Investigating Mechanisms of RBM20 Liquid-liquid Phase Separation Driving Cardiomyocyte Physiology and Dilated Cardiomyopathy - PROJECT SUMMARY/ABSTRACT
Mutations in the muscle-specific RNA 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. We recently uncovered the existence of a nuclear RBM20 splicing
factory that brings into proximity multiple co-regulated DNA loci from different chromosomes. Formation of this
three-dimensional chromatin structure relies on RBM20 foci that are nucleated by its main splicing target, the
pre-mRNA encoding for the giant protein titin (TTN). My preliminary data suggests that RBM20 foci undergo
liquid-liquid phase separation (LLPS), a mechanism thought to contribute to the segregation and regulation of
sub-nuclear compartments. My preliminary data also suggests RBM20 LLPS is perturbed in RBM20 DCM
mutants. Thus, the central hypothesis tested in this proposal is that RBM20 assembles membrane-less
macromolecular condensates required for homeostatic function in the heart. My specific aims are to: (1)
determine the biophysical properties of the RBM20 splicing factory and impact of DCM mutations; and (2)
determine whether RBM20 LLPS is required for RBM20 function, and how RBM20 mutations drive DCM
phenotypes. I will perform in vitro experiments using purified human RBM20, and cellular experiments using
human pluripotent stem cell-derived cardiomyocytes (HPSC-CMs), to determine the mechanisms of RBM20
LLPS and impact of DCM mutations. I will leverage a combination of super-resolution microscopy, photo-
bleaching and photo-conversion microscopy, and molecular biology, to study LLPS. To correlate biophysical and
topological changes due to perturbation of RBM20 LLPS with cardiac function, I will characterize three-
dimensional engineered heart tissues (3D-EHTs), a cardiac organoid model. To test whether LLPS is required
for RBM20 function, I will perform DNA fluorescent in situ hybridization and RT-qPCR of target genes, to measure
splice factory assembly and alternative splicing, respectively. To reveal how RBM20 mutations drive DCM
phenotypes, I will measure electrophysiological and contractile performance of WT and mutant 3D-EHTs.
Collectively, these experiments will exhaustively characterize how RBM20 mutations, which drive aggressive
DCM, affect the biophysical properties, cellular dynamics, and function of RBM20. In addition, these experiments
will reveal how RBM20 DCM mutants affect cardiac physiology and drive disease, with the potential to reveal
new therapeutic options for RBM20 DCM. More broadly, our work will enhance our understanding of how LLPS-
mediated compartmentalization drives human health and how perturbation of LLPS contributes to disease. This
project will take place in the highly supportive and collaborative environment of the Institute for Stem Cell and
Regenerative Medicine at the University of Washington. With the mentorship of my Sponsor and Co-Sponsor
(Dr. Charles E. Murry and Dr. Nathan J Sniadecki, respectively), this project will provide the training required for
my career goal of establishing an independent research laboratory.
