In our previous, RNF114protein was identified in the proteomic map of MIIoocyte. It is lack of functional studies in the oocyte. And other study has proved thatRNF114protein expressed in embryo stem cells and decreased obviously after stemcells undergoing differentiation. Our study had confirmed that the level of mRNAexpression levels in oocytes and zygotes is highest and then decreased significantlyafter cleavage and maintained a low level at the following stages.Immunofluorescence showed the persistent expression of RNF114protein inthe cytoplasm from GV to the blastocyst stage. These results indicated the lowtranscriptional activity of rnf114after zygoticgeneactivation（ZGA）in mice andRNF114mRNA and/or proteins had been gathered during the maturation of oocytes.So it might play an essential role as a maternal factor in early embryonic development.In addition, we found that RNF114protein has a RING finger and two of C2H2zincfinger domains by domain analysis,which suggested us it might play a biologicaleffect as an E3ligase during the early embryonic development by ubiquitinating thedownstream substrates and regulating their degradation or transcriptional activity.Through the microinjection of two specific pairs of siRNA, the function ofRNF114protein was blocked in early embryos. Our results proved that during theprocess of early embryo development, the defect of RNF114would cause thedecrease of both formation rate and the cell number of blastocyst, and the ratio ofinner cell mass and trophoblast cells in blastocyst has changed（showed moredecrease of inner cell mass）.Besides,our research had verified the E3activity ofRNF114protein by in vitro substrate-independent ubiquitination reaction andscreened40kinds of substrate protein by protein array technology. Then, part of thesubstrates（Cd74、Optn、Psat1、Rab3il1、Tnip1）had been confirmed bysubstrate-dependent ubiquitination reaction in vivo and in vitro. Besides it seemedthat RNF114，as an E3，mediated degradation of the substrates.In particular，it hasbeen reported that overexpression of Tnip1and Optn could inhibit TNF-inducedNF-kB activation to causes the retardation of cell cycle progress，cell proliferationand increase cell apoptosis.And overexpression of Cd74was recently shown tomediate positive feedback to ERK activity which could destruct ESC totipotency andpromote its differentiation.The above results led us to speculated that RNF114mightparticipate in cell proliferation and differentiation, cell apoptosis, maintenance of embryonic totipotency or other biological response during early embyonicdevelopment by mediating ubiquitination of some factors involved in the ERK/NF-kB signaling pathway.In summary, our study proved the key function of RNF114protein as an E3ubiquitin ligase in early embryonic development and its role in maintenance ofembryonic totipotency through the regulation of its downstream substrates. Theexploration of biological function and mechanism of RNF114can not only providesus a better understanding of early embryonic developmental events and regulatorymechanisms, but also offers some new ideas to improve the success probability ofembryos cultured in vitro.