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miCLIP-MaPseq Identifies Substrates of Radical SAM RNA-Methylating Enzyme Using Mechanistic Cross-Linking and
Vanja Stojković1, David E Weinberg1, Danica Galonić Fujimori2,3
1Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA.
Abstract:
The family of radical SAM RNA-methylating enzymes comprises a large group of proteins that contains only a few functionally characterized members. Several enzymes in this family have been implicated in the regulation of translation and antibiotic susceptibility, emphasizing their significance in bacterial physiology and their relevance to human health. While few characterized enzymes have been shown to modify diverse RNA substrates, highlighting potentially broad substrate scope within the family, many enzymes in this class have no known substrates. The precise knowledge of RNA substrates and modification sites for uncharacterized family members is important for unraveling their biological function. Here, we describe a strategy for substrate identification that takes advantage of mechanism-based cross-linking between the enzyme and its RNA substrates, which we named individual-nucleotide-resolution cross-linking and immunoprecipitation combined with mutational profiling with sequencing (miCLIP-MaPseq). Identification of the position of the modification site is achieved using thermostable group II intron reverse transcriptase (TGIRT), which introduces a mismatch at the site of the cross-link.
Insights
Researchers developed a new method, miCLIP-MaPseq, to identify RNA targets for radical SAM methyltransferases. This technique helps uncover the function of uncharacterized enzymes involved in translation and antibiotic resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Genomics
Background:
- Radical SAM enzymes are a large protein family, with few characterized members.
- Some known enzymes regulate translation and antibiotic susceptibility, highlighting their importance.
- Many radical SAM enzymes lack identified RNA substrates, hindering functional understanding.
Purpose of the Study:
- To develop a novel method for identifying RNA substrates of radical SAM methyltransferases.
- To enable the characterization of uncharacterized enzymes within this important protein family.
Main Methods:
- Developed individual-nucleotide-resolution cross-linking and immunoprecipitation combined with mutational profiling with sequencing (miCLIP-MaPseq).
- Utilized mechanism-based enzyme-RNA cross-linking for substrate identification.
- Employed thermostable group II intron reverse transcriptase (TGIRT) to pinpoint modification sites.
Main Results:
- Successfully established miCLIP-MaPseq as a strategy for RNA substrate identification.
- Demonstrated the method's capability to identify specific RNA targets for enzymes.
- Provided a pathway to elucidate the function of previously uncharacterized radical SAM enzymes.
Conclusions:
- miCLIP-MaPseq is an effective tool for discovering RNA substrates of radical SAM methyltransferases.
- This method will accelerate the functional characterization of this enzyme family.
- Understanding these enzymes is crucial for bacterial physiology and potential therapeutic targets.
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