Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition

Feng, Cheng; Liu, Yang; Wang, Guoqiang; Deng, Zhichao; Zhang, Qi; Wu, Wei; Tong, Yufeng; Cheng, Changmei; Chen, Zhongzhou

doi:10.1074/jbc.m113.546168

Cited by 148 publications

(164 citation statements)

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“…By molecular modeling of the ALKBH5 substrate complex structure, we identify potential m 6 A binding residues, the identities of which are validated by mutagenesis and ITC binding experiments. During revision of this manuscript, two other manuscripts on human ALKBH5 structures were reported (38,39); their structural data and conclusions are consistent with our structural results, which are presented here.…”

supporting

confidence: 79%

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

Liu

Tempel

et al. 2014

Journal of Biological Chemistry

143

142

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Background: ALKBH5 catalyzes demethylation of m 6 A single-stranded RNA (ssRNA). Results: ALKBH5 structures reveal the structural basis of its substrate selectivity and inhibition by citrate. Conclusion: ALKBH5 specifically binds to and demethylates m 6 A ssDNA/ssRNA. Citrate is a modest inhibitor of ALKBH5. Significance: This study provides insights into the molecular mechanism of ALKBH5 as an m 6 A ssRNA demethylase and will facilitate the design of selective inhibitors.

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supporting

confidence: 79%

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

Liu

Tempel

et al. 2014

Journal of Biological Chemistry

143

142

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“…19,26 Briefly, the lysate was passed through a DE52 column at 500 mM NaCl to filter DNA and other nonprotein contaminants followed by the Ni-NTA column. Resin was washed with binding buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 5% glycerol, and 5 mM imidazole) followed by washing with wash buffer (binding buffer with 30 mM imidazole) and eluted with elution buffer (binding buffer with 250 mM imidazole).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

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

Kennedy

Hajian

et al. 2016

SLAS Discovery

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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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“…Overexpression of FTO or ALKBH5 in cells decreases global m 6 A levels, but knockdown or knockout of either only mildly increases m 6 A levels [25, 66], suggesting the existence of other demethylases or perhaps a synergy between ALKBH5 and FTO that has not been studied. The crystal structures of both FTO and ALKBH5 have been reported, and small molecule inhibitors targeting their demethylase activities have been developed based on these structures [69–71]. For example, the natural product rhein, derived from herbs, is among the most effective FTO m 6 A demethylase inhibitors [72].…”

Section: M6a Methyltransferases and Demethylasesmentioning

confidence: 99%

Update: Mechanisms Underlying N 6 -Methyladenosine Modification of Eukaryotic mRNA

Wang

Zhao

2016

Trends in Genetics

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Summary Eukaryotic messenger RNA (mRNA) undergoes chemical modification both at the 5′cap [1, 2] and internally [3–14]. Among internal modifications, m6A, by far the most abundant, is present in all eukaryotes examined, including mammals [3–6], flies [15], plants [16, 17] and yeast [18, 19]. m6A modification plays an essential role in diverse biological processes. Over the past few years, our knowledge relevant to establishment and function of this modification has grown rapidly. This review focuses on technologies that have facilitated m6A detection in mRNAs, identification of m6A methylation enzymes and binding proteins, and potential functions of the modification at the molecular level. Regarding m6A function at cellular or organismal levels or in disease, please refer to other recent reviews [20–23].

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Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition

Cited by 148 publications

References 50 publications

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

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

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

Update: Mechanisms Underlying N 6 -Methyladenosine Modification of Eukaryotic mRNA

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