Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

Xu, Chao; Liu, Ke; Tempel, W.; Demetriades, Marina; Aik, W.S.; Schofield, Christopher J.; Min, Jinrong

doi:10.1074/jbc.m114.550350

Cited by 151 publications

(158 citation statements)

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“…Demethylation of the m 6 A mark is carried out by fat mass and obesity protein (FTO) and ALKBH5, both of which belong to the ALKB family that includes ALKBH1-8 and FTO. 11,19,20 Both FTO and ALKBH5 are Fe(II)-and 2-oxoglutarate (2-OG)-dependent enzymes that remove m 6 A marks through an oxidative process. 21 Knockdown of ALKBH5 was shown to affect splicing in tissue culture cells and displayed a sterility phenotype in male mice.…”

Section: Introductionmentioning

confidence: 99%

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

et al. 2016

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N6 -methyladenosine (m 6 A) is the most common reversible internal modification in mammalian RNA. Changes in m 6 A levels have been implicated in a variety of cellular processes, including nuclear RNA export, control of protein translation, and protein splicing, and they have been linked to obesity, cancer, and other human diseases. METTL3 and METTL14 are N 6 -adenosine methyltransferases that work more efficiently in a stable METTL3-METTL14 heterodimer complex (METTL3-14). ALKBH5 is an m 6 A-RNA demethylase that belongs to the AlkB family of dioxygenases. We report the development of radioactivity-based assays for kinetic characterization of m 6 A-RNA modifications by METTL3-14 complex and ALKBH5 and provide optimal assay conditions suitable for screening for ligands in a 384-well format with Z′ factors of 0.78 and 0.77, respectively.

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Section: Introductionmentioning

confidence: 99%

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

et al. 2016

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“…METTL3 contains an S-adenosyl methionine (SAM) binding domain, and utilizes SAM as a substrate to methylate target mRNAs that contain a DRACH m 6 A consensus sequence, often found in 3 ′ UTR's and around stop codons (Meyer et al 2012;, while METTL14 lacks catalytic activity but participates in mRNA binding/targeting (Sĺedźand Jinek 2016;Wang et al 2016a,b). m 6 A methylation of RNA is reversible and can be removed by alkylation repair homolog 5 (ALKBH5) or fat mass and obesity related protein (FTO) (Jia et al 2011Fu et al 2013Fu et al , 2014Zheng et al 2013;Meyer and Jaffrey 2014;Xu et al 2014a). Methylated mRNA is transported out of the nucleus and bound by RNA binding proteins, including most members of the YTH family (Dominissini et al 2012;Wang et al 2014a;Xu et al 2014b).…”

Section: Introductionmentioning

confidence: 99%

N⁶-methyladenosine is required for the hypoxic stabilization of specific mRNAs

Fry

Law

Ilkayeva

et al. 2017

RNA

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Post-transcriptional regulation of mRNA during oxygen deprivation, or hypoxia, can affect the survivability of cells. Hypoxia has been shown to increase stability of a subset of ischemia-related mRNAs, including VEGF. RNA binding proteins and miRNAs have been identified as important for post-transcriptional regulation of individual mRNAs, but corresponding mechanisms that regulate global stability are not well understood. Recently, mRNA modification by N 6 -methyladenosine (m 6 A) has been shown to be involved in post-transcriptional regulation processes including mRNA stability and promotion of translation, but the role of m 6 A in the hypoxia response is unknown. In this study, we investigate the effect of hypoxia on RNA modifications including m 6 A. Our results show hypoxia increases m 6 A content of poly(A) + messenger RNA (mRNA), but not in total or ribosomal RNA in HEK293T cells. Using m 6 A mRNA immunoprecipitation, we identify specific hypoxia-modified mRNAs, including glucose transporter 1 (Glut1) and c-Myc, which show increased m 6 A levels under hypoxic conditions. Many of these mRNAs also exhibit increased stability, which was blocked by knockdown of m 6 A-specific methyltransferases METTL3/14. However, the increase in mRNA stability did not correlate with a change in translational efficiency or the steady-state amount of their proteins. Knockdown of METTL3/14 did reveal that m 6 A is involved in recovery of translational efficiency after hypoxic stress. Therefore, our results suggest that an increase in m 6 A mRNA during hypoxic exposure leads to post-transcriptional stabilization of specific mRNAs and contributes to the recovery of translational efficiency after hypoxic stress.

