Although the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterisation is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: Localisation of RNA (LoRNA) and a streamlined density-based Localisation of Proteins by Isotope Tagging (dLOPIT) to map RNA and protein to organelles (nucleus, ER and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localisation dynamics for 31839 transcripts and 5314 proteins during the unfolded protein response (UPR), revealing that ER-localised transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview to date of RNA and protein subcellular localisation dynamics.
