R255 Methylation Of METTL14 Regulates RNA M~6A Modification And Endoderm Differentiation Of Mouse Embryonic Stem Cells

There are more than 100 modifications of RNA,among which N6-methyladenosine(N6-methyladenosine,m6A)is the most common modification on mRNA and lncRNA.RNA m6A is catalyzed by the methyltransferase complex METTL3/METTL14/WTAP(Writer),removed by the demethylases FTO,ALKBH5(Eraser),and can be combined with the methylation recognition protein YTHDF family members(Reader),etc.And make a difference.Arginine methylation modification is an important type of post-translational modification,which is catalyzed by arginine methylase.Arginine methylation can regulate the activity of corresponding proteins by affecting their ability to bind to RNA or other proteins.Although phosphorylation of METTL14 and SUMO modification of METTL13 have been reported,arginine methylation modification has not been reported in the methylation enzyme METTL3/METTL14 protein complex.Based on this,we first identified by mass spectrometry and found that METTL14 arginine(R255)was methylated.Then we treated the cells with AdOx,an arginine methylase inhibitor,and found that the content of arginine methylation on METTL14 decreased after treatment.After METTL14 R255 was mutated to K(R255K),the arginine methylation level was also reduced.Because R255 is conserved in many species and is located at the interface between the methylase complex and RNA,suggesting that R255 has a function.In order to further study the function of the R255 site,we established the METTL14 R255K mutant cell line through CRISPR-cas9 gene editing technology.Through LC-MS/MS technology,it was found that m6A abundance on mRNA in mutant cells decreased.Then,meRIP-seq was used to identify m6A sites on the whole genome,and it was found that m6A in R255K was downregulated overall.MeRIP-qPCR further confirmed the reduction of m6A level on the gene.Genes regulated by R255 m6A generally have lower methylation levels and higher expression levels.In order to study the mechanism by which the R255 site regulates the level of m6A,we investigated the methyl transfer in R255K cellsThe stability of the enzyme complex components,intracellular localization,etc.were analyzed and found to be insignificant.Then the overall stability of the methyltransferase complex was studied,and it was found that after the R255 modification was removed,the WTAP content in the methylase complex interacting with METTL14 decreased,and the free WTAP content increased,while the interaction with METTL14 There was no significant change in METTL3 protein content.Since R255 is on the binding interface of two proteins,METTL3 and METTL14,and RNA,we found through RNA pull-down and RIP-qPCR that the loss of R255 methylation will weaken the binding ability of RNA and protein.In summary,we have concluded that R255 methylation affects the stability of the protein complex and affects its ability to bind RNA.To determine the biological function of R255 methylation modification,we analyzed by RNA-seq data and found that pluripotency maintenance gene expression was reduced in R255K cells.Further alkaline phosphatase staining(AP staining)found that R255K cells had reduced pluripotency compared to WT cells.Then we studied the differentiation potential of R255.Through the EB differentiation experiment,we found that the expression of the endoderm differentiation marker gene in R255K did not increase,while the other two germ layer differentiation were not affected,suggesting that R255 methylation may be specific Regulates endoderm differentiation of embryonic stem cells.In summary,our research shows that METTL14 R255 is methylated,and R255 methylation can promote the generation of m6A modification by stabilizing the methyltransferase complex and promote the combination of RNA and METTL14,and affect the embryonic stem cells.Endoderm differentiation process.