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In Vitro Assays for RNA Methyltransferase Activity

  • Sara Haag
  • Katherine E. Sloan
  • Claudia Höbartner
  • Markus T. BohnsackEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)

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 if 3H–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.

Key words

Methyltransferase RNA modification Methylation S-Adenosyl methionine Epitranscriptome Fluorography RP-HPLC 

Notes

Acknowledgments

We thank Ahmed Warda for comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SPP 1784: BO3442/2-1 to M.T.B.; HO4436/2-1 to C.H.) and the Faculty of Medicine, Georg-August-University Göttingen (M.T.B. and “Startförderung” to S.H.).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Sara Haag
    • 1
  • Katherine E. Sloan
    • 1
  • Claudia Höbartner
    • 2
  • Markus T. Bohnsack
    • 1
    • 3
    Email author
  1. 1.Centre for Biochemistry and Molecular Cell BiologyInstitute for Molecular Biology, Georg-August-UniversityGöttingenGermany
  2. 2.Institute for Organic and Biomolecular ChemistryGeorg-August-UniversityGöttingenGermany
  3. 3.Göttingen Centre for Molecular BiosciencesGeorg-August-UniversityGöttingenGermany

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