Global characterization of RNA modifying enzymes with RNA-mediated activity-based protein profiling (RNABPP)

Post-transcriptional RNA modifications can regulate RNA function and play an important role in gene expression. Studying RNA modifying enzymes and their associated modifications remains a considerable challenge. Here we describe the RNA-mediated activity-based protein profiling (RNABPP) methodology, a chemoproteomic strategy for profiling the activity of RNA modifying enzymes in their native context. RNABPP relies upon metabolic RNA labeling with modified ribonucleoside-based probes, combined with protein-RNA enrichment and quantitative proteomics. The RNABPP approach is a general strategy based on chemical reactivity and enzyme mechanism, making it suitable for probing multiple classes of RNA modifying enzymes across diverse biological systems.