Distinguishing the Fat Mass and Obesity Associated Protein FTO as a RNA N6-Methyl Hydroxylase

Chemical modifications on ribonucleic acids (RNA), also termed RNA epigenetics, have diverse functions in gene regulation. The most well-characterised modification is N6 - methyladenosine (m 6 A), which affects gene regulation and is associated with cancer and viral infection. m 6 A can be removed by demethylases. Two human m 6 A demethylases have been identified to date: they are the fat mass and obesity associated protein (FTO) and AlkB homologue 5 (ALKBH5). Both FTO and ALKBH5 are potential anticancer targets because they were shown to promote certain cancers such as acute myeloid leukemia by FTO and glioblastoma by ALKBH5. Despite their importance, the differences in catalytic mechanisms and physiological roles between FTO and ALKBH5 remain unclear. Understanding these gaps of knowledge will allow us to piece together how the catalytic activities of FTO and ALKBH5 lead to cancer development thus allowing for better tailored anti-cancer therapies. Reports on activities of FTO and ALKBH5 have begun to suggest they have distinct biological roles. FTO has been reported to produce a mixture of products, including N6 -hydroxymethyladenosine (hm 6 A), N6 -formyladenosine (f6 A) and the demethylated adenosine (A). Our preliminary data using high throughput mass spectrometry and NMR showed that in the initial stages of FTO catalysis, hm 6 A was the only product while demethylated A was not observed, suggesting that FTO is a RNA hydroxylase instead of a demethylase. Recently, FTO has been shown to demethylate N6 ,2′O-dimethyladenosine (m 6 Am) near the 5′cap. Consistent with published studies, we also showed that FTO has a significantly higher activity on m6 Am near the RNA 5′-cap than internal m 6 A. Combining these findings, we hypothesise that FTO is a m 6 Am hydroxylase near the RNA 5′cap, which generates a novel RNA modification N6 - hydroxymethyl-2′O-methyladenosine (hm 6 Am), which could have important gene regulatory functions. hm 6 Am near the 5′cap may be the missing link between FTO’s catalytic function and cancer development. In this proposed study, we will (1) ascertain FTO’s in vitro hydroxylase activity against m6 Am near the RNA 5′cap, (2) perform structural and biophysical studies on FTO-m 6 Am substrate complexes, (3) investigate the potential roles of 5′cap-hm 6 Am on gene regulation, such as RNA stability, splicing and translation. Results from this study will allow us to distinguish the physiological role of FTO from ALKBH5, thus understanding how different cancers linked to each demethylase develop. This research will also aid in selective inhibitor design for RNA demethylases. Importantly, success of this project will open new lines of investigation on gene regulation via RNA hydroxylation.