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Function, composition, and mechanism of RNA splicing factories in cardiomyopathy

Award Number: R01HL160825
ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 12/10/2022
PERIOD OF PERFORMANCE END DATE: 08/09/2024

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Function, composition, and mechanism of RNA splicing factories in cardiomyopathy - 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $522,500 )
2025	2025	UNIVERSITY OF SOUTHERN CALIFORNIA	3720 S FLOWER ST FL 3	LOS ANGELES	CA	90033	LOS ANGELES	USA	Cardiovascular Diseases Research	001	3	1/18/2025	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$522,500
2025	2025	UNIVERSITY OF SOUTHERN CALIFORNIA	3720 S FLOWER ST FL 3	LOS ANGELES	CA	90033	LOS ANGELES	USA	Cardiovascular Diseases Research	003	3	4/15/2025	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$404,821
2025	2024	UNIVERSITY OF WASHINGTON	4333 BROOKLYN AVE NE	SEATTLE	WA	98195	KING	USA	Cardiovascular Diseases Research	002	2	4/15/2025	NON-COMPETING CONTINUATION	-$404,821
2025	2024	UNIVERSITY OF WASHINGTON	4333 BROOKLYN AVE NE	SEATTLE	WA	98195	KING	USA	Cardiovascular Diseases Research	000	2	1/17/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $522,500

Issue Date FY: 2024 ( Subtotal = $574,078 )
2024	2024	UNIVERSITY OF WASHINGTON	4333 BROOKLYN AVE NE	SEATTLE	WA	98195	KING	USA	Cardiovascular Diseases Research	000	2	11/15/2023	NON-COMPETING CONTINUATION	$527,216
2024	2024	UNIVERSITY OF WASHINGTON	4333 BROOKLYN AVE NE	SEATTLE	WA	98195	KING	USA	Cardiovascular Diseases Research	001	2	6/26/2024	NON-COMPETING CONTINUATION	$46,862
														Subtotal = $574,078

Issue Date FY: 2023 ( Subtotal = $586,606 )
2023	2023	UNIVERSITY OF WASHINGTON	4333 BROOKLYN AVE NE	SEATTLE	WA	98195	KING	USA	Cardiovascular Diseases Research	000	1	12/10/2022	NEW	$586,606
														Subtotal = $586,606

Grand Total All Awards = $1,683,184

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Function, composition, and mechanism of RNA splicing factories in cardiomyopathy

Award Number: R01HL160825

ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 12/10/2022

PERIOD OF PERFORMANCE END DATE: 08/09/2024

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