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“…Although the exact cellular functions of this modification are still not completely understood, m 6 A has been linked to the regulation of the circadian clock (8) and m 6 A levels are highest during yeast meiosis (1). The m 6 A methyl group can be removed by the dioxygenases FTO (9) and ALKBH5 (10,11), suggesting that m 6 A is a reversible modification on the RNA.…”

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Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

Luo

Tong

2014

Proc. Natl. Acad. Sci. U.S.A.

208

237

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Methylation of the N6 position of selected internal adenines (m 6 A) in mRNAs and noncoding RNAs is widespread in eukaryotes, and the YTH domain in a collection of proteins recognizes this modification. We report the crystal structure of the splicing factor YT521-B homology (YTH) domain of Zygosaccharomyces rouxii MRB1 in complex with a heptaribonucleotide with an m 6 A residue in the center. The m 6 A modification is recognized by an aromatic cage, being sandwiched between a Trp and Tyr residue and with the methyl group pointed toward another Trp residue. Mutations of YTH domain residues in the RNA binding site can abolish the formation of the complex, confirming the structural observations. These residues are conserved in the human YTH proteins that also bind m 6 A RNA, suggesting a conserved mode of recognition. Overall, our structural and biochemical studies have defined the molecular basis for how the YTH domain functions as a reader of methylated adenines.T he methylation of the N6 position of selected internal adenines (m 6 A) modification is widespread in eukaryotic mRNAs and noncoding RNAs (1-3), reviewed in refs. 4-6. Recent studies have linked this modification to the regulation of alternative splicing (2), RNA processing, and mRNA degradation (7). Although the exact cellular functions of this modification are still not completely understood, m 6 A has been linked to the regulation of the circadian clock (8) and m 6 A levels are highest during yeast meiosis (1). The m 6 A methyl group can be removed by the dioxygenases FTO (9) and ALKBH5 (10, 11), suggesting that m 6 A is a reversible modification on the RNA.A consensus sequence G(m 6 A)C has been identified for this modification based on transcriptome-wide mapping (1, 2), which is consistent with that identified from earlier biochemical studies (4-6). Several YTH domain (12) family members, YTHDF1, YTHDF2, and YTHDF3 in humans (2, 7) and methylated RNAbinding protein 1 (MRB1) in yeast (1), have been shown to bind RNAs with m 6 A modification, and the binding consensus for the YTH domain of YTHDF2 is also G(m 6 A)C (7), consistent with that found by transcriptome-wide mapping.The YTH domain contains ∼160 residues and is found in yeast, plants, and animals ( Fig. S1) (12, 13). The domain is located at the C-terminal end of yeast MRB1 and human YTHDF1-3 (Fig. 1A), and its sequence is well conserved among these proteins (Fig. 1B and Fig. S1). The N-terminal regions of these proteins are poorly conserved, although that of YTHDF2 mediates its function in regulating mRNA localization and degradation (7). Yeast MRB1 regulates phosphate metabolism by destabilizing the mRNA of a transcription factor of the pathway and, hence, it is also known as Pho92 (14), although direct evidence of MRB1 regulating m 6 A-containing mRNAs in yeast cells is lacking.The structures of the YTH domains of two related proteins, human YTH domain containing protein 1 [YTHDC1; Protein Data Bank (PDB) ID code 2YUD] and YTHDC2 (2YU6), have been reported. Human YTHDC1 has 29% sequence i...

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Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

Cited by 151 publications

References 60 publications

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

N⁶-methyladenosine is required for the hypoxic stabilization of specific mRNAs

Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

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Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

Cited by 151 publications

References 60 publications

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

N6-methyladenosine is required for the hypoxic stabilization of specific mRNAs

Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

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N⁶-methyladenosine is required for the hypoxic stabilization of specific mRNAs