Articles pubs.acs.org/acschemicalbiology
Diazirine Photocrosslinking Recruits Activated FTO Demethylase Complexes for Specific N6‑methyladenosine Recognition Hyun Seok Jeong,† Gosuke Hayashi,‡ and Akimitsu Okamoto*,†,‡ †
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan ‡ Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *
ABSTRACT: N6-methyladenosine (m6A) is a prevalent modification of RNAs. m6A exists in mRNA and plays an important role in RNA biological pathways and in RNA epigenetic regulation. We applied diazirine photocrosslinking to the event of m6A recognition mediated by the fat mass and obesity associated (FTO) demethylase. A highly photoreactive diazirine adjacent to m6A on the RNA successfully recruited activated FTO complexes with an m6A preference. The process of recognition of m6A via FTO using diazirine photocrosslinking was controlled by the α-ketoglutarate (α-KG) cosubstrate and the Fe(II) cofactor, which are involved in m6A oxidative demethylation. In addition, FTO bound to ssRNAs prior to the m6A recognition process. Diazirine photocrosslinking contributes to increasing the chances of capturing activated FTO complexes with specific m6A recognition and provides new insights into the dynamic FTO oxidative demethylation process. The process of recognition of m6A in RNA via FTO may depend on the kinetics of the dynamic binding between m6Acontaining RNAs and FTO. These incomplete binding kinetics render it difficult to maintain the binding. Therefore, the use of new chemical techniques to capture the intermediate reactions between m6A-containing RNAs and FTO is necessary. Photocrosslinking is a powerful tool in these studies as it induces covalent bonding for desired targets, independent of incomplete binding kinetics.11,12 It can also help identify binding partners or unveil biological mechanisms. A wide range of photocrosslinking groups have been used for smallmolecule−protein interactions, protein−protein interactions, and oligonucleotide−protein interactions.13,14 We expect that photocrosslinking can be applied to capture the event of m6A recognition by FTO, which is the main process of FTO mediated m6A oxidative demethylation. Here, we describe the photocrosslinking between m6Acontaining RNAs and FTO. The photocrosslinking of m6Acontaining RNAs may capture the event of m6A recognition using FTO. In addition, photocrosslinking may contribute to enriching the desired target, the m6A-containing RNA, which is difficult to capture using conventional chemical reactions.
N6-methyladenosine (m6A) is a common modification of many types of RNAs, including mRNA, snRNA, and rRNA.1,2 Recently, it has been reported that m6A modifications are widely present in thousands of mRNAs, with an estimated frequency of 3−5 m6A per mRNA. m6A is highly involved in posttranscriptional regulation, e.g., mRNA splicing, translation, nuclear export, mRNA decay, subcellular localization, and tissue-specific regulation.3−5 The m6A modification is dynamically regulated by m6A methyltransferases (METTL3 and METTL14)6 and m6A demethylases (FTO and ALKBH5).7,8 The m6A methylation is induced by m6A methyltransferases within a general consensus of Pu(G > A)m6AC(A/C/U). Conversely, the fat mass and obesity-associated (FTO) protein, which is a member of the nonheme Fe(II) and α-ketoglutarate (α-KG)-dependent dioxygenase AlkB family, converts m6A back to the original A via that m6A demethylation process.7 A previous study revealed that the crystal structure of FTO might provide a preference for single-stranded nucleic acids.9 Although m6A was confirmed as a major target of FTO induced demethylation, the process of substrate recognition of ssRNAs during m6A demethylation remains unknown. In addition, the m6A demethylation mechanism was suggested only at the level of the process of molecular recognition of m6A, which is mediated by Fe(IV)-oxo intermediates and oxidizes the C−H bonds of the substrates.10 FTO is mainly expressed in the nucleoplasm, suggesting that nuclear RNAs are the main substrates of FTO.5,7 However, the process of substrate selection and m6A recognition in ssRNA in FTO-mediated demethylation remains unknown. © XXXX American Chemical Society
Received: December 12, 2014 Accepted: March 9, 2015
