title: In Vitro Assays for RNA Methyltransferase Activity.
abstract: RNA methyltransferases (MTases) are responsible for co- and posttranscriptional methylation of nucleotides in a wide variety of RNA substrates. Examination of the target specificity, catalytic activity, and function of these enzymes requires in vitro methylation assays. Here, we provide a detailed protocol for the methylation of in vitro transcripts, synthetic RNAs, and total cellular RNA using recombinant RNA methyltransferases and S-adenosylmethionine (SAM) as a methyl group donor. We describe how this method can be coupled to fluorographic detection of RNA methylation if3H-labeled SAM is used, and discuss alternative chromatography-based methods for the detection of methylated nucleotides, focusing on reversed-phase high-performance liquid chromatography (RP-HPLC). In both cases, mutagenesis of the methyltransferase or the RNA substrate can be employed to elucidate the catalytic mechanisms and target specificity of the enzymes. Together these approaches provide valuable insight into the action of RNA methyltransferases on the molecular level and serve as a basis for further functional characterization of RNA methyltransferases in vivo.
authors.affiliation: Centre for Biochemistry and Molecular Cell Biology, Institute for Molecular Biology, Georg-August-University, Humboldtallee 23, 37073, Göttingen, Germany.
authors.forename: Sara
authors.initials: S
authors.lastname: Haag
authors.affiliation: Centre for Biochemistry and Molecular Cell Biology, Institute for Molecular Biology, Georg-August-University, Humboldtallee 23, 37073, Göttingen, Germany.
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Sloan
authors.affiliation: Institute for Organic and Biomolecular Chemistry, Georg-August-University, 37077, Göttingen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Höbartner
authors.affiliation: Centre for Biochemistry and Molecular Cell Biology, Institute for Molecular Biology, Georg-August-University, Humboldtallee 23, 37073, Göttingen, Germany. markus.bohnsack@med.uni-goettingen.de.|Göttingen Centre for Molecular Biosciences, Georg-August-University, 37073, Göttingen, Germany. markus.bohnsack@med.uni-goettingen.de.
authors.forename: Markus T
authors.initials: MT
authors.lastname: Bohnsack
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods; D066298:In Vitro Techniques; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012313:RNA; D008780:Methyltransferases
keywords: Epitranscriptome; Fluorography; Methylation; Methyltransferase; RNA modification; RP-HPLC; S-Adenosyl methionine