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

Abstract

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.

Structural Basis for Cooperative Function of Mettl3 and Mettl14 Methyltransferases

“…The in vitro methylation assay was carried out in triplicate with a 15 uL reaction mixture containing: 200 nM RNA oligonucleotides (“GGACU”= 5′-UACACUCGAUCU GGACU AAAGCUGCUC; “GGAUU” = 5′-UACACUCGAUC UGGAUU AAAGCUGCUC), 20 mM Tris (pH 7.5), 0.01% Triton-X, 1 mM DTT, 50 uM ZnCl2, 0.2 U/uL RNasin, 1% glycerol and 460 nM [ 3 H]-SAM (Li et al, 2016). All recombinant Mettl3/Mettl14 complexes purified from E. coli were analyzed by SDS-PAGE (Figures S1B and S6B), quantified by UV absorbance at 280nm, and used at 50nM in the final reaction.…”

Interactions, localization, and phosphorylation of the m6A generating METTL3–METTL14–WTAP complex

“…For m 6 A methylation assay a RNA probe of four repetitive RRACH sequences 5 ′ -GGACUGGACUGGACUGGACU-3 ′ (Wang et al 2016b) was used. The reaction mixture of 40 µL contained 20 mM Tris/HCl (pH 7.5), 0.01% (v/v) Triton-X, 1 mM DTT, 50 µM ZnCl 2 , 0.2 U/mL RiboLock (Thermo Fisher Scientific), 1% (v/v) glycerol, 50 nM purified protein (Li et al 2016) and 2.25 × 10 −3 µCi [3H]-SAM. The methylation reactions were incubated at 30°C for 1 h. The methylation reaction was stopped at 65°C for at least 5 min and purified afterwards using MicroSpin G-25 columns (GE Healthcare Life Sciences) according to the manufacturer's protocol.…”

Discovery of Small Molecules that Activate RNA Methylation through Cooperative Binding to the METTL3-14-WTAP Complex Active Site

“…The enzymatic assay was modified from Li et al (2016). The experiments were conducted in reaction buffer (20 mM Tris pH 7.5, 1 mM DTT, 0.01% Triton X-100, 40U/100ml buffer RNaseOUT).…”