Screening And Identification Of The Downstream Genes Of M6A Demethylase FTO In Hela Cells

N6-methyl-adenosine(m6A)is one of the most common and abundant modifications of eukaryotic RNA molecules.The m6 A modification process is dynamically reversible and is catalyzed by the methyltransferase complex consisting of METTL3,WTAP and METTL14 and can be "erased" under the demethylase FTO and ALKBH5.m6 A co-immunoprecipitation high-throughput sequencing(Me RIP-Seq)found that M6 A modification was distributed in the 5'UTR,CDS and 3'UTR of m RNA,but mainly concentrated in the 3'UTR region,especially near the stop codon.highest.And the m6 A modification mainly occurs in the DRACH motif(R is G or A,H is A,C,or U),and the most common motif is GGACU.A large number of studies have shown that m6 A regulates the expression of genes by regulating m RNA structure,splicing,stability and translation efficiency,and regulating the processing of micro RNAs,and thus participates in biological processes including stem cell differentiation,DNA damage repair,and tumorigenesis.FTO is the major m6 A demethylase gene that regulates the protein levels of its downstream genes by modulating the methylation level of specific m6 A sites in its downstream genes.Abnormal expression of the FTO gene and abnormal changes in the corresponding gene m6 A levels have been found in a variety of diseases including tumors.However,the downstream genes of FTO demethylation function and the conservation of m6 A modifications of these downstream genes in different cells remain unclear.This study first screened eight candidate FTO demethylation downstream genes by published FTO gene knockdown and overexpressed RNA-seq and m6A-seq data.Then knock down and overexpress FTO gene in Hela cells and detect the expression changes of 8 potential genes after FTO knockdown and overexpression.The results showed that FTO had a positive regulatory effect on the expression of C21orf59 gene,but negatively regulated the expression of MZF1,PPARD,KCNG1,RARA and ASB2 genes.Finally,Me-RIP was used to detect the methylation level of specific m6 A sites after FTO knockdown.The results showed that there was an m6 A site with a significantly increased methyl level on the m RNA of the 6 downstream genes regulated by FTO,thereby confirming that the 6 genes are downstream genes of FTO demethylation function.The implementation of this study has the following two aspects:(1)Provide important reference data for the pathogenesis of diseases caused by abnormal expression of FTO.(2)This study shows that m6 A has positive regulation and negative regulation in gene expression.Therefore,the two types of downstream genes identified in this study can be used as a model to study the molecular mechanism of m6A-regulated gene expression.