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

This figshare project contains original microscopy images and image analysis code used in our study entitled "Enhanced hybridization-proximity labeling discovers protein interactomes of single RNA molecules". The ability of RNAs to associate with diverse protein partners and compartmentalize within specific subcellular environments is fundamental to eukaryotic cell function. Elucidating the protein interactomes of ribonucleoprotein assemblies containing a limited number of RNA molecules remains a considerable experimental challenge. We present an enhanced hybridization-proximity labeling (HyPro) technology for mapping the proteomes of endogenously expressed RNA microcompartments. By re-engineering the recombinant HyPro enzyme – the core of our labeling approach – and optimizing the efficiency and specificity of the proximity biotinylation reaction, we developed a method that identifies proteins associated with compact RNA-containing nuclear bodies, small clusters of pre-mRNAs at transcription sites, and, crucially, individual transcripts. When applied to C9orf72 RNA with expanded G4C2 hexanucleotide repeats, retained as single-molecule foci in the nuclei of amyotrophic lateral sclerosis (ALS) patient-derived induced pluripotent stem cells, our technology identified extensive interactions between mutant transcripts and disease-linked paraspeckle markers, as well as a specific set of pre-mRNA splicing factors. These findings highlight early RNA processing and localization defects in ALS that might contribute to this late-onset neurodegenerative disorder. Overall, our approach provides a powerful tool for profiling RNA-protein interactions, with strong potential to advance studies across the life sciences and biomedicine.