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DOI: 10.1021/cb5010096 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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RESULTS AND DISCUSSION In photocrosslinking studies, diazirine is one of the commonly used photoreactive groups.15 Diazirine is a three-membered ring system that contains two nitrogen atoms and one carbon atom and is widely applicable in biology as a photoaffinity labeling probe.16−21 Its relatively small size allows the minimization of the possibility of interfering with the interactions. It is also chemically stable under wide reaction conditions. Most importantly, it can be cleaved by longwavelength (360 nm) light to produce high photoaffinity for desired targets. Diazirine generates a reactive carbene upon UV irradiation, and the carbene rapidly forms a covalent bond with the nearby protein residues. Hence, we selected diazirine as the photocrosslinking group to capture the event of m6A recognition by FTO with enhanced binding. We designed a diazirine photocrosslinking reaction between an m6A-containing RNA and FTO, as described in the scheme of Figure 1a. In the FTO m6A demethylation process, FTO
The reactive carbene induced by diazirine photolysis under UV irradiation may attack nearby ssRNA binding sites located around FTO active sites, resulting in stable covalent bonding between ssRNA and FTO, to produce ssRNA−FTO photocrosslinking complexes. We predicted that m6A-containing RNAs might be enriched in the presence of FTO via strong covalent bonding. To accomplish this strategy, we incorporated the photoactivatable diazirine group into the m6A ssRNA. To conjugate the diazirine photocrosslinking group with the RNA, we applied the Suzuki−Miyaura cross-coupling method, which has been used in DNA postsynthetic modification,22 in the RNA platform (Figure 1b). We selected a 15 nt ssRNA containing a central N6-methyladenosine (m6A) as an FTO recognition site and 5-iodoridine (I5U) as a diazirine conjugation site (as described in FITC-I5U-m6A), as well as a control ssRNA containing a central A and I5U (FITC-I5U-A; Table 1). The selected distance between m6A and I5U was 2 nt Table 1. ssRNA Sequences Used in This Study ssRNA FITC-I5U-m6A FITC-I5U-A FITC-Dz5U-m6A FITC-Dz5U-A FITC-m6A FITC-A
sequences (5′-3′) FITC-AUU FITC-AUU FITC-AUU FITC-AUU FITC-AUU FITC-AUU
GI5UC Am6AC AGC AGC GI5UC AAC AGC AGC GDz5UC Am6AC AGC AGC GDz5UC AAC AGC AGC GUC Am6AC AGC AGC GUC AAC AGC AGC
to minimize the perturbation of the interaction between the FTO m6A recognition site and the ssRNA. In addition, FITC was labeled at the 5′-end of the ssRNA, for sensitive detection based on fluorescence. The corresponding m6A, I5U, and FITC phosphoramidites (Supporting Information Figure S1) were incorporated into ssRNAs using an automated DNA/RNA synthesizer. The synthesized ssRNAs were treated with postsynthetic processing, followed by HPLC purification (Supporting Information Figure S2). The FITC-I5U-m6A and FITC-I5U-A ssRNAs were conjugated with diazirine containing boronic esters via Suzuki−Miyaura cross-coupling to produce FITC-Dz5U-m6A and FITC-Dz5U-A ssRNAs, respectively. The resultant ssRNAs containing the diazirine photocrosslinking group (Dz5U) were also purified by HPLC (Supporting Information Figure S3). The diazirine coupling reactions in RNA platforms were performed successfully, yielding major peaks on HPLC. Control RNAs that did not contain Dz5U (FITC-m6A and FITC-A) were also synthesized, to confirm the photocrosslinking effects (Table 1). The diazirine-conjugated RNAs (FITC-Dz5U-m6A and FITC-Dz5U-A) and the control ssRNAs without diazirine (FITC-m6A and FITC-A) were coincubated with FTO individually in the presence of the α-KG cosubstrate and the Fe(II) cofactor. The coincubated solutions were immediately exposed to UV for a short period (≤10 min). The photo-crosslinked products were separated using polyacrylamide gel electrophoresis (PAGE) and visualized using the VersaDoc imaging system and an FITC filter. The photo-cross-linked ssRNA−FTO complexes were detected by FITC fluorescent signals around 65 kDa, which was similar to the total molecular weight of the ssRNA−FTO complex (the size was predicted based on the 5 kDa size of the ssRNA and the 60 kDa size of hFTO; Figure 2a). The ssRNAs containing Dz5U (FITC-Dz5U-m6A and FITC-Dz5U-A) were
Figure 1. Diazirine photocrosslinking between an N6-methyladenosine (m6A)-containing RNA and the m6A-specific demethylase FTO. (a) FTO recognition mechanism of m6A and the design of diazirine photocrosslinking between the m6A-containing RNA and FTO. (b) Synthesis of the diazirine-labeled m6A-containing ssRNA.
