Li Fengling, Kennedy Steven, Hajian Taraneh, Gibson Elisa, Seitova Alma, Xu Chao, Arrowsmith Cheryl H, Vedadi Masoud
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
J Biomol Screen. 2016 Mar;21(3):290-7. doi: 10.1177/1087057115623264. Epub 2015 Dec 23.
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.
N⁶-甲基腺苷（m⁶A）是哺乳动物RNA中最常见的可逆性内部修饰。m⁶A水平的变化与多种细胞过程有关，包括核RNA输出、蛋白质翻译控制和蛋白质剪接，并且它们与肥胖、癌症和其他人类疾病有关。METTL3和METTL14是N⁶-腺苷甲基转移酶，它们在稳定的METTL3-METTL14异二聚体复合物（METTL3-14）中工作效率更高。ALKBH5是一种m⁶A-RNA去甲基化酶，属于双加氧酶的AlkB家族。我们报告了基于放射性的检测方法的开发，用于对METTL3-14复合物和ALKBH5的m⁶A-RNA修饰进行动力学表征，并提供了适合在384孔板中筛选配体的最佳检测条件，Z'因子分别为0.78和0.77。
