Functional Study Of Endogenous Small Interfering RNA In Mouse Maternal MRNA Degradation

Early embryonic development in mammals refers to the whole process of oocyte fertilization and development to blastocysts,in which several landmark key events occur,such as maternal factor degradation,zygote gene activation,blastomere compaction,and morula to blastocyst transition.Clarification of the mechanisms of early embryonic development in mammals is crucial for the applications of assisted reproduction.Most of matured oocytes in vitro are morphologically normal,but developmental defects after fertilization,in which abnormal degradation of maternal factor is one of the reasons.Maternal factor refers to RNA and protein synthesized and stored in the cytoplasm during oocyte development.It is essential for regulation of early embryo nic development.As embryonic development progresses,it is gradually replaced by the product s of zygotic genome,and the maternal factors are gradually degraded.The degradation of maternal m RNAs is important and mainly achieved by the maternal pathway and the zygote pathwa y.Previous studies have revealed the functional roles of RNA-binding proteins,micro RNAs(mi RNAs)and Piwi-interacting RNAs(pi RNAs)on the degradation of maternal m RNA.However,the function of endogenous small interfering RNA(endo-si RNA)in the process is still elusive.Endo-si RNA can negatively regulate gene expression at the post-transcriptional level,indicating that it may have important functions on the degradation of maternal m RNA.Thus,in the study,we investigated the function of endo-si RNA on the degradation of maternal m RNA in early mouse embryos.Firstly,we screened and identified maternal genes and corresponding small RNAs by bioinformatics,and identified 2,730 maternal genes and corresponding 9,148 endo-si RNAs and 72 mi RNAs.Expression pattern showed that the expression level of endo-si RNAs was mainly expressed at high levels before the two-cell stage and significantly downregulated in the four-cell stage,while the mi RNA was up-regulated in the two-cell stage embryo,when most of the maternal m RNAs were degradated.Endo-si RNA is mainly present in oocytes,one-cell and two-cell embryos,and its expression pattern is negatively correlated with maternal m RNA,indicating that endo-si RNA may play a key role on maternal m RNA degradation.The biogensis of endo-si RNA mainly depends on Dicer.Next,we injected the Dicer si RNA and antibody into the zygote to knock down the expression of Dicer,and investigated the effect of the deletion of endo-si RNA on the degradation of maternal m RNA.Single-cell transcriptome sequencing showed that Dicer knockdown resulted in abnormal expression of 1164 genes,and 462 genes were up-regulated and 71 genes were maternal genes,suggesting that endo-si RNA has limited function in the zygote pathway and may function mainly through the maternal pathway.Furthermore,we examined the expression of the maternal genes Spin,Zfp277 and Brip and their corresponding endo-si RNAs by RT-PCR and q PCR.The results showed that the expression pattern s of the three maternal m RNAs were negatively correlated with the endo-si RNAs,and,by injection of mimics and inhibitors of the endo-si RNAs,we verified the endo-si RNAs repressed the expression of the maternal m RNAs,indicating endo-si RNA could promote the degradation of maternal m RNA.And,by m RNA degradation assay,it was proved that endo-si RNA inhibited the expression of the maternal m RNAs at the post-transcriptional level.We also investigated the mechanism of endo-si RNA biogensis.We found that lnc521 was specifically expressed in zygote and complement with zfp277 m RNA.We also observed that lnc521 was specifically expressed in the cytoplasm,indicating it can form ds RNA with zfp277 m RNA.By RNA interference,we found that the formation of Zfp277 corresponding endo-si RNAs was significantly affected upon knockdown of lnc521,and microinjection of 322 bp sequence of lnc521,which was complement with zfp277 m RNA,could effectively rescue the formation of the endo-si RNAs and promote the degradation of the maternal m RNA.It indicates that the formation of Zfp277 corresponding endo-si RNAs is lnc521-dependent.In early embryonic development of mammals,the conversion of maternal factors to zygot ic transcripts is of great significance.In the process,maternal factors are rapidly cleaned and replaced by new substances produced by embryos.The results indicate that endo-si RNA promotes the degradation of maternal m RNA at the post-transcriptional level,opening a new direction for the study of maternal-zygotic transition.