Exploration Of The Role And Molecular Mechanism Of RNF114 Protein In Mouse Early Embryo Development

In human and many organisms,the changes between maternal to zygotic transition(MZT)include the shift from meiosis to mitosis and the shift from a germ cell to a zygote.The zygote can produce both somatic and germ cells.The degradation of maternal proteins and RNAs together with the zygotic genome activation(ZGA)are the most important issues during MZT.In mammals,transcription of germ cells stops during meiotic maturation and the embryonic genome of mouse is not widely activated until late of 2-cell stage.The embryonic genome of human shows transcriptional activity until 4-8 cells satge.That is to say,MZT occurs in the absence of mRNA transcription.Therefore,early embryonic development rely on maternal mRNAs and proteins which synthesized during oogenesis.Far away,the known maternal factor is very limited.It’s a great encouragement to study futher about maternal factors which will help us to understand more about the important events in the process of early embryonic development.RNF114(RING finger protein 114)protein was a potential maternal protein.In the previous study,through the microinjection of two specific pairs of siRNA to reduce the level of RNF114 protein,we found that the siRNA-injected zygotes exhibit significantly two-cell developmental arrest.Through domain analysis of RNF 114 protein,we found that there is a RING finger domain in the protein structure.Finally,early studies confirmed that TAB1、RALGPS1、CD74、TNIP1 can be degraded by RNF 114 protein through its E3 activity.One of them,TAB 1’ overexpression cause two-cell stage arrest,too.It has been reported that the zygotic genome of mouse activated during late of 2-cell stage.So we speculate that the reduce of RNF114 protein may inhibit the zygotic genome activation through accumulation of TAB1 protein.To detect newly synthesized RNA after microinjected of two Rnf114 siRNAs or Tab1 mRNA,we added EU to the KSOM medium for about two hours when the embryo reached late of 2-cell stage.Compared to control groups,the incorporation of EU is sharply reduced in the microinjected groups.And when we added EDU to detect the cell cycle of the arrested 2-cells,we found that they had incorporation of EDU and whole nuclear membrane which indicated that they were arrested in G2 stage of second mitosis.According to the reports,TAB1 can inhibit the activity of NF-κB pathway which was important for early embryonic development of mouse.Previous study confirmed the NF-κB pathway was gradually activated from M Ⅱ oocytes to 4-cell stage.To further analyze the involvement of TAB1 and RNF114 in the regulation of NF-κB activity and ZGA in this study.First of all,we added NF-κB pathway inhibitor Bay 11-7082 to KSOM,we found that most of the embryo arrested in 2 cell satge.According the test of EU incorporation,we found that zygotic genome activation inhibited in the same situation.Then we microinjected two Rnf114 siRNAs or Tab1 mRNA into the zygotes.In contrast to the control group,most p65 nuclear translocation was blocked and the expression level of p-IκB reduced after Rnf114 siRNAs or Tab1 mRNA injected.All these results suggest that NF-κB pathway was inhibited.In summary,our study verified that RNF114 protein could degrade TAB 1 protein through its E3 ubiquitin ligase activity,and involved in the activation of NF-κB pathway,thus play an vital role in zygotic gene activation and early embryonic development.Exploration of biological function and mechanism of RNF114 will help us further understand the relationship between maternal factors and important events,such as,ZGA and MZT,happened in early embryonic development.