Base-resolution quantitative DAMM-seq for mapping RNA methylations in tRNA and mitochondrial polycistronic RNA.

The human AlkB family proteins, such as FTO and ALKBH5, are known to mediate RNA m6A demethylation. However, although ALKBH7 localizes in mitochondria and affects metabolism, the detailed biological function and mechanism have remained unknown for years. We developed Demethylation-Assisted Multiple Methylation sequencing (DAMM-seq) to simultaneously detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) methylations in both steady-state RNA and nascent RNA, and discovered that human ALKBH7 demethylates m22G and m1A within mt-Ile and mt-Leu1 pre-tRNA regions, respectively, in mitochondrial polycistronic RNA. DAMM-seq quantitatively and sensitively monitors the methylation stoichiometry change at pre-tRNA junctions within nascent mt-RNA, revealing the target region where ALKBH7 regulates RNA processing and local structural switch of polycistronic mt-RNAs. A new RNA demethylase in human cells was characterized through the base-resolution quantification of multiple RNA methylations in nascent mt-RNA, resolving the long-standing question about the functional substrate of ALKBH7.

Discovery of a potent, selective and cell active inhibitor of m6A demethylase ALKBH5.

AlkB homolog 5 (ALKBH5) is an RNA m6A demethylase involved in the regulation of genes transcription, translation and metabolism and has been considered as a promising therapeutic target for various human diseases, especially cancers. However, there is still a lack of potent and selective ALKBH5 inhibitors. Herein, we report a new class of ALKBH5 inhibitors containing the 1-aryl-1H-pyrazole scaffold, which were obtained through fluorescence polarization-based screening, structural optimization and structure-activity relationship analysis. Among these compounds, 20m was the most potent one, which showed an IC50 value of 0.021 µM in fluorescence polarization assay. Compound 20m exhibited high selectivity towards ALKBH5 versus FTO as well as other AlkB subfamily members, indicating good selectivity for ALKBH5. Cellular thermal shift assay (CETSA) analysis showed that 20m could efficiently stabilize ALKBH5 in HepG2 cells. Dot blot assay demonstrated that 20m could increase m6A level in intact cells. Collectively, 20m is a potent, selective and cell active ALKBH5 inhibitor and could be used as a versatile chemical probe to explore the biological function of ALKBH5.

Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5-RNA complexes.

AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5'-3' orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.

RNA demethylase ALKBH5 in cancer: from mechanisms to therapeutic potential.

RNA demethylase ALKBH5 takes part in the modulation of N6-methyladenosine (m6A) modification and controls various cell processes. ALKBH5-mediated m6A demethylation regulates gene expression by affecting multiple events in RNA metabolism, e.g., pre-mRNA processing, mRNA decay and translation. Mounting evidence shows that ALKBH5 plays critical roles in a variety of human malignancies, mostly via post-transcriptional regulation of oncogenes or tumor suppressors in an m6A-dependent manner. Meanwhile, increasing non-coding RNAs are recognized as functional targets of ALKBH5 in cancers. Here we reviewed up-to-date findings about the pathological roles of ALKBH5 in cancer, the molecular mechanisms by which it exerts its functions, as well as the underlying mechanism of its dysregulation. We also discussed the therapeutic implications of targeting ALKBH5 in cancer and potential ALKBH5-targeting strategies.

Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates.

The AlkB family of nonheme Fe(II)/2-oxoglutarate-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6-methyladenosine (m6A) but furnish different major products, N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a direct m6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for nonredundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.

A fluorescent nanobiosensor for the facile analysis of m6A RNA demethylase activity.

RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.

Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.

FTO demethylates internal N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate-in vitro and in vivo-that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.

Active Site Breathing of Human Alkbh5 Revealed by Solution NMR and Accelerated Molecular Dynamics.

