mTRACE: Modification-enabled Tracking of RNA-associated Cellular Events [ac4C-seq]

However, existing methods face limitations in continuously recording these events in living cells. Here we introduce mTRACE (Modification-enabled Tracking of RNA-associated Cellular Events), a molecular recording framework that leverages RNA modification enzymes to capture the history of RNA-associated cellular processes.

By systematically screening a pool of 34 candidate enzymes, we identified N6-adenosine methyltransferase M.EcoGII as a highly efficient and unbiased modifier. mTRACE exploits M.EcoGII to introduce natural and non-disruptive RNA modification, writing durable marks on transcripts undergoing specific molecular events. We demonstrate that mTRACE enables precise mapping of RNA-protein interactions, including tracking and retrospective detection of ALYREF binding; quantification of both translational activity and efficiency by coupling M.EcoGII to ribosomal proteins; and spatial recording of RNA residence across subcellular compartments using engineered M.EcoGII variants. mTRACE provides a powerful solution for dissecting RNA dynamic regulation and opens a new avenue for multi-dimensional recording of RNA biology with spatiotemporal resolution.