Study On The Molecular Mechanism Of RNA M~6A Modification Level In Donor Cells Affecting Development Ability Of Porcine Somatic Cell Nuclear Transfer Embryos

The differentiation state and epigenetic modification mode in donor cells are one of the important factors affecting somatic cell nuclear transfer(SCNT)embryos development.RNA m~6A modification is one of the most abundant base modifications on eukaryotic m RNA.A large number of studies have shown that m~6A modification participated in animal gametogenesis,embryonic development and diseases formation by regulating m RNA stability,degradation,transport,splicing and translation.However,it is unclear whether there are differences in the RNA m~6A modification level between different types of donor cells and the difference will affect the development of porcine SCNT embryos.In this study,we compared the RNA m~6A modification level of different types of donor cells by immunofluorescence staining and Me RIP-seq.We revealed that DNA methylation affected the binding of transcription factor SP1 on METTL14 thus regulating the RNA m~6A level by dual luciferase experiment,point mutation,Ch IP and in vitro methylation assay.Then we analyzed the effects of the RNA m~6A level on the development ability of SCNT embryos by overexpressing or knockdown the expression level of RNA m~6A methyltransferase METTL14 in donor cells.Combined with single-cell transcriptome sequencing,we identified a candidate gene TOP2B,which affecting the DNA damage in porcine SCNT embryos in an m~6A dependent manner.Finally,our study provides a new clue of RNA m~6A modification in the development of SCNT embryos.The main findings are as follows:1.The effect of RNA m~6A methylation level of different types of donor cells on the developmental ability of SCNT embryos.In this study,porcine bone marrow mesenchymal stem cells(p BMSCs)and fetal fibroblasts(p EFs)were used as donor cells.We firstly detected and compared the RNA m~6A levels in different donor cells.The results showed that RNA m~6A level of p BMSCs was significantly higher than that of p EFs.The results of q PCR and WB showed that the expression level of RNA m~6A methylation-related enzymes in p BMSCs was also significantly higher than that in p EFs.Then we used Me RIP-seq technology to detect the RNA m~6A level in the whole transcriptome of different types of donor cells.our result found that there were obvious differences in the RNA m~6A modification of different donor cells.Compared with p EFs,p BMSCs showed a higher m RNA m~6A methylation levels.Those differential m~6A methylated genes are mainly enriched in ribosome processing,cell replication pathways.A large number of genes were expressed positively correlated with RNA m~6A methylation level.Then,we compared the development ability of SCNT embryos derived from different donor cells.Our results suggested that compared with p EFs,SCNT embryos with p BMSCs as donor cells had higher RNA m~6A methylation levels and embryonic development ability.2.DNA methylation of the METTL14 promoter regulated the RNA m~6A methylation level of donor cell through the transcription factor SP1.In order to study the causes of differences in the RNA m~6A methylation levels in different types of donor cells,BSP experiment was used to analyze the promoter DNA methylation level of the METL14 in different donor cells.The results showed that the promoter DNA methylation level of METTL14 in p BMSCs is lower than that in p EFs.Then we found that the transcription factor SP1 directly bound to the METTL14promoter region through identifying the core promoter region,site mutation of the transcription factor and Ch IP experiments.We peformed in vitro methylated experiments,and treated donor cells with DNMTs inhibitor,SP1 inhibitors or SP1overexpression,then we confirmed that DNA methylation inhibits the transcription factor SP1 from binding to METL14,thus regulating RNA m~6A methylation modification.What's more,the addition of RG108 decreased the DNA methylation level of donor cells but increased the RNA m~6A methylation level.This study provided an experimental basis for further studying the regulatory relationship between DNA methylation and RNA methylation.3.The molecular mechanism that changing the RNA m~6A methylation level of donor cell affected the development of SCNT embryos.To further investigate whether the RNA m~6A methylation level in donor cells affects the development of porcine SCNT embryos,we constructed lentiviral vectors that overexpression or interference of METTL14 in p EFs and p BMSCs to change the RNA m~6A methylation modification level in donor cells.The results showed that changing the expression of METTL14 could affect the RNA m~6A level in donor cells.We got reconstructed embryos by SCNT technology,and found that changing the RNA m~6A modification levels in donor cells could affected the m~6A modification levels of SCNT embryos at different developmental stages.Transcriptome sequencing showed that metabolic pathways,ribosomal biosynthesis,splicing bodies,RNA degradation and protein transport signaling pathways were regulated.And by m RNA stability assay,double luciferase experiment,SELECT assay and microinjection of TOP2B si RNA,we finally found that m~6A modification can regulate DNA damage by changing the stability of TOP2B m RNA and protein expression during SCNT embryo development.