binds the α-KG cosubstrate and the Fe(II) cofactor at the H231, D233, and H307 amino acid residues to produce an activated FTO complex. Subsequently, the activated FTO complex might exhibit preference for m6A-containing ssRNAs over natural A-containing ssRNAs. Then, FTO might recognize m6A to form an m6A-containing ssRNA−FTO intermediate complex, which can be captured by diazirine photocrosslinking. B
DOI: 10.1021/cb5010096 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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between m6A and A. This result indicates that activated FTO recognized m6A on the FTO bound ssRNA. The photocrosslinking results obtained from gradually increasing FTO/ RNA ratios suggested that the ability of FTO to discriminate between m6A and A was concentration dependent (Supporting Information Figure S7). We also confirmed other RNAs containing m6A within the specific sequence consensus (RRACH, R = G or A; H = A, C, or U; Supporting Information Figure S8).6 In these RRACH motif-containing RNA sequences, the photocrosslinking results were similar to those obtained for the 15 nt ssRNAs (FITC-Dz5U-m6A, FITCDz5U-A, and control RNAs). As it is known that FTO performs m6A demethylation independent of sequence consensus,7,23−25 FTO might bind to each m6A on ssRNAs. Our photocrosslinking results obtained using different sequence consensuses indicated that FTO recognizes each m6A on ssRNAs to form ssRNA−FTO complexes with an m6A preference. The previously reported FTO crystal structures revealed the presence of an extra loop for ssDNA or ssRNA recognition.9 However, the long-term maintenance of ssRNA−FTO was not confirmed by other reports of FTO demethylation7,23−25 or our experimental results obtained using control RNAs (FITC-m6A and FITC-A). In addition, FTO has no sequence preferences for m6A recognition, according to those reports. These results suggest that FTO binds m6A during the recognition process only, thus making it difficult to observe the long-term presence of native ssRNA−FTO complexes, which can be recognized using a general gel shift assay. Although it might be difficult to detect the long-term interaction between ssRNA and FTO, diazirine photocrosslinking contributed to the formation of stable and durable ssRNA−FTO complexes with m6A preference via covalent bonding. The diazirine group did not disturb the α-KG and Fe(II)dependent FTO demethylation. FITC-Dz5U-m6A was treated with FTO without photoirradiation at RT for 3 h, followed by P1 nuclease digestion and dephosphorylation (Figure 2b). The resultant products were analyzed by HPLC and showed the complete consumption of m6A. Thus, FTO converted the internal m6A of FITC-Dz5U-m6A into adenosine, independent of Dz5U, which confirmed that the diazirine group did not affect the FTO demethylation functions. FTO is an Fe(II) and α-KG-dependent dioxygenase that uses Fe(II) and α-KG to recruit dioxygen for m6A oxidation.7,23 To confirm that the ability of FTO to recognize m6A is associated with oxidative demethylation, we tested the enrichment of the ssRNA−FTO complex using FTO HD (H231A and D233A double mutants) and Fe(II)-binding site loss mutant (Figure 3). After UV photoirradiation, FTO HD did not discriminate FITC-Dz5U-m6A from FITC-Dz5U-A, in contrast to FTO WT, which is the original FTO protein. These results indicate that the Fe(II) cofactor, which bound to FTO mutation sites in this study, was critical for the FTO-based recognition of m6A. The α-KG cosubstrate and Fe(II) cofactor strongly affected the ability of FTO to recognize m6A. We incubated FITCDz5U-m6A and FITC-Dz5U-A in diverse buffers. FTO bound to ssRNAs increasingly in the absence of α-KG and Fe(II) or in the absence of Fe(II) (Figure 4a). However, FTO did not discriminate between m6A and A in this condition (Figure 4a, lanes 1−4). Interestingly, ssRNA might bind to the FTO extra loop even in the absence of α-KG and Fe(II) or in the absence of Fe(II), and we observed the enrichment of the ssRNA−FTO complex compared with that detected in the presence of α-KG and Fe(II) together. The capacity of FTO to discriminate
Figure 2. Diazirine photocrosslinking between ssRNAs and FTO. (a) Detection of ssRNA−FTO complexes in the presence or absence of UV. ssRNAs (0.1 nmol) were incubated with FTO (0.4 nmol) under UV irradiation at 365 nm (UV (+)) or dark (UV (−)) in the presence of binding buffer. The binding buffer contained 50 mM pH 7.5 HEPES-KOH, 8 mM L-ascorbic acid (L-AA), 300 μM α-ketoglutaric acid (α-KG), 283 μM Fe(NH4)2(SO4)2·6H2O. The fluorescence intensity of the ssRNA−FTO complex was quantified using graphs. The fluorescence intensities were normalized using the fluorescence intensity of FITC-Dz5U-A in the absence of UV, which was set as 1.0. Error bars, mean ± SEM for n = 3 experiments. Stars indicate significance in Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. (b) HPLC profiles of the FTO demethylation assay. FITC-Dz5U-m6A was treated in the absence or presence of FTO, followed by treatment with P1 nuclease and alkaline phosphatase. The final products were analyzed by HPLC using the profile of standard nucleosides composed of A, C, G, U, and m6A (Supporting Information Figure S11).
