HCR-Proxy analysis of site-specific pre-rRNA proximal proteomes.

We developed a novel method termed HCR-Proxy, a proximity labeling strategy that couples Hybridization Chain Reaction (HCR)-mediated signal amplification with in situ free radical-based biotinylation (Proxy-based labelling), allowing proteomic mapping of RNA-associated proteins with subcompartmental spatial resolution. To benchmark the method, we applied HCR-Proxy to specific sequences of nascent pre-ribosomal RNA (pre-rRNA), enabling the dissection of distinct proteomic landscapes across nucleolar subcompartments associated with distinct pre-rRNA maturation and processing steps.