期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 57, 期 21, 页码 6342-6346出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201800188
关键词
metabolic labeling; N-6-methyladenosine; ribose methylation; RNA; transferases
资金
- SPP1784 of the DFG [RE2796/3-1, LE 3260/2-1]
- EXC 1003 Cells in Motion-Cluster of Excellence
- Graduate School of the Cells-in-Motion Cluster of Excellence, University of Munster, Germany [EXC 1003-CiM]
- Fonds der Chemischen Industrie
m(6)A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome-wide assignment of m(6)A sites is of utmost importance. However, m(6)A does not interfere with Watson-Crick base pairing, making polymerase-based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl groups can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65% at m(6)A sites after bioconjugation and purification, hence enabling detection of METTL3-METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl-L-selenohomocysteine and validated different types of known rRNA methylation sites.
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