The Function And Mechanism Study Of Maternal BTG4 During Mouse Early Embryogenesis

Maternal mRNAs decay is a critical process during the early embryonic development.However,the mechanism and regulators governing maternal mRNAs clearance remains unclear in mammals.In general,the CCR4-NOT complex plays predominant roles in mRNAs deadenylation that is the first and often rate-limiting step accounting for mRNA turnover.But there are no RNA-binding protein in CCR4-NOT complex,other RNA-binding protein or adaptor protein are needed to mediate it's deadenylation function.The anti-proliferative BTG/TOB proteins as adaptor protein are important players in CCR4-NOT mediated mRNAs deadenylation and subsequent degradation in somatic cell.Although BTG/TOB protein family plays an important function in mRNA deadenylation,there has been reported five members(Tob1?Tob2?Btg1?Btg2?Btg3)of the knockout mice have normal viability and fertility.In addition,compared with the five members,Btg4 specificity high expression in mouse oocytes and early embryo according to the published gene expression database.These indicated that Btg4 may plays an important regulatory role in maternal mRNA degradation during the process of maternal to zygote transition.This project studied the functions and mechanism of Btg4 with knockout mice during the process of maternal to zygote transition.Real-time quantitative RT-PCR showed that Btg4 highly expressed in ovary and testis,appear in the oocytes and early embryo with maternal expression patterns;At present study,we found that BTG4 protein showed a dominant and specific expression pattern during mouse maternal to zygotic transition(MZT).This suggests that it may play important function in this particular development stage.To research the Btg4 physiological function,this study established the gene knockout mice.The specificity of the expression model of BTG4 protein is controlled by the cis element of 3 '-UTR and the corresponding trans role factor.Disruption of Btg4 had no effect on mouse viability and the male's fertility,but led to the infertility of female mice.The absence of Btg4 does not affect the female's ovulation and oocyte maturation.However,maternal Btg4 knockout lead to the early embryo developmental failure,even the fertilized eggs formed by the wild type sperm and maternal Btg4 loss of eggs.Although the transcriptome profile of GV oocytes was comparable to that of control,over 46%(6401/13770)and 20%(2898/14058)mRNAs were up-regulated in Btg4-/-MII oocytes and 1-cell embryos,respectively.Using the qRT-PCR technology,we choose some representative maternal genes to verify the high-throughput sequencing results.To further explore reason Btg4 loss up-regulated the maternal mRNA,Poly(A)tail length assay(PAT)showed that the representative transcripts in Btg4-/-MII oocytes and the 1-cell embryos failed to be deadenylated and were thus resistant to be degraded.Furthermore,we found that BTG4 might bridge the CCR4-NOT complex and PABPC1L,an oocyte-and-early-embryo-specific poly(A)binding protein,to control maternal mRNAs elimination during mouse MZT.Deletion of two conservative Motif(BoxA and B)of BTG4 protein affected the BTG4 and CNOT7 interaction.Finally,We also carried out BTG4 rescue experiment,the results showed that only the full-length BTG4 can rescue abnormal phenotypes caused by Btg4 loss.Altogether,our results demonstrate that Btg4 is a key regulator for the degradation of maternal mRNAs in mouse early embryonic development,suggesting it function through bridging CCR4-Not complex and poly(A)binding protein PABPC1L during this process.Moreover,Our research not only increased the understanding of the molecular regulatory mechanism during maternal to zygote transition,but can also provide some reference for the pathogenesis of human infertility and so on.