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Antibody-Free Assay for RNA Methyltransferase Activity Analysis
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RNA Cap Methyltransferase Activity Assay.

Jackson B Trotman1,2,3, Daniel R Schoenberg1,2

  • 1Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA.

Bio-Protocol
|April 13, 2018
PubMed
Summary
This summary is machine-generated.

This study details a biochemical assay for analyzing RNA cap methyltransferase activity. The method uses a radiolabeled RNA substrate and S-adenosylmethionine to quantify N7-methylation in various biological samples.

Keywords:
5′ CapCap methyltransferaseEnzyme activity assayP1 nucleaseRNARNMTSubcellular fractionationThin-layer chromatography

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Virology

Background:

  • RNA cap methyltransferases are crucial enzymes found in eukaryotes and viruses.
  • These enzymes modify the guanine-N7 position of the mRNA 5' cap structure.
  • Understanding their activity is vital for studying gene regulation and viral replication.

Purpose of the Study:

  • To provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity.
  • To enable the quantification of N7-methylation in diverse biological samples.
  • To facilitate the study of these enzymes in various biological contexts.

Main Methods:

  • Incubation of biological samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM).
  • Analysis of N7-methylated caps via P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging.
  • Includes steps for substrate generation and preparation of nuclear/cytoplasmic extracts.

Main Results:

  • The protocol successfully quantifies RNA cap methyltransferase activity.
  • Demonstrates applicability to various sample types, including cell extracts and recombinant proteins.
  • Provides a robust method for assessing N7-methylation extent.

Conclusions:

  • This assay offers a reliable method for biochemical analysis of RNA cap methyltransferases.
  • The protocol is versatile and adaptable for different biological sample types.
  • Enables detailed investigation into the function and regulation of these enzymes.