AlkB homolog 5 (Alkbh5) is one of nine members of the AlkB family, which are nonheme Fe2+/α-ketoglutarate-dependent dioxygenases that catalyze the oxidative demethylation of modified nucleotides and amino acids. Alkbh5 is highly selective for the N6-methyladenosine modification, an epigenetic mark that has spawned significant biological and pharmacological interest because of its involvement in important physiological processes, such as carcinogenesis and stem cell differentiation. Herein, we investigate the structure and dynamics of human Alkbh5 in solution. By using 15N and 13Cmethyl relaxation dispersion and 15N-R1 and R1ρ NMR experiments, we show that the active site of apo Alkbh5 experiences conformational dynamics on multiple timescales. Consistent with this observation, backbone amide residual dipolar couplings measured for Alkbh5 in phage pf1 are inconsistent with the static crystal structure of the enzyme. We developed a simple approach that combines residual dipolar coupling data and accelerated molecular dynamics simulations to calculate a conformational ensemble of Alkbh5 that is fully consistent with the experimental NMR data. Our structural model reveals that Alkbh5 is more disordered in solution than what is observed in the crystal state and undergoes breathing motions that expand the active site and allow access to α-ketoglutarate. Disordered-to-ordered conformational changes induced by sequential substrate/cofactor binding events have been often invoked to interpret biochemical data on the activity and specificity of AlkB proteins. The structural ensemble reported in this work provides the first atomic-resolution model of an AlkB protein in its disordered conformational state to our knowledge.

Multiprotein Dynamic Combinatorial Chemistry: A Strategy for the Simultaneous Discovery of Subfamily-Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3.

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self-assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic "thermal tag" with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily-selective probes against two clinically important epigenetic enzymes: FTO (7; IC50 =2.6 µm) and ALKBH3 (8; IC50 =3.7 µm). To date, this is the first report of a subfamily-selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.

1H, 15N, 13C backbone resonance assignment of human Alkbh5.

N6-methyladenosine (m6A) is the most abundant and reversible post-transcriptional modification in eukaryotic mRNA and long non-coding RNA (lncRNA). The central role of m6A in various physiological processes has generated considerable biological and pharmacological interest. Alkbh5 (AlkB homologue 5) belongs to the AlkB family and is a non-heme Fe(II)/α-ketoglutarate-dependent dioxygenase that selectively catalyzes the oxidative demethylation of m6A. Herein, we report the backbone 1H, 15N, 13C chemical shift assignment of a fully active, 26 kDa construct of human Alkbh5. Experiments were acquired at 25 °C by heteronuclear multidimensional NMR spectroscopy. Collectively, 92% of all backbone resonances were assigned, with 195 out of a possible 212 residues assigned in the 1H-15N TROSY spectrum. Using the program TALOS+, a secondary structure prediction was generated from the assigned backbone resonance that is consistent with the previously reported X-ray structure of the enzyme. The reported assignment will permit investigations of the protein structural dynamics anticipated to provide crucial insight regarding fundamental aspects in the recognition and enzyme regulation processes.

N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5.

N(6)-Methyladenosine (m6A) is currently one of the most intensively studied post-transcriptional modifications in RNA. Due to its critical role in epigenetics and physiological links to several human diseases, it is also of tremendous biological and medical interest. The m6A mark is dynamically reversed by human demethylases FTO and ALKBH5, however the mechanism by which these enzymes selectively recognise their target transcripts remains unclear. Here, we report combined biophysical and biochemical studies on the specificity determinants of m6A demethylases, which led to the identification of an m6A-mediated substrate discrimination mechanism. Our results reveal that m6A itself serves as a 'conformational marker', which induces different conformational outcomes in RNAs depending on sequence context. This critically impacts its interactions with several m6A-recognising proteins, including FTO and ALKBH5. Remarkably, through the RNA-remodelling effects of m6A, the demethylases were able to discriminate substrates with very similar nucleotide sequences. Our findings provide novel insights into the biological functions of m6A modifications. The mechanism identified in this work is likely of significance to other m6A-recognising proteins.