SON Affects Mouse Early Embryo Development By Regulating Pre-mRNA Splicing And Histone Methylation

Embryonic development is an orderly and dynamic process composed of a series of complex cell biological events,including cell division,growth and destiny differentiation to specific functional cells.The smooth progress of early embryonic development is an essential prerequisite for the generation of normal organisms.Therefore,the illumination of molecular mechanisms regulating the development of pre-implantation embryos is of great significance for improving the production efficiency of cloned animals,promoting the establishment of animal models of human diseases and promoting the progress of assisted reproduction technology.Zygotic Genome Activation(ZGA)was activated after the combination of sperm-egg,which transformed the transcriptional static oocytes into omnipotent cleavage cells and prepared the embryos for differentiation and further development.There are thousands of genes activated during this time,which means a lot of pre-mRNA is produced.These pre-mRNAs must be precisely spliced to ensure that the mature mRNA is properly translated into a functional protein.It has been shown that variable splicing plays a crucial role in early embryonic development in mammals.However,the specific mechanism in early embryonic development and its associated factors regulating splicing are currently unknown.Moreover,histone modification plays a very important role in the activation of embryonic genes and the determination of cell fate in pre implantation embryos.Here,we report a larger Ser/Arg(SR)associated protein,SON.The effects of Son on early embryonic development of mice were studied by injection of si RNA to construct the early embryo model of knockdown Son gene,including the changes of embryonic development,nuclear spot morphology,pre-mRNA splicing,transcriptomics and histone methylation modification.The main research results are as follows:1.SON affects early mouse embryo development by regulating pre-mRNA splicingThis study takes the fertilized eggs of ICR mice as the research object.(1)We used RT-PCR to detect the pre-mRNA content of embryos(zygotes and late 2-cell)before and after ZGA.The results showed that there were higher levels of pre-mRNA of Klf5,Nid2 and Mxra7 genes in the zygote than in the late 2 cells.(2)We detected the assembly of nuclear spots in embryos(early,middle,and late 2-cell)before and after ZGA by immunofluorescence staining of pre-mRNA splicing related factor SC35.The results showed that the nuclear spots of pre-ZGA embryos(early and middle 2-cell)were not completely assembled,while there were complete nuclear spots in post-ZGA embryos(late 2-cell).(3)We analyzed the co-localization of SON protein and SC35 in early mouse embryos by immunofluorescence technique.The results show that SON and SC35 are co-located in mouse embryos,which indicates that Son may play a crucial role in the assembly of nuclear spots and pre-mRNA splicing during the development of mouse embryos.(4)We analyzed the effect of down-regulation of Son on mouse early embryo development by means of cytoplasmic injection of si RNA.The results showed that si RNA injection significantly reduced the mRNA level of Son in mouse embryos,and significantly reduced the blastocyst rate of mouse early embryo development.Further analysis showed that the down-regulation of Son did not affect the cleavage rate,but most embryos stagnated in the morula stage.More importantly,we found that the mulberry embryos of the Son interference group were smaller than those of the NC control group and the blank control group.The results of nuclear staining showed that the number of cells in the mulberry embryo in the Son interference group was significantly reduced,and most of the mulberry embryos were abnormal in cleavage.(5)We analyzed the effect of Son down-regulation on the assembly of nuclear spots in mouse embryos by immunofluorescence staining of SC35.The results showed that the down-regulation of Son affected the assembly of SC35,resulting in a completely rounded morphology of SC35 in the nucleus.(6)We used RT-PCR to analyze the effect of Son down-regulation on pre-mRNA splicing.The results showed that the down-regulation of Son resulted in an increase in the pre-mRNA content of Tubg1,Akt1,Katnb1 and Rad23 A genes in the embryo.The above results show that the pre-mRNA splicing of ZGA preembryos is not mature,and Son regulates the pre-mRNA splicing by promoting the nuclear spot assembly of early embryos and ultimately participates in the development of mouse early embryos.2.Effects of SON on transcriptome and histone methylation in mouse embryosSON is reported to be involved not only in pre-mRNA splicing regulation,but also directly in gene transcription regulation.(1)First of all,we found that the down-regulation of Son did not affect the overall transcription level of ZGA embryos(late 2 cells)by using 5-ethynyluridine(EU)labeling experiment.(2)The effects of Son down-regulation on the transcriptome of 4-cell embryos were analyzed by RNA-Seq technique.The results showed that compared with the control group,there were 3527 significant genes in the 4-cell embryos after Son down-regulation,of which2128 genes were up-regulated and 1399 genes were down-regulated.It includes development-related genes(Nanog,Pou5f1 and Prdm14),histone methylation-related genes(Kdm5d and Setd7),cell cycle-related genes(Aurkb)and DNA replication,recombination and repair related genes(Pnkp and Smc1a).The results of q PCR were consistent with the results of RNA-Seq.The expression of embryodevelopment-related genes(Nanog,Pou5f1 and Prdm14)in 4-cell embryos decreased after down-regulation of Son.(3)The results of RNA-Seq and q PCR showed that the down-regulation of Son resulted in the down-regulation of cell cycle-related genes(Aurkb)and DNA replication,recombination and repair related genes(Pnkp and Smc1a),and the abnormal expression of these genes might lead to DNA damage.So we use γH2A.X staining showed that down-regulation of Son resulted in increased DNA damage in early mouse embryos.(4)The down-regulation of Son also led to a decrease in the expression of histone demethyltransferase Kdm5 d and an increase in the expression of histone methyltransferase Setd7.Therefore,we further tested the methylation of histone in early mouse embryos after down-regulation of Son.The results showed that the down-regulation of Son resulted in a significant increase in the levels of H3K4me3,H3K9me3 and H3K27me3 in early mouse embryos.The above results showed that the down-regulation of Son seriously affected the whole genome transcription level of mouse early embryos,enhanced the DNA damage of early embryos and up-regulated the histone methylation modification level of early embryos.