Thursday, March 13, 2025
3/13/2025
PROJECT SUMMARY Mutations in the muscle-specific splicing factor RBM20 are a recently identified cause of aggressive dilated cardiomyopathy (DCM) characterized by severe arrhythmias. However, the underlying mechanisms are still unclear, and thus no therapies are available. Our group recently discovered a nuclear “splicing factory” involving RBM20 hotspots, which brings into proximity multiple co-regulated loci from different chromosomes. Formation of this three-dimensional (3D) chromatin structure relies the nucleation of RBM20 foci by its main splicing target, the pre-mRNA encoding for the giant protein titin (TTN). Ablating TTN transcription disrupts the RBM20 splicing factory and dysregulates the alternative splicing of genes involved in calcium handling, including the L-type calcium channel (CACNA1C) and calcium/calmodulin-dependent protein kinase II delta (CAMK2D). Thus, the central hypothesis tested in this proposal is that RBM20 assembles membraneless macromolecular condensates that control alternative splicing to centrally regulate cardiac development and disease. Our specific aims are: (1) Identify the functional consequences of dysregulating the RBM20 splicing factory; (2) Define the biophysical properties that drive assembly of the RBM20 splicing factory; (3) Define the key components of the RBM20 splicing factory. In Aims 1 and 2, we will perform cellular experiments to clarify the mechanisms and disease relevance of RBM20 focus formation, as well as functional studies using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Complementing hPSC-CM monolayer cultures, we will characterize 3D engineered heart tissues (3D-EHTs) and human myocardial grafts. We will utilize CRISPR/Cas9 genome editing to study the effect of RBM20 DCM mutations, as well as to generate fluorescent reporter lines to study focus dynamics in physiologically relevant models. We will characterize disease- associated changes in 3D chromatin topology within the RBM20 splicing factory using a combination of established sequencing- and imaging-based methods. We will then synthesize these biophysical, topological, and biochemical changes with functional genome-wide alterations in alternative splicing, to understand the dysregulation of cardiac electrophysiological and contractile properties. Aiming to elucidate the disease mechanism, we will probe the pathogenic role of specific RBM20-regulated splicing isoforms for CACNA1C and CAMK2D. In Aim 3, we will apply a novel interaction-discovery method developed in the Shechner laboratory, oligonucleotide-directed biotinylation (ODB), to perform an unbiased “multi-omic” analysis of the composition of the splicing factory. We will then determine the role of the newly identified splicing targets in RBM20 DCM, and of the putative RBM20 cofactors in the regulation of RBM20 puncta architecture and of cardiac genes' alternative splicing. Collectively, these experiments will elucidate the mechanisms by which LLPS and subnuclear architecture collaborate to drive alternative splicing in DCM, potentially revealing novel therapeutic targets.
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