enriched as the forms of ssRNA−FTO complexes formed in the presence of UV irradiation and compared with those formed in the absence of UV irradiation (Figure 2a, lanes 1 and 2 and lanes 5 and 6). The dramatically enriched FITC-RNAs including Dz5U, observed in the presence of UV irradiation, indicated that the detected ssRNA−FTO complexes were induced by the covalent bonding between the ssRNA and FTO, which was mediated by reactive carbenes from diazirine photolysis under UV irradiation. The photocrosslinking activity of the diazirine group (Dz5U) was also confirmed via the comparison between Dz5U-containing ssRNAs (FITC-Dz5U-m6A) and control RNAs that did not include Dz5U (FITC-m6A and FITC-A; Figure 2a, lanes 1 and 3 and lanes 2 and 4). FITC-Dz5U-m6A specifically reacted with FTO. For example, when FTO was replaced with BSA protein, the reaction yield was very low (Figures S5 and S6). In addition, there is no difference in the reaction efficiency between the methylated RNA and the unmethylated RNA. Therefore, the ssRNA−FTO complex formation was not disturbed by BSA (Supporting Information Figure S5 and S6). The m6A-containing ssRNA (FITC-Dz5U-m6A) was more enriched compared with an A-containing ssRNA (FITC-Dz5UA) in the presence of α-KG and Fe(II) (Figure 2a, lanes 1 and 2), which was related to the ability of FTO to discriminate C
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between m6A and A was maximized in the presence of α-KG and Fe(II), although the overall binding affinity was decreased in the presence of Fe(II) (Figure 4a, lanes 7 and 8). A high concentration of Fe(II) ions might interfere with the loading of ssRNA onto FTO because of the strong binding to ssRNA instead of the FTO protein in the in vitro experiment. However, it is still possible that ssRNA binds to FTO, and that m6A demethylation occurs in the subsequent processes. To confirm that the FTO demethylation activity is dependent on α-KG or Fe(II), we performed an FTO demethylation assay in the absence of α-KG or Fe(II). As a result, FTO did not induce m6A demethylation activity in the absence of α-KG/Fe(II), Fe(II), or α-KG (Figure 4b). According to these results, α-KG and Fe(II) might play a role in the FTO-specific recognition of m6A ssRNA. In addition, FTO inhibitor rhein,26,27 which binds to the α-KG binding sites of FTO, reduced the binding affinity of FTO to m6A ssRNA, compared to the original binding affinity in the absence of rhein (Supporting Information Figure S9). The diazirine photocrosslinking results revealed that FTO recruited a sufficient amount of ssRNA, regardless of the presence of m6A on the ssRNA, although the recruitment exhibited an m6A preference. It is suggested that the ssRNAbinding event of FTO should occur prior to the m6A recognition event. Taken together, these results suggest the existence of an FTO oxidative demethylation process of m6A to A (Figure 5). First, activated FTO binds to ssRNA at the extra
Figure 3. Diazirine photocrosslinking of FITC-Dz5U-m6A or FITCDz5U ssRNAs with FTO WT (wild type) and HD (H231A/D233A mutant) in the presence of binding buffer. The binding buffer contains 50 mM pH 7.5 HEPES-KOH, 8 mM L-ascorbic acid (L-AA), 300 μM α-ketoglutaric acid (α-KG), 283 μM Fe(NH4)2(SO4)2·6H2O. The ssRNA−FTO complex was identified by FITC filter, and FTO protein was quantified by coomassie brilliant blue staining. The fluorescence intensities were quantified using graphs. The fluorescence intensity values were normalized using the fluorescence intensity of the ssRNAFTO HD mutant which was set as 1.0. Error bars, mean ± SEM for n = 3 experiments. Stars indicate significance in Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4. Diazirine photocrosslinking of the FITC-Dz5U-m6A or FITC-Dz5U ssRNA in the presence of various binding buffer conditions. (a) The FITC-Dz5U-m6A or FITC-Dz5U-A ssRNA was coincubated with FTO and exposed to UV in the presence of L-AA (lanes 1 and 2), L-AA and α-KG (lanes 3 and 4), L-AA and Fe(II) (lanes 5 and 6), L-AA, α-KG, and Fe(II) (lanes 7 and 8). The ssRNA− FTO complexes were identified using an FITC filter and FTO proteins were quantified by coomassie brilliant