ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/005/SRR6497885/SRR6497885.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/007/SRR6497887/SRR6497887.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/003/SRR6497883/SRR6497883.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/006/SRR6497876/SRR6497876.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/001/SRR6497891/SRR6497891.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/001/SRR6497881/SRR6497881.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/008/SRR6497878/SRR6497878.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/009/SRR6497889/SRR6497889.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/005/SRR6497875/SRR6497875.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/006/SRR6497886/SRR6497886.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/000/SRR6497890/SRR6497890.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/003/SRR6497873/SRR6497873.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/008/SRR6497888/SRR6497888.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/004/SRR6497884/SRR6497884.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/007/SRR6497877/SRR6497877.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/002/SRR6497882/SRR6497882.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/000/SRR6497880/SRR6497880.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/009/SRR6497879/SRR6497879.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR649/004/SRR6497874/SRR6497874.fastq.gzprimary200OKGenomicsRNA Biology Group, Max Planck Institute for Molecular Biomedicinehttps://www.ebi.ac.uk/ena/browser/view/PRJNA430372m6A 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 m6A sites is of utmost importance. However, m6A 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 can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m6A 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. Overall design: enrichment of methylated nucleotides by bioconjugation of propagyl groups in synthetic oligos and rRNAxref:PubMed:29461645ENAribonucleic acid, Acid, RNA, Ribonucleic, Reactions, Click Chemical, Chemical Reaction, Click, ribose nucleic acid, Non Polyadenylated RNA, ribonucleic acids, Non-Polyadenylated, Chemical Technique, Chemical Reactions, Click Chemical Reaction, RNS, Ribonucleic Acid, Click Chemical Reactions, RNA Gene Products., Non-Polyadenylated RNA, Click Chemical Techniques, Chemical Techniques, Click Chemistries, Techniques, Reaction, yeast nucleic acid, Click Chemical Technique, Ribonukleinsaeure, Gene Products, Chemistry, pentosenucleic acids, Ribonucleic acids, Chemistries, Technique, Non Polyadenylatedribonucleic acid, Acid, RNA, Ribonucleic, Reactions, Click Chemical, Chemical Reaction, Click, ribose nucleic acid, Non Polyadenylated RNA, ribonucleic acids, Non-Polyadenylated, Chemical Technique, Chemical Reactions, Click Chemical Reaction, RNS, Ribonucleic Acid, Click Chemical Reactions, RNA Gene Products., Non-Polyadenylated RNA, Click Chemical Techniques, Chemical Techniques, Click Chemistries, Techniques, Reaction, yeast nucleic acid, Click Chemical Technique, Ribonukleinsaeure, Gene Products, Chemistry, pentosenucleic acids, Ribonucleic acids, Chemistries, Technique, Non PolyadenylatedfalseEnzymatic or in vivo installation of propargyl groups in combination with click chemistry enables enrichment and detection of methyltransferase target sites in RNAEnzymatic or in vivo installation of propargyl groups in combination with click chemistry enables enrichment and detection of methyltransferase target sites in RNA2023-05-172018-03-01PRJNA430372GSE10929829461645