CAPturAM, a Chemo-Enzymatic Strategy for Selective Enrichment and Detection of Physiological CAPAM-Targets

Abstract

Modified nucleotides impact all aspects of eukaryotic mRNAs and contribute to regulation of gene expression at the transcriptional and translational level. At the 5′ cap, adenosine as first transcribed nucleotide is often N6-methyl-2′-O-methyl adenosine (m6Am). This modification is tissue dependent and reversible, pointing to a regulatory function. CAPAM was recently identified as methyltransferase responsible for m6Am formation, however, the direct assignment of its target transcripts proves difficult. Antibodies do not discriminate between internal N6-methyl adenosine (m6A) and m6Am. Here we present CAPturAM, an antibody-free chemical biology approach for direct enrichment and probing of physiological CAPAM-targets. We harness CAPAM's cosubstrate promiscuity to install propargyl groups on its targets. Subsequent functionalization with an affinity handle allows for their enrichment. Using wildtype and CAPAM−/− cells, we successfully applied CAPturAM to confirm or disprove CAPAM-targets, facilitating the verification and identification of CAPAM targets.