blue staining. The fluorescence intensities of ssRNA−FTO complexes were quantified as m6A/A fluorescence intensity ratios. Error bars, mean ± SEM for n = 3 experiments. Stars indicate significance in Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. (b) HPLC profiles of the FTO demethylation assay in the various binding buffer conditions. FITCDz5U-m6A was treated with FTO in the presence of L-AA only, in the presence of L-AA, and α-KG, and in the presence of L-AA and Fe(II), followed by P1 nuclease digest and alkaline phosphatase treatment. The final products were analyzed by HPLC using the profile of standard nucleosides composed of A, C, G, U, and m6A (Supporting Information Figure S11).
Figure 5. Suggested FTO m6A oxidation demethylation process based on experimental data. FTO binds to A RNA or m6A RNA at the extra loop first. If FTO binds to m6A-containing RNA, FTO recognizes m6A on ssRNA and mediates m6A oxidative methylation. In contrast, if FTO binds to A RNA, the A RNA is released from FTO quickly. Diazirine photocrosslinking might capture A RNA with FTO, m6A RNA with FTO, and oxidative demethylated intermediates: hm6A RNA with FTO, and f6A RNA with FTO.
loop.9 Subsequently, if FTO recognizes m6A on the bound ssRNA during the molecular recognition process, FTO might induce the oxidative demethylation of m6A into N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A) as intermediates to form the final product, demethylated A. It is possible that ssRNA−FTO intermediates contain hm6A or f6A, because hm6A is present as an oxidative intermediate of m6A within FTO and f6A has a short half-life of ∼3 h in aqueous solution.25 However, ssRNAs containing A might be quickly released from FTO, similar to completely demethylated D
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thiogalactopyranoside (IPTG) and further incubated for 12 h at 18 °C. The cells were harvested by centrifugation. From this step, all work should be in ice or 4 °C to protect protein from degradation. The cells were lysed by a lysis buffer (50 mM HEPES-KOH (pH 8.0), 2 mM βmercaptoethanol, 5% (v/v) glycerol, 300 mM NaCl, lysozyme (2 mg mL−1)) with a sonicator (power 20%, 1 min × 6 times). Then the lysate was centrifuged with 12 000g for 30 min. The supernatant was purified by His GraviTrap (GE healthcare). The column was equilibrated with 10 mM imidazole containing a lysis buffer and washed by 10 mM and 40 mM containing an imidazole lysis buffer. FTO was eluted by an elution buffer containing 250 mM imidazole. The fractions were collected and confirmed by SDS-PAGE. FTO was further purified with gel filtration/size exclusion chromatography (Superdex 200 Increase 5/150 GL column, FTO was eluted with the buffer containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl) using the AKTA FPLC chromatography system. Photocrosslinking between FTO and ssRNA. A total of 0.1−0.4 nmol of FTO (1× to 4 × ) and 0.1 nmol of RNAs (1×) were coincubated with an appropriate binding buffer (50 mM pH 7.5 HEPES-KOH, 8 mM L-ascorbic acid (L-AA), 300 μM α-ketoglutaric acid (α-KG), 283 μM Fe(NH4)2(SO4)2·6H2O). The mixed solution was quickly exposed to UV irradiation (MAX-302, Asahi Spectra) for specific time intervals (10 min) on the parafilm and at a distance of 5 cm. Then, the protein−RNA complexes were mixed with the sample buffer and analyzed by SDS-PAGE. The protein-bound RNAs were detected by FITC filter during UV exposure (VersaDoc). The amounts of proteins were also detected by coomassie blue staining for quantitative normalization. FTO Demethylation Assay. A total of 0.5 nmol of m6Acontaining ssRNA and 0.5 nmol of FTO were mixed in the binding buffer (50 mM HEPES-KOH (pH 7.5), 8 mM L-AA, 300 μM α-KG, 283 μM Fe(NH4)2(SO4)2·6H2O, 50 μg mL−1 of BSA) in a total of 100 μL for 3 h at RT. The reaction mixture was quenched by the addition of 5 mM EDTA followed by heating for 5 min at 95 °C. To analyze the reaction mixture, the solution was treated with P1 nuclease and alkaline phosphatase. The solutions were analyzed on a HPLC system (Nacalai COSMOSIL 5C18-PAQ, 4.6 mm I.D. × 150 mm) with eluted buffer A (25 mM NaH2PO4) with a flow rate 2 mL min−1 at RT.
