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Antibody-Free Assay for RNA Methyltransferase Activity Analysis
Published on: July 9, 2019
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Precise identification of an RNA methyltransferase's substrate modification site
Siying Xiang1, Minsong Gao1, Jie Cao1
1MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, China. liujz@zju.edu.cn.
Chemical Communications (Cambridge, England)
|February 15, 2021
Summary
We developed a nonradioactive assay to pinpoint RNA methylation sites for METTL3/METTL14 and METTL16 enzymes. This method uses chemical labeling and sequencing to identify methylation locations on complex RNA substrates.
Area of Science:
- Biochemistry
- Molecular Biology
- RNA Biology
Background:
- RNA methylation is crucial for gene regulation.
- Identifying methylation sites is essential for understanding RNA function.
- Current methods for site identification can be complex or rely on radioactivity.
Purpose of the Study:
- To develop a simple, nonradioactive biochemical assay for determining RNA methylation sites.
- To enable accurate site determination for human RNA N6-methyladenosine methyltransferases (METTL3/METTL14 and METTL16).
- To provide a platform for studying complex RNA substrates and discovering new methyltransferases.
Main Methods:
- Enzyme-assisted chemical labeling of RNA substrates.
- Utilizing an allyl-substituted methyltransferase cofactor.
- Identifying the labeling site via mutation signals from standard nucleic acid sequencing.
Main Results:
- Accurate determination of substrate methylation sites for METTL3/METTL14 and METTL16.
- Successful application to long and structurally complex RNA substrates.
- Demonstrated a nonradioactive and simple assay procedure.
Conclusions:
- The developed assay is a valuable tool for RNA methylation research.
- Facilitates the investigation of enzymatic methylations on diverse RNA substrates.
- Aids in the discovery of novel RNA methyltransferases and expands understanding of RNA epigenetics.
