Related Experiment Video

Updated: Apr 30, 2026

Dot Blot Assay for Detecting Global N6-Methyladenosine RNA Modification Levels
08:40

Dot Blot Assay for Detecting Global N6-Methyladenosine RNA Modification Levels

Published on: February 6, 2026

253

Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine

Chao Xu1, Ke Liu2, Wolfram Tempel1

  • 1the Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.

The Journal of Biological Chemistry
|April 30, 2014
PubMed

Insights

N(6)-Methyladenosine (m(6)A) demethylase ALKBH5

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Epigenetics

Background:

  • N(6)-Methyladenosine (m(6)A) is the most abundant internal RNA modification in eukaryotes.
  • ALKBH5 is an AlkB family dioxygenase known to demethylate m(6)A in single-stranded RNA.

Purpose of the Study:

  • To elucidate the structural and biochemical properties of ALKBH5.
  • To identify the m(6)A binding pocket and key residues involved in recognition.
  • To investigate the inhibitory effects of citrate on ALKBH5 activity.

Main Methods:

  • X-ray crystallography was used to determine the structures of ALKBH5.
  • Catalytic assays were performed to assess demethylation activity on RNA and DNA.
  • Mutagenesis and Isothermal Titration Calorimetry (ITC) were employed to study m(6)A binding.

Main Results:

  • Crystal structures of ALKBH5 were obtained with cofactors and the inhibitor citrate.
  • ALKBH5 demethylates both single-stranded RNA and single-stranded DNA.
  • Citrate was identified as a modest inhibitor of ALKBH5 (IC50 ≈ 488 µM).
  • A disulfide bond in ALKBH5 immobilizes a loop, preventing dsDNA binding.
  • Key residues in the m(6)A binding pocket were identified.

Conclusions:

  • ALKBH5 possesses a structural mechanism to exclude dsDNA binding.
  • The study provides structural insights into m(6)A recognition by ALKBH5.
  • Citrate represents a potential, albeit modest, inhibitor for ALKBH5.
Keywords:
Crystal StructureDioxygenaseIsothermal Titration Calorimetry (ITC)RNA CatalysisRNA MethylationTricarboxylic Acid Cycle (TCA Cycle) (Krebs Cycle)

More Related Videos

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues08:56

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

Published on: December 5, 2016

12.0K
Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis08:50

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Published on: May 14, 2020

6.8K

Related Concept Videos

RNA Stability01:53

RNA Stability

Intact DNA strands can be found in fossils, while scientists sometimes struggle to keep RNA intact under laboratory conditions. The structural variations between RNA and DNA underlie the differences in their stability and longevity. Because DNA is double-stranded, it is inherently more stable. The single-stranded structure of RNA is less stable but also more flexible and can form weak internal bonds. Additionally, most RNAs in the cell are relatively short, while DNA can be up to 250 million...
31.6K
RNA Editing02:23

RNA Editing

RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
8.3K
Nucleic Acid Structure01:25

Nucleic Acid Structure

The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms  a 5′ to 3′ phosphodiester linkage.
DNA Structure
DNA...
8.1K
Transfer RNA Synthesis02:36

Transfer RNA Synthesis

One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
10.2K
Chromatin Structure Regulates pre-mRNA Processing02:41

Chromatin Structure Regulates pre-mRNA Processing

In eukaryotic cells, nascent mRNA transcripts need to undergo many post-transcriptional modifications to reach the cell cytoplasm and translate into functional proteins. For a long time, transcription and pre-mRNA processing were considered two independent events that occur sequentially in the cell. However, it has now been well established that transcription and pre-mRNA processing are two simultaneous processes that are precisely regulated inside the cell.
The chromatin structure, especially...
6.6K