Genome-wide mapping of mesoscale neuronal RNA organization and condensation

Subcellular RNA organization can affect critical cellular functions. However, our understanding of RNA microenvironments, particularly biomolecular condensates, remains limited, largely due to a lack of technologies to comprehensively interrogate mesoscale RNA organization. Here, we adapt Split-Pool Recognition of Interactions by Tag Extension to map micron-scale RNA-RNA spatial proximity genome-wide across cell regions (RNA-SPRITE). Deploying RNA-SPRITE, we find extensive, conserved organization of mature mRNAs, with increased colocalization between mRNAs that share RNA-binding protein (RBP) motifs or encode functionally related proteins. Both effects are especially strong in dendrites and axons, suggesting prevalent mRNA co-regulation. Moreover, mRNAs with less compact folding, lower translation efficiency, and specific RBP motifs are more likely to be in RNA-rich condensates. However, perturbations that broadly dissolve or enhance condensation reveal that RBP motif and encoded protein-mediated colocalizations largely remain intact, independent of condensation. These results demonstrate the power of RNA-SPRITE in revealing critical aspects of RNAs functional organization. In BriefUnbiased, genome-wide maps of RNA-RNA mesoscale spatial proximity uncover extensive subcellular organization and its governing principles. HighlightsO_LIRNA-SPRITE reveals micron-scale RNA colocalization genome-wide across cell regions C_LIO_LImRNA colocalization specificity is driven by shared motifs and encoded protein function C_LIO_LImRNAs with less compact folding, lower translation efficiency, and distinct protein-binding motifs are more likely to be in condensates C_LIO_LINeurites have a particularly high degree of sequence and function-dependent mRNA organization C_LI