Fig 1.
Characterization of several kinds of nucleoside modification in DNA and RNA.
(a) Proposed oxidative demethylation of m6A to N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A) in RNA by FTO and oxidation of 5mdC to 5hmdC and 5-formylcytosine (5fC) in DNA by TET2. (b) Coomassie staining and western blot of His-tagged full-length human FTO proteins purified from BL21(DE3)-PlysS E. coli. (c) Base ion mass transitions for LC-MS-MS analysis of A, m6A, dC, 5mdC and 5hmdC standard. The MRM transitions were monitored as follows: 267.9 to 136.1 (A); 282.1 to 150.1 (m6A); 228.0 to 112.0 (dC); 258.0 to 142.0 (5hmdC); 242.1 to 126.1 (5mdC). (d) LC-MS-MS standards curves of A, m6A, dC, 5mdC and 5hmdC.
Fig 2.
LC-MS-MS assay measures the demethylation activity of FTO in vitro.
(a) (left) LC-MS-MS profiles of nucleosides derived from enzymatic hydrolysis of RNA samples containing m6A upon treatment with the FTO protein. The upper LC-MS-MS profile shows nucleoside A and m6A standards. (Right) Quantitation of m6A in vitro reaction system of FTO and synthetic RNA substrates. (b) (left) LC-MS-MS profiles of nucleosides derived from enzymatic hydrolysis of DNA samples containing 5mdC upon treatment with the FTO proteins. The upper LC-MS-MS profile shows nucleoside 5hmdC, dC and 5mdC standards. (Right) Quantitation of 5mdC in vitro reaction system of FTO and synthetic DNA. (c) FTO catalyzes the demethylation of m6A in a dose- and time-dependent manner. (d) FTO shows no obvious converting 5mdC to 5hmdC in DNA with the increase of the dose of FTO and reaction time. *p < 0.05; N.S.: No Significance. Error bars, mean ± S.E.M. for triplicate experiments.
Table 1.
Kinetic constants for FTO demethylation of m6A in ssRNA and 5mdC in DNA.
Fig 3.
Immunofluorescence and LC-MS-MS experiments measure the demethylation activity of FTO in vivo.
(a) Immunofluorescence analysis of 5hmdC level generated from 5mdC in FTO or TET2 overexpressed Hela cells. Cells were stained with anti-5hmdC antibody (green), showed that 5hmdC signal is obvious in the TET2 overpexpressed cells, instead of TET2 mutant or FTO gene transfected cells. Nuclei are stained by DAPI. Scale bar: 0–50 μm. (b) LC-MS-MS quantification analysis showed percentage of m6A/A in mRNA and total RNA isolated from control and FTO overexpressed cells. (c) LC-MS-MS quantification analysis showed percentage of 5hmdC/dC, 5hmdC/5mdC in DNA isolated from control, FTO and TET2 overexpressed cells. *p < 0.05; N.S.: No Significance. Error bars, mean ± S.E.M. for triplicate experiments.