Records of RNA locations in living yeast revealed through covalent marks

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a new strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly and progressively marks RNAs with 3' terminal uridines. This Localized RNA Recording approach yields a record of RNA locations, is neither toxic nor limited by diffusion of chemical probes, and here is validated through identification of many mRNAs localized selectively to ER or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria indicating coordination of complex assembly. Non-coding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.