Scintillation proximity assay for measurement of RNA methylation.
Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA-protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay. We have adapted and optimized a scintillation proximity assay (SPA) to replace the more costly, wasteful and cumbersome filter binding assay and demonstrate its utility in studies of three distinct methyltransferases, RmtA, KsgA and ErmC'. In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC' most efficiently methylates protein-depleted or protein-free 23S rRNA. This assay does not utilize engineered affinity tags that are often required in SPA, and is capable of detecting either radiolabeled RNA or RNA-protein complex. We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors. This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format.
Baker
Matthew R
MR
Department of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23298-0133, USA.
Zarubica
Tamara
T
Wright
H Tonie
HT
Rife
Jason P
JP
D002498:Centrifugation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism*; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012588:Scintillation Counting
D004791:Enzyme Inhibitors; D012313:RNA; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C047346:16S rRNA (adenine(1518)-N(6)-adenine(1519)-N(6))-dimethyltransferase; C035181:rRNA (adenosine-O-2'-)methyltransferase