Molecular characterization of Nuclear Body assembly and pathological misassembly

Understanding how the three-dimensional architecture of the nucleus regulates genomic functions remains a central challenge in cell biology. Nuclear bodies (NBs)—membraneless organelles like nucleoli, nuclear speckles, and Cajal Bodies—play a foundational role in nuclear organization that has long been observed cytologically, but which has been challenging to characterize at the molecular level. NBs are thought to partition the genome into thousands of functionally distinct compartments, spatially controlling core genomic functions like ribosome biogenesis, pre-mRNA splicing, and DNA repair. Dysregulation of this compartmental architecture is a pervasive disease driver implicated in many human pathologies, ranging from neurodegenerative disorders to cancer. Remarkably, these complex and essential structures are often built de novo in response to cellular needs, assembling hundreds of distinct proteins, RNAs, and co-regulated loci over the course of mere minutes. These NB-assembly pathways are orchestrated by RNA molecules—which initiate the assembly process and scaffold the intact compartment—and evidence suggests that these programs can be functionally modulated to meet the distinct demands of different tissue-types. Yet, probing the molecular events underlying these phenomena remains a longstanding challenge, since NB architecture is opaque to most conventional genomics tools (e.g., Hi-C), and it is too dynamic and fragile to survive standard biochemical methods (e.g., pulldown). As a result, the molecular architecture of most NBs, how this architecture varies across cell-types and disease states, and its assembly over time, have eluded characterization for decades. This proposal will address these critical knowledge gaps by utilizing O-MAP—a new RNA-targeted spatial-omics tool—to establish general-use methods for probing NB architecture and dynamics. O-MAP is a high-resolution proximity-biotinylation technique that uses programmable, RNA-FISH-like oligo probes to deliver biotinylating enzymes to a target RNA, in situ. This enables systematic discovery of all factors near that RNA, without complex cell engineering of biochemical fractionation. We propose adapting this "off-the-shelf" proximity- omics tool to probe NB architecture by targeting the RNA scaffolds on which NBs are assembled, using the Cajal Body (CB)–a key disease-relevant NB–as a model. In (Aim 1), we generate a high-confidence reference catalog of CB proteins, transcripts, and genomic loci, and characterize the organization of these components using SIM super-resolution imaging. Parallel experiments in a suite of cell-types will reveal how this molecular architecture is adapted across cellular states and in cancer. In (Aim 2), we elucidate the molecular details of the CB biogenesis pathway, using a drug-inducible CB-assembly system, and applying O-MAP and SIM at time points during assembly. This work will yield unprecedented insight into an important disease-relevant nuclear body and establish a versatile methodological framework applicable to other NBs and a wide range of biological contexts. Project Number: 1R21HG014903-01 | Fiscal Year: 2026 | NIH Institute/Center: National Human Genome Research Institute (NHGRI) | Principal Investigator: David Shechner (+1 co-PI) | Institution: UNIVERSITY OF WASHINGTON, SEATTLE, WA | Award Amount: $444,794 | Activity Code: R21 | Study Section: Molecular Genetics Study Section[MG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11285808