The full length mFTO and hFTO were cloned, expressed, and purified (Fig. S2). A restriction endonuclease digestion assay with the use of Dpn
II was adopted to evaluate the repair activities of both hFTO and mFTO towards 1-meA, 1-meG and εA in ssDNA or dsDNA [13
]. A modified 49 mer DNA with 1-meA, 1-meG, or εA incorporated into the GATC sequence is resistant to Dpn
II cleavage. Removal of the base lesion by FTO allows Dpn
II to cleave the 49 mer DNA probe into two fragments, providing an assay for the repair activity (). As reported previously [21
], we found very low activities of both proteins toward repairing 1-meA in 49 mer ssDNA (). Both proteins failed to repair 1-meA in dsDNA (stoichiometric amount) after a 12 h incubation at either 16°C () or 37°C (data not shown); whereas, purified ABH2 exhibited good repair activities for 1-meA and εA in dsDNA under the same conditions (). In addition, we did not observe any noticeable activity of mFTO or hFTO toward εA and 1-meG in either ssDNA or dsDNA. The 3-meT base lesion, when incorporated into the Dpn
II cleavage sequence, could not block the enzymatic digestion of the DNA probe. Thus, a different assay was used to evaluate repair of this base lesion.
Fig. 1 A restriction enzyme digestion assay for repair of 1-meA, εA, and 1-meG by ABH2, mFTO, and hFTO. (A) A 49 mer ssDNA (0.2 nmol) with 1-meA, εA, or 1-meG incorporated into a GATC sequence (can be recognized and cleaved by DpnII) was used (more ...)
To probe 3-meT demethylation by FTO proteins, a 15 mer ssDNA [5′-CTTGTCA(3-meT)CAGCAGA-3′] with 3-meT incorporated in the middle was synthesized and purified. This DNA was digested into nucleosides by nuclease P1 and alkaline phosphatase, and subsequently analyzed by HPLC. As shown in , nucleosides dC, dG, dT, dA and 3-medT could be cleanly separated. The change in intensity of the 3-medT peak was monitored with other nucleoside peaks as internal references. The 3-meT-containing ssDNA was incubated with either mFTO or hFTO at 16°C for 12 h. After quenching the reaction, the content of 3-meT was measured by the digestion assay and HPLC analysis. The results indicated complete demethylation of 3-meT in ssDNA by both mFTO and hFTO (). When a 15 mer 3-meC-containing ssDNA was subjected to the same repair assay, only ~15% of 3-meC was demethylated after 12 h under the same conditions (), showing strong preference of FTO proteins toward 3-meT over 3-meC.
Fig. 2 HPLC chromatograms of digested nucleosides from 3-meT-containing DNA (1 nmol), 3-meC-containing DNA (1 nmol) and 3-meU-containing RNA (1 nmol). (A) Complete demethylation of 3-meT in a 15 mer ssDNA by mFTO and hFTO. All reactions were run for 12 h at (more ...)
However, almost negligible demethylation activities (< 5% after 12 h) were observed for both proteins () when a 3-meT-containing dsDNA (obtained by annealing the 15 mer ssDNA with its complementary strand) was subjected to the same repair procedure. Thus, we conclude that recombinant mFTO and hFTO are not likely to catalyze demethylation of dsDNA substrates in vitro and perhaps in vivo.
The strong preference for 3-meT in ssDNA by the FTO proteins raised the question of whether 3-meU in ssRNA could also serve as a substrate for these proteins. We synthetically prepared 3-meU-CE phosphoramidite (Fig. S3) and incorporated it into a 15 mer ssRNA via solid state synthesis (Fig. S4). When this RNA substrate was incubated with either mFTO or hFTO, complete removal of the methyl group on 3-meU was observed in both cases (). Thus, both mFTO and hFTO are capable of repairing 3-meT in ssDNA and 3-meU in ssRNA in vitro.
The inhibition effect of α-KG at millimolar concentrations was evaluated as noted previously [29
] (Fig. S5), and 300 μM of α-KG was chosen for further studies. Next, the pH-activity profiles were estimated for the demethylation of 3-meT in ssDNA and 3-meU in ssRNA. In the case of mFTO, a decrease of the demethylation activity was observed with increasing pH for both ssDNA and ssRNA substrates (). However, hFTO exhibited the highest activity at pH 6.0 for both ssDNA and ssRNA substrates (). Thus, we chose pH 6.0 for detailed kinetic analysis.
Fig. 3 The pH-activity profiles for demethylation reactions of 3-meT in ssDNA and 3-meU in ssRNA by mFTO and hFTO. All reactions were run in triplicate at 16°C for 12 h. (A) Demethylation of 3-meT in the 15 mer ssDNA (1 nmol) by mFTO (0.05 nmol). (B) (more ...)
The kinetic studies were performed at 20°C with varying concentrations of ssDNA or ssRNA substrates. The results are shown in and summarized in . The mFTO protein showed higher activities toward both ssDNA and ssRNA substrates than hFTO. It should be noted that this result may not reflect the in vivo activities of these proteins because the purified recombinant mFTO appears to be more stable than the purified recombinant hFTO in vitro. The kinetic studies revealed that both mFTO and hFTO exhibit a 2-fold preference for 3-meU in ssRNA as the substrate over 3-meT in ssDNA. The experiments were repeated in triplicate with similar results obtained.
Fig. 4 Kinetics of demethylation reactions catalyzed by mFTO and hFTO. All reactions were run in triplicate at 20°C and pH 6.0. (A) Demethylation of 3-meT in the 15 mer ssDNA by mFTO (0.5 μM). (B) Demethylation of 3-meU in the 15 mer ssRNA by (more ...)
Kinetic constants for 3-meT and 3-meU demethylation by mFTO and hFTO at 20°C and pH 6.0
In summary, this study has established the DNA/RNA demethylation activity of the recombinant human FTO for the first time (). Both mFTO and hFTO showed no observable activity towards εA and 1-meG in ssDNA. A very low demethylation activity of 1-meA in ssDNA was observed for these two proteins. They could also catalyze demethylation of 3-meC in ssDNA, but with a much lower efficiency as compared to 3-meT in ssDNA. Both mFTO and hFTO failed to repair base lesions in dsDNA, strongly suggesting that they are involved in ssDNA or ssRNA processing.
FTO can demethylate both 3-meT from ssDNA and 3-meU from ssRNA.
Importantly, we showed that both recombinant hFTO and mFTO can demethylate 3-meU in ssRNA in vitro
(). The FTO protein’s RNA demethylation activity is slightly more efficient than the demethylation of 3-meT in ssDNA mediated by the same proteins. Considering the negligible repair of dsDNA substrates by the FTO proteins and their slight preferences for 3-meU in ssRNA over 3-meT in ssDNA, it is attractive to suggest FTO as a RNA demethylase [21
]. Perhaps it catalyzes the reverse reaction of a previously unrecognized RNA methylation and exerts gene regulation function at the RNA level. Disruption of this regulatory role of FTO may lead to the obesity phenotype linked to this protein. Of course, detailed in vivo
experiments are required to further test this hypothesis. This current work serves as the first comprehensive evaluation of the activities of both mFTO and hFTO in vitro
, and provides a foundation for further inquiry into the role of this very interesting nucleic acid demethylase.