RNA Cap Methyltransferase Activity Assay

Methyltransferases that methylate the guanine-N7 position of the mRNA 5' cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involv...

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Bibliographic Details
Published inBio-protocol Vol. 8; no. 6
Main Authors Trotman, Jackson B, Schoenberg, Daniel R
Format Journal Article
LanguageEnglish
Published United States Bio-Protocol 20.03.2018
Bio-protocol LLC
Subjects
Biology
Methods
Cap methyltransferase
P1 nuclease
Subcellular fractionation
RNA
RNMT
Thin-layer chromatography
5′ Cap
Enzyme activity assay
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Summary:Methyltransferases that methylate the guanine-N7 position of the mRNA 5' cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [ P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [ P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments.
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ISSN:2331-8325
2331-8325
DOI:10.21769/BioProtoc.2767