ssRNAs. This may explain the observation that ssRNAs containing A were also enriched by diazirine photocrosslinking. To date, m6A-binding proteins were mainly discovered using photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) based on the photoreactive nucleoside analog, 4-thiouridine.28 One of the proteins discovered in this fashion, YTHDF2, showed enrichment between m6A RNAs and A RNAs, and it was confirmed by LC/ MS-MS and gel shift assay, although the enrichment factor was relatively small.28 However, FTO does not exhibit high binding affinity to m6A in the native state in the case of YTHDF2. We suggest that FTO establishes contact with m6A for a short time to induce m6A demethylation; however, after demethylation, FTO does not maintain the binding to m6A and is released from m6A-containing RNA. Thus, diazirine photocrosslinking contributes to the recruitment of FTO with incomplete binding kinetics, as well as of the activated FTO recognition complex. Conclusions. In this work, we inserted a diazirine photoaffinity group into an m6A-containing RNA at a specific site using Suzuki−Miyaura cross-coupling and successfully recruited FTO with m6A preference. The cross-coupling reaction on RNA was performed under mild conditions; therefore, we expect that diazirine can be easily inserted into mRNA in cells incubated with I5U. Diazirine photocrosslinking dramatically enriched activated FTO-based m6A recognition complexes, which formed within the first 10 min of the reaction. The resultant photocrosslinking data indicated that the cofactor Fe(II), accompanied by α-KG, determines the ability of FTO to discriminate between m6A and A. The α-KG cosubstrate and the Fe(II) cofactor formed activated FTO complexes that contributed to capturing dioxygen and recognizing m6A. In addition, the reaction mechanism of FTO revealed that ssRNA binds to FTO first independent of the m6A substrate, followed by the occurrence of the m6A recognition and by subsequent oxidative demethylation. This process explained the observation that the enrichment of photo-cross-linked ssRNA−FTO complexes was relatively independent of the m6A or A substrates. Finally, diazirine photocrosslinking represents a useful tool to capture activated FTO at the initiation step and provides new insights on dynamic FTO-mediated oxidative demethylation.
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ASSOCIATED CONTENT
S Supporting Information *
Supporting Information Figures S1−S11, Supporting Information Methods, and 1H NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
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METHODS
RNA Oligonucleotide Cross-Coupling. First, 100 μM RNA oligonucleotides (10 nmol, 1 eq, FITC-I5U-m6A or FlTC-I5U-A), 50 mM Tris-HCl (pH 8.5), 100 μM Pd(OAc)2X2 (10 nmol, 1 eq, X = a ligand derived from 2-aminopyrimidine), and 10 mM diazirine boronic ester were combined in an e-tube and incubated at 37 °C for 6 h. Then, the e-tube was spun down and purified using HPLC with the gradient between 0.1 M triethylammonium acetate (TEAA; pH 7.0) and acetonitrile and a linear gradient over 50 min from 5% to 35% (v/ v) acetonitrile at a flow rate of 3.0 mL min−1. This condition was optimized for a longer retention time compared to original condition in RNA HPLC purification. FTO Expression and Purification. The expression vector of hFTO wild type (PET302-hFTO) and HD mutant (PET302-hFTOH231A/D233A) were kindly provided from Prof. Y. G. Yang. These vectors contained hFTO and 6 × His tags for metal affinity chromatography purification. FTO was overexpressed in E. coli strain BL21 Gold(DE3) (Stratagene),29 and the bacteria were grown on LBagar plates containing 100 mg L−1 ampicillin. Overnight cultures, which were grown aerobically at 37 °C with a shaking speed of 180 rpm, were used to inoculate 500 mL of LB medium with 100 mg L−1 ampicillin and grown at 37 °C and 180 rpm until OD600 reached at A600−1.0. FTO was induced by 0.5 mM of isopropyl-β-D-
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by International Research Fellow of the Japan Society for the Promotion of Science (25-03342) and the Funding Program for Next Generation World-Leading Researchers, Japan (LR036). We would like to gratefully thank Y. G. Yang (Chinese Academy of Sciences) for providing us FTO expression vectors.
