Figure 2: Formation of new RNA modifications during oxidative demethylation of m6A in RNA by FTO.
From: FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA
(a) A 9mer ssRNA containing a 5′ m6A is used for HPLC-MS/MS analysis. After the RNA is treated with FTO, it is digested by nuclease P1 to release the first base as a nucleoside for further HPLC-MS/MS analysis. The rest of the bases are left as nucleoside 5′-monophosphate (5′-NMP). (b) HPLC analysis of nuclease P1-digested 5′m6A-9mer RNA oligo (5 μM) after treating with FTO. m6A was converted to hm6A and f6A after the treatment with 2.5 μM of FTO for 2 min at room temperature; substantial conversion of hm6A to f6A can be observed in 20 min. These newly formed peaks coelute with hm6A and f6A standards, respectively. The last peak in hm6A standard is N6-methyleneadenosine, a dehydration product that coexists with hm6A. (c,d) Comparison of MS/MS profile of m6A-oxidation products with hm6A and f6A standards. (c) The initial product matches the elution time and MS/MS fragmentation pattern of hm6A. (d) The later-formed product matches the elution time and MS/MS fragmentation pattern of f6A. (e) Kinetic behaviour of the reaction indicates that the formation rate of f6A is slower than the oxidation rate from m6A to hm6A by a factor of 12.3±1.9. Error bars, mean±s.e.m. for n=4 experiments.
