title: An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity in E. coli cell lysate.
abstract: Post-transcriptional modifications of RNA diversify the genetically encoded sequences and play important roles in fundamental cellular processes like mRNA and rRNA maturation. Most RNA methylations are catalyzed by S-adenosylmethionine-dependent RNA methyltransferases. An assay for rapid and easy detection of this enzymatic activity would be highly desirable to identify novel RNA methyltransferases, test S-adenosylmethionine analogs or screen RNA methyltransferase inhibitors. We have developed an enzyme-coupled assay to determine the activity of trimethylguanosine synthases, a class of RNA methyltransferases responsible for RNA cap hypermethylation in eukaryotes. We show that this assay can be used in E. coli cell lysate to measure the activity of recombinantly produced trimethylguanosine synthase. Potential hits are validated for formation of hypermethylated product using HPLC and MALDI-TOF-MS. Furthermore, we demonstrate how this assay can be implemented to screen methyltransferase inhibitors.
authors.affiliation: Department of Chemistry, School of Mathematics, Informatics and Natural Sciences, University of Hamburg, Hamburg, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Schulz
authors.forename: Andrea
authors.initials: A
authors.lastname: Rentmeister
mesh_terms: D002109:Caffeic Acids / Q000737:chemistry; D002392:Catechin / Q000031:analogs & derivatives / Q000737:chemistry; D002726:Chlorogenic Acid / Q000737:chemistry; D002863:Chromogenic Compounds / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli; D057166:High-Throughput Screening Assays; D006710:Homocysteine / Q000737:chemistry; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D010928:Placental Lactogen; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002109:Caffeic Acids; D002863:Chromogenic Compounds; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006710:Homocysteine; D002726:Chlorogenic Acid; D002392:Catechin; D010928:Placental Lactogen; D004228:Dithionitrobenzoic Acid; C045651:epigallocatechin gallate; D008780:Methyltransferases; C456271:trimethylguanosine synthase; C040048:caffeic acid