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REFERENCES
(1) Pan, T. (2013) N6-methyl-adenosine modification in messenger and long non-coding RNA. Trends Biochem. Sci. 38, 204−209. (2) Heinrichs, A. (2014) m6A RNA methylation in mammals. Nat. Struct. Mol. Biol. 21, 117−117. (3) Jia, G., Fu, Y., and He, C. (2013) Reversible RNA adenosine methylation in biological regulation. Trends Genet. 29, 108−115. E
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2-Oxoglutarate-Dependent Nucleic Acid Demethylase. Science 318, 1469−1472. (24) Ma, M., Harding, H. P., O’Rahilly, S., Ron, D., and Yeo, G. S. H. (2012) Kinetic analysis of FTO (fat mass and obesity-associated) reveals that it is unlikely to function as a sensor for 2-oxoglutarate. Biochem. J. 444, 183−187. (25) Fu, Y., Jia, G., Pang, X., Wang, R. N., Wang, X., Li, C. J., Smemo, S., Dai, Q., Bailey, K. A., Nobrega, M. A., Han, K.-L., Cui, Q., and He, C. (2013) FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA. Nat. Commun. 4, 1798− 1798. (26) Aik, W., Demetriades, M., Hamdan, M. K., Bagg, E. A., Yeoh, K. K., Lejeune, C., Zhang, Z., McDonough, M. A., and Schofield, C. J. (2013) Structural basis for inhibition of the fat mass and obesity associated protein (FTO). J. Med. Chem. 56, 3680−3688. (27) Chen, B., Ye, F., Yu, L., Jia, G., Huang, X., Zhang, X., Peng, S., Chen, K., Wang, M., Gong, S., Zhang, R., Yin, J., Li, H., Yang, Y., Liu, H., Zhang, J., Zhang, H., Zhang, A., Jiang, H., Luo, C., and Yang, C. G. (2012) Development of cell-active N6-methyladenosine RNA demethylase FTO inhibitor. J. Am. Chem. Soc. 134, 17963−17971. (28) Wang, X., Lu, Z., Gomez, A., Hon, G. C., Yue, Y., Han, D., Fu, Y., Parisien, M., Dai, Q., Jia, G., Ren, B., Pan, T., and He, C. (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117−120. (29) Meyre, D., Proulx, K., Kawagoe-Takaki, H., Vatin, V., GutierrezAguilar, R., Lyon, D., Ma, M., Choquet, H., Horber, F., Van Hul, W., Van Gaal, L., Balkau, B., Visvikis-Siest, S., Pattou, F., Farooqi, I. S., Saudek, V., O’Rahilly, S., Froguel, P., Sedgwick, B., and Yeo, G. S. H. (2009) Prevalence of Loss-of-Function FTO Mutations in Lean and Obese Individuals. Diabetes 59, 311−318.
