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

## TL;DR

A new method called HyPro identifies proteins interacting with specific RNA molecules, revealing early defects in a neurodegenerative disease.

## Contribution

An improved HyPro technology enables in situ proteome profiling of low-abundance RNA microcompartments and single RNA molecules.

## Key findings

- HyPro identifies proteins associated with compact RNA compartments and individual transcripts.
- The method reveals interactions with disease-linked markers and splicing factors in ALS-related RNA foci.

## Abstract

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.

Systematic discovery of proteins interacting with low-abundance RNAs is challenging. Here, the authors develop an enhanced HyPro technology to identify proteins associated with compact RNA compartments and single RNA molecules, revealing early defects in ALS-linked mutant C9orf72 transcripts.