Nascent transcript O-MAP reveals the molecular architecture of an RBM20-regulated single-locus subnuclear compartment organized around the TTN RNA

Abstract

Eukaryotic gene expression is regulated by highly organized subnuclear compartmentalization, yet the molecular architecture of locus-specific nuclear microenvironments remains difficult to dissect. Here, we adapt oligonucleotide-mediated proximity-interactome mapping (O-MAP) to map genomic loci, RNAs, and proteins within discrete nuclear neighborhoods, using a cardiac-specific RNA factory organized around nascent TTN RNA as a model. We find that the TTN-associated microenvironment is embedded within a developmentally regulated hub of chromosomal interactions, including cis-interacting active domains and trans-interacting poised and repressed domains. In addition to chromatin contacts, the factory contains diverse RNAs in a pattern consistent with substantial post-transcriptional recruitment and is enriched for RNA-metabolic and chromatin-regulatory proteins. These include the splicing factor RBM20 and chromatin-scaffolding factor SAFB, which exert opposing effects on factory-associated gene expression. Disruption of factory architecture through loss of TTN RNA or RBM20 leads to widespread splicing defects and mislocalization of transcripts. Notably, many splicing defects caused by RBM20 loss are attenuated by TTN RNA depletion, indicating that TTN's unbound transcript exerts toxic gain-of-function effects on RNA metabolism in the absence of RBM20. Collectively, this work establishes O-MAP as a powerful approach for biochemically dissecting single-locus compartments and reveals how coding RNAs can influence nuclear architecture and gene regulation.