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PMID: 26701100 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Journal of biomolecular screening ·Vol. 21 ·No. 3 ·2016-03-00 ·Pages 290-7

Li F, Kennedy S, Hajian T, Gibson E, Seitova A, Xu C, Arrowsmith CH, Vedadi M

Abstract

N(6)-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.

Keywords

METTL14 METTL3 RNA demethylase RNA methyltransferase epigenetics

MeSH Terms

AlkB Homolog 5, RNA Demethylase/metabolism Biological Assay/methods Drug Discovery/methods Humans Kinetics Ligands Methyltransferases/metabolism Protein Binding Radioligand Assay/methods

Chemicals

Ligands ALKBH5 protein, human AlkB Homolog 5, RNA Demethylase METTL14 protein, human Methyltransferases METTL3 protein, human

Authors & Affiliations

8 authors, click to expand affiliations / ORCID

Li Fengling