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Antibody-Free Assay for RNA Methyltransferase Activity Analysis
Published on: July 9, 2019
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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
Summary
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.
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.
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