Cell-type-resolved RNP topologies reveal dynamic structural mechanisms of splicing and therapeutic targets

Resolving RNA conformations in native ribonucleoprotein (RNP) complexes remains a fundamental challenge. Here, we introduce spatial hydroxyl acylation reversible crosslinking with immunoprecipitation (SHARCLIP) to simultaneously capture RNA-RNA, RNA-protein and protein-protein contacts in cells. SHARCLIP profiling of HNRNPC-associated RNA conformations established a global phased map of ribonucleosomes, resolving a decades-old debate on heterogenous nuclear (hn)RNP assembly. We built a dynamic structural atlas across seven cell lineages for >10,000 RNAs, generating ~200 million contacts, and identifying millions of dynamic loops, steric blockers and conformational switches that control splicing outcome. Deciphering the structural logic of mutually exclusive exons (MXEs) enabled rational design of structure-breaking and stabilizing antisense oligonucleotides (ASOs). We demonstrate effective isoform swapping in 12 genes linked to genetic disorders. SHARCLIP provides a comprehensive roadmap for cellular RNA structural biology and structure-guided RNA therapeutics.