Mechanistic Studies Of N6-methyladenosine (m’A)Demethylase FTO In Regulation Of Pre-mRNA Splicing Processing

FTO (fat mass and obesity associated) was identified as one of the genes associated with fat and energy metabolism. FTO genetic variants are not only related to obesity, but also associated with diabetes, cancer and other chronic diseases. The findings from FTO overexpressed or knockout mice have shown its important role in adipose accumulation and energy metabolism. In addition, in vitro biochemical data demonstrated that the dioxygenase FTO possesses the ability to remove N6-methyladenosine (m6A) methyl group in RNA. However, how FTO influences obesity-related metabolic pathways through the regulation of m6A in mRNA remains largely unknown.In this study, we demonstrated a novel role of FTO-mediated m6A modification in mRNA splicing and adipogenesis by using preadipocytes3T3-L1cell model system. During the process of adipocytic differentiation, both FTO mRNA and protein levels are gradually declined, while METTL3expression is no changed. Consistently, m6A level is significantly increased. FTO deficient, but not another m6A demethylase ALKBH5, leads to inhibition of adipogenesis. METTL3depletion promotes adipogenesis that can only be reversed by catalytically active FTO.Transcriptome and m6A-seq analyses revealed that both expression and mRNA splicing pattern of many genes with multi-isoforms are regulated by FTO. Specifically, m6A is enriched in5’and3’exonic sequences flanking the splice site, spatially overlapping with the ESE binding clusters of mRNA splicing regulatory SRSF1and SRSF2, but not SRSF3and SRSF4. FTO knock-down promotes RNA binding ability of SRSF2, and increases the retention level of specific exons of a set of genes, such as adipogenic factor RUNX1T1. FTO deletion promotes the sixth-exon skipping of RUNX1T1, while METTL3knockdown could reverse its role. Further, a totally opposite effects on adipocyte differentiation were observed by overexpression of RUNX1T1transcript with either inhibiting in the presence of the sixth exon or promoting in the absence of the sixth exon. Similar as the change of FTO expression, expression of RUNX1T1without the sixth exon also tends to be decreased during adipogenesis. These data suggest that FTO regulates adipogenesis through the regulation of RUNX1T1m6A level and its alternative splicing.Collectively, our findings provide the novel evidence that FTO-dependent m6A modification may regulate adipocyte differentiation through influencing the process of mRNA splicing. Additionally, m6A modification in mRNA may function as a new cis-acting element potentially significant in the process of mRNA alternative splicing, which provides important clues for further investigations on FTO function in obesity, and new insight into RNA epitranscriptome regulation of cellular process.