Novel tools for multi-omic characterization of subnuclear RNA-scaffolded structures in development and disease

Abstract

RNA molecules are increasingly recognized as fundamental regulators of subcellular organization. In the nucleus, RNAs scaffold numerous structures that mediate diverse and essential genomic functions. These range from small-scale chromatin interactions that co-regulate handfuls of genes, to subnuclear organelles that collectively control cellular gene-expression, epigenetic, and metabolic programs. RNA-scaffolded structures are characteristically dysregulated in, and thought to be causally linked to, a plethora of human diseases, including developmental and neurodegenerative disorders, retroviral infections, and cancer. Nuclear architectural RNAs may therefore represent a large, untapped pool of novel therapeutic targets. However, elucidating and characterizing these targets—identifying the proteins, RNAs, and genomic loci with which a given RNA interacts—remains challenging.The studies presented in this thesis represent groundbreaking advancements in technologies I generated to capture and characterize nuclear RNA-scaffolded structures and their associated genomic loci, proteins, and RNAs, even those with low abundance. Employing these innovative techniques on gene silencing long non-coding RNAs (lncRNAs), I identified the biomolecular constituents of the nuclear structures they scaffold. Notably, this work shed light on the molecular constituents of the X-chromosome inactivation center, scaffolded by the nascent Xist transcript, and provided a better understanding of gene silencing lncRNA function. Moreover, I also applied the suite of technologies I developed to investigate a severe form of dilated cardiomyopathy caused by RBM20 mutations (RBM20-DCM), focusing on the nascent TTN transcript that naturally scaffolds nuclear RBM20 foci. These analyses revealed key biomolecules crucial for the native function of RBM20 foci and unveiled aberrations in alternative splicing and mitochondrial reactive oxygen species handling upon loss of RBM20, offering insight into the pathogenesis of RBM20-DCM. Collectively, this thesis lays the foundation for exploring nuclear RNA interactomes in various contexts, facilitating a deeper understanding of these structures in both native biological and aberrant disease states. The newly developed technologies hold significant potential for broader applications in understanding the composition and localization of other RNA-scaffolded structures, providing invaluable information for future research and potential therapeutic interventions. By delving into the complexities of nuclear RNA-scaffolded structures, these studies not only advance our understanding of cellular regulation but also open up exciting possibilities for targeted therapies against numerous human diseases. These findings represent a significant advancement in the field of RNA biology and lay the foundation for further investigations in the realm of RNA-based therapeutics and precision medicine. Ultimately, the discoveries made here pave the way for future breakthroughs in deciphering the mechanisms governing RNA-mediated subcellular organization and the promising utilization of RNA scaffolded structures and their constituents as therapeutic targets.