Systematic discovery of subcellular RNA patterns in the gut epithelium

Subcellular RNA localization is crucial for the spatio-temporal control of protein synthesis and underlies key processes during development, homeostasis and disease. In epithelial cells, RNA can localize asymmetrically along the apico-basal axis. Yet, we ignore the localization of most transcripts as well as the diversity of patterns that they adopt. Here, we utilized proximity labeling (APEX-seq) and spatial transcriptomics (MERFISH) to map subcellular transcript localization in intestinal organoids and adult mice tissue. Many transcripts presented localization bias, often localizing in granular structures. We described the intrinsic and environmental factors that influence the formation of these patterns. We identified translation-dependent and -independent localization patterns and pintointed the role of 3' UTRs and RNA-binding proteins. This subcellular RNA atlas can be referenced to extract new biological insights and provide hypothesis-free observation on spatial transcriptomics. Overall design: In order to capture RNA in its cognate cellular environment, we utilized APEX2. DPP4 gene was fused with APEX2 to investigate apical localization in small intestinal organoids from mouse. As controls, GFP and ACTB genes were fused with APEX2. 4 samples of each fusion were sequenced for their RNA abundance and DPP4 samples were compared against GFP and ACTB samples separately to test for apically enriched RNAs.