title: A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5.
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.
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Fengling
authors.initials: F
authors.lastname: Li
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Steven
authors.initials: S
authors.lastname: Kennedy
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Taraneh
authors.initials: T
authors.lastname: Hajian
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Elisa
authors.initials: E
authors.lastname: Gibson
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Alma
authors.initials: A
authors.lastname: Seitova
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Chao
authors.initials: C
authors.lastname: Xu
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowsmith
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada m.vedadi@utoronto.ca.
authors.forename: Masoud
authors.initials: M
authors.lastname: Vedadi
mesh_terms: D000071502:AlkB Homolog 5, RNA Demethylase / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D055808:Drug Discovery / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008780:Methyltransferases / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*
chemical_list: D008024:Ligands; C559920:ALKBH5 protein, human; D000071502:AlkB Homolog 5, RNA Demethylase; C587355:METTL14 protein, human; D008780:Methyltransferases; C468275:METTL3 protein, human
keywords: METTL14; METTL3; RNA demethylase; RNA methyltransferase; epigenetics