M~6A Demethylase FTO Regulates MZF1 Gene Verification And Identification Of Its Action Sites

N6-methyladenosine(m~6A)is one of the most common and abundant epigenetic modifications in eukaryotic mRNA sequences,catalyzed by methyltransferases METTL3,WTAP and METTL14,and can be removed by the action of demethylase FTO and ALKBH5.m~6A modification can regulate mRNA alternative splicing,translation and stability,and abnormal levels of m~6A modification can lead to a variety of diseases,including a variety of malignant tumors.Although the m~6A modification is found in the5'UTR,coding region and 3'UTR of mRNA but mainly enriched near the stop codon and plays a regulatory role in a variety of biological processes,including regulation of mRNA stability.m~6A is mainly exist in the RRACH motif(R is G or A,H is A,C or U)and its regulation mainly depends on the protein to which it binds.Currently,m~6A binding protein and its downstream metabolic pathway have been studied extensively,but the sequence characteristics required for these proteins to bind to m~6A are not known,but the analysis of these sequence features is indispensable for the final disclosure of the function and regulation mechanism of m~6A.On the basis of previous work,we screened the candidate target gene MZF1 of the m~6A demethylase FTO and preliminarily determined the site of action near the stop codon of the gene mRNA.Then we constructing FTO knockdown and overexpressing cell lines further determind the regulatory effect of FTO on the MZF1.Then,Using FTO to regulate the stability of MZF1 gene mRNA as a model,by using the double luciferase assay to study the MZF1 gene by FTO by mutation and substitution of GGACU at the site of action stop of MZF1 gene mRNA and its flanking sequence The regulated core sequence and the efficiency of regulation of different RRACH motifs.The core sequence of FTO regulation and the regulation efficiency of different RRACH motifs.Our study can elucidate the effect of RRCAH motif type and its flanking sequences on the regulatory function in the process of m~6A regulation of mRNA stability,and reveal the molecular mechanism of m~6A regulation of mRNA stability,which provides important for the molecular mechanism of m~6A regulatory gene expression.