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J Am Chem Soc. 2018 Jun 13;140(23):7135-7143. doi: 10.1021/jacs.8b02618. Epub 2018 Jun 5.

miCLIP-MaPseq, a Substrate Identification Approach for Radical SAM RNA Methylating Enzymes.

Author information

1
Department of Cellular and Molecular Pharmacology , University of California , San Francisco , California 94158 , United States.
2
Department of Pharmaceutical Chemistry , University of California , 600 16th Street, MC2280 San Francisco , California 94158 , United States.

Abstract

Although present across bacteria, the large family of radical SAM RNA methylating enzymes is largely uncharacterized. Escherichia coli RlmN, the founding member of the family, methylates an adenosine in 23S rRNA and several tRNAs to yield 2-methyladenosine (m2A). However, varied RNA substrate specificity among RlmN enzymes, combined with the ability of certain family members to generate 8-methyladenosine (m8A), makes functional predictions across this family challenging. Here, we present a method for unbiased substrate identification that exploits highly efficient, mechanism-based cross-linking between the enzyme and its RNA substrates. Additionally, by determining that the thermostable group II intron reverse transcriptase introduces mismatches at the site of the cross-link, we have identified the precise positions of RNA modification using mismatch profiling. These results illustrate the capability of our method to define enzyme-substrate pairs and determine modification sites of the largely uncharacterized radical SAM RNA methylating enzyme family.

PMID:
29782154
DOI:
10.1021/jacs.8b02618
[Indexed for MEDLINE]

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