Kinetic analysis of FTO
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Genomewide-association studies have revealed that SNPs in FTO (Fat mass and obesity-associated transcript) are robustly associated with BMI and obesity. FTO is an Fe(II) 2-oxoglutarate (2OG) dependent dioxygenase that can demethylate 3-methylthymine (3-meT) in single-stranded DNA, as well as 3-methyluracil (3-meU) and N6-methyl adenosine in RNA. Here, we describe the development of an RNase cleavage assay measuring FTO's demethylation activity on 3-methyluracil. RNase A cleaves at the 3'end of pyrimidines, including uracil, and a methyl group at position three of uracil inhibits cleavage. An oligonucleotide probe was designed consisting of a DNA stem, a RNA loop containing a single 3meU as the only RNase A cleavage site, a fluorescent reporter on one end and a quencher at the other. FTO demethylation of the unique 3-methyluracil enables RNase A cleavage, releasing the quencher and enabling a fluorescent signal. In the presence of excess RNase A, FTO activity is limiting to the development of fluorescent signal, which can be read continuously and is able to discriminate between wildtype and the catalytically dead R316Q FTO. 2-oxoglutarate is a co-substrate of FTO and as a metabolite in the citric-acid cycle is a marker of intracellular nutritional status. Our assay was used to measure, for the first time, the Km of FTO for 2-oxoglutarate. The Km of 2.88uM is up to 10-fold lower than the estimated intracellular concentrations of 2-oxoglutarate, rendering it unlikely that FTO functions as a sensor for 2-oxoglutarate levels.
Biochem J (2012) Epub ahead of print. PMID 22435707