Abstract
Eukaryotic nuclei adopt a highly compartmentalized architecture that influences nearly all genomic processes. Understanding how this architecture impacts gene expression has been hindered by a lack of tools for elucidating the molecular interactions at individual genomic loci. Here, we adapt oligonucleotide-mediated proximity-interactome mapping (O-MAP) to biochemically characterize discrete, micron-scale nuclear neighborhoods. By targeting O-MAP to introns within the TTN pre-mRNA, we systematically map the chromatin loci, RNAs, and proteins within a muscle-specific RNA factory organized around the TTN locus. This reveals an unanticipated compartmental architecture that organizes cis- and trans-interacting chromosomal domains, including a hub of transcriptionally silenced chromatin. The factory also recruits dozens of unique RNA-binding and chromatin-scaffolding factors, including QKI and SAFB, along with their target transcripts. Loss of the cardiac-specific splicing factor RBM20-a master regulator of TTN splicing that is mutated in dilated cardiomyopathy-remodels nearly every facet of this architecture. This establishes O-MAP as a pioneering method for probing single-locus, microcompartment-level interactions that are opaque to conventional tools. Our findings suggest new mechanisms by which coding genes can "moonlight" in nuclear-architectural roles.