Single-cell profiling reveals pervasive heterogeneity in subcellular RNA localization

Subcellular RNA localization is a fundamental layer of gene regulation, yet its heterogeneity across individual cells remains poorly understood. Here, we introduce the RNA Localization Profiler (RLP), a proximity-based RNA-editing strategy that maps compartment-specific RNAs in living cells. Across the cytoplasm, endoplasmic reticulum (ER), and plasma membrane, RLP identifies robust and highly specific RNA localization programs linked to translation and membrane organization. Single-cell RLP (scRLP) reveals that individual cells harbor roughly 5,000-7,000 cytoplasmic RNAs, with <10% associated with the ER. These measurements uncover pervasive subcellular heterogeneity in RNA localization that is undetectable by bulk assays. Spatial RNA patterns define an orthogonal axis of cell-state identity that is independent of gene expression. For example, ZWINT mRNA relocalizes to the cytoplasm in a cell cycle-dependent manner. These findings establish heterogeneous levels of subcellular RNA localization as a variable dimension of intracellular organization and cell identity.