Enhanced hybridization-proximity labeling discovers protein interactomes of single RNA molecules.

RNAs engage diverse protein partners and localize to specific subcellular compartments, yet dissecting proteomes associated with low-abundance or dispersed RNA molecules remains a challenge. We present an enhanced hybridization-proximity labeling (HyPro) technology for in situ proteome profiling of endogenously expressed RNA microcompartments. We re-engineer the HyPro enzyme and optimize proximity biotinylation conditions to identify proteins associated with compact RNA-containing nuclear bodies, small pre-mRNA clusters, and individual transcripts. Applying this approach to pathogenic G4C2 repeat-containing C9orf72 RNAs, retained as single-molecule foci in the nuclei of amyotrophic lateral sclerosis (ALS) patient-derived pluripotent stem cells, we reveal extensive interactions with disease-linked paraspeckle markers and a specific set of pre-mRNA splicing factors. These findings highlight early RNA processing and localization defects in ALS that may contribute to this late-onset neurodegenerative disorder. Overall, HyPro provides a broadly applicable platform for mapping RNA-protein interactions, enabling insights into RNA biology and its dysregulation in disease.