(4) Meyer, K. D., and Jaffrey, S. R. (2014) The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat. Rev. Mol. Cell Biol. 15, 313−326. (5) Fu, Y., Dominissini, D., Rechavi, G., and He, C. (2014) Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, 293−306. (6) Liu, J., Yue, Y., Han, D., Wang, X., Fu, Y., Zhang, L., Jia, G., Yu, M., Lu, Z., Deng, X., Dai, Q., Chen, W., and He, C. (2014) A METTL3−METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93−95. (7) Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., Yang, Y.-G., and He, C. (2011) N6Methyladenosine in nuclear RNA is a major substrate of the obesityassociated FTO. Nat. Chem. Biol. 7, 885−887. (8) Zheng, G., Dahl, J. A., Niu, Y., Fedorcsak, P., Huang, C.-M., Li, C. J., Vågbø, C. B., Shi, Y., Wang, W.-L., Song, S.-H., Lu, Z., Bosmans, R. P. G., Dai, Q., Hao, Y.-J., Yang, X., Zhao, W.-M., Tong, W.-M., Wang, X.-J., Bogdan, F., Furu, K., Fu, Y., Jia, G., Zhao, X., Liu, J., Krokan, H. E., Klungland, A., Yang, Y.-G., and He, C. (2013) ALKBH5 Is a Mammalian RNA Demethylase that Impacts RNA Metabolism and Mouse Fertility. Mol. Cell 49, 18−29. (9) Han, Z., Niu, T., Chang, J., Lei, X., Zhao, M., Wang, Q., Cheng, W., Wang, J., Feng, Y., and Chai, J. (2010) Crystal structure of the FTO protein reveals basis for its substrate specificity. Nature 464, 1205−1209. (10) Fu, Y., and He, C. (2012) Nucleic acid modifications with epigenetic significance. Curr. Opin. Chem. Biol. 16, 516−524. (11) Pendergrast, P. S. (1992) Determination of the orientation of a DNA binding motif in a protein−DNA complex by photocrosslinking. Proc. Natl. Acad. Sci. U.S.A. 89, 10287−10291. (12) Burdine, L., and Kodadek, T. (2004) Target Identification in Chemical Genetics. Chem. Biol. 11, 593−597. (13) Sumranjit, J., and Chung, S. J. (2013) Recent advances in target characterization and identification by photoaffinity probes. Molecules 18, 10425−10451. (14) Lapinsky, D. J. (2012) Tandem photoaffinity labelingbioorthogonal conjugation in medicinal chemistry. Bioorg. Med. Chem. 20, 6237−6247. (15) Das, J. (2011) Aliphatic Diazirines as Photoaffinity Probes for Proteins: Recent Developments. Chem. Rev. 111, 4405−4417. (16) Winnacker, M., Breeger, S., Strasser, R., and Carell, T. (2009) Novel diazirine-containing DNA photoaffinity probes for the investigation of DNA-protein-interactions. Chembiochem 10, 109−118. (17) MacKinnon, A. L., and Taunton, J. (2009) Target Identification by Diazirine Photocrosslinking and Click Chemistry. Curr. Protoc. Chem. Biol. 1, 55−73. (18) Song, C.-X., and He, C. (2011) Bioorthogonal Labeling of 5Hydroxymethylcytosine in Genomic DNA and Diazirine-Based DNA Photocrosslinking Probes. Acc. Chem. Res. 44, 709−717. (19) Chou, C., Uprety, R., Davis, L., Chin, J. W., and Deiters, A. (2011) Genetically encoding an aliphatic diazirine for protein photocrosslinking. Chem. Sci. 2, 480−480. (20) Li, G., Liu, Y. Y. Y., Chen, L., Wu, S., and Li, X. (2013) Photoaffinity Labeling of Small-Molecule-Binding Proteins by DNATemplated Chemistry. Angew. Chem., Int. Ed. 52, 9544−9549. (21) Zhang, H., Song, Y., Zou, Y., Ge, Y., An, Y., Ma, Y., Zhu, Z., and Yang, C. J. (2014) A diazirine-based photoaffinity probe for facile and efficient aptamer-protein covalent conjugation. Chem. Commun. 50, 4891−4894. (22) Lercher, L., McGouran, J. F., Kessler, B. M., Schofield, C. J., and Davis, B. G. (2013) DNA modification under mild conditions by Suzuki-Miyaura cross-coupling for the generation of functional probes. Angew. Chem., Int. Ed. 52, 10553−10558. (23) Gerken, T., Girard, C. A., Tung, Y. C. L., Webby, C. J., Saudek, V., Hewitson, K. S., Yeo, G. S. H., McDonough, M. A., Cunliffe, S., McNeill, L. A., Galvanovskis, J., Rorsman, P., Robins, P., Prieur, X., Coll, A. P., Ma, M., Jovanovic, Z., Farooqi, I. S., Sedgwick, B., Barroso, I., Lindahl, T., Ponting, C. P., Ashcroft, F. M., O’Rahilly, S., and Schofield, C. J. (2007) The Obesity-Associated FTO Gene Encodes a F
DOI: 10.1021/cb5010096 ACS Chem. Biol. XXXX, XXX, XXX−XXX
