Dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in the western world. RBM20 (RNA binding motif protein 20) is a recently described cardiac-specific, RNA splicing protein that is mutated in 2-3% of DCM. RBM20 directly binds to the RNA transcripts of many cardiomyopathy-associated genes and ensures the production of cardiac-specific protein isoforms. Human studies revealed a striking, unexplained, recurring pattern of seven tightly clustered single amino acid DCM-associated substitutions near the RS domain of RBM20. The tight pattern of heterozygous mutations in DCM patients and biochemical studies of RBM20 binding proteins suggest that human DCM mutations could have dominant negative interfering effects on the RBM20 splicing complex. Our central hypothesis is that the RBM20 RS domain mutants cause dominant negative interference resulting in pathophysiological RNA splicing that are distinct from loss-of-function mutations. We plan to develop a series of isogenic human induced pluripotent stem cell (iPSC)-cardiomyocytes (iPS-CMs). We have made multiple single-base RBM20 mutations without antibiotic selection (scarless) in iPSCs. We can efficiently produce RBM20 RS domain mutant (R636S) iPS-CMs, and the cells display cellular pathology consistent with DCM. RNA-Seq studies of R636S mutant iPS-CMs reveal >360 alternative splicing events, including many that have been previously reported.
Our aims are:
Aim 1. To test the hypothesis that RBM20 DCM-causing point mutations have a dominant negative effect, by analyzing pathological changes in splicing by RNA-Seq. We will engineer the seven recurring human DCM mutations into isogenic iPSCs, as well as RBM20 null mutations. The RNA-Seq studies will focus on identifying the most pathological alternative splicing events that could explain the DCM pathology.
Aim 2. To test the hypothesis that RBM20 RS domain mutation (R636S) has different RNA- and protein- binding, we will use FLAG and APEX epitope tagging respectively, to selectively identify binding partners. We will use FLAG-RBM20-RNA binding assays (CLIP-Seq) to determine the exact RBM20 RNA binding sites. FLAG- and APEX-APMS will identify putative binding partners and regulators of RBM20.
Aim 3. To determine if the putative binding partners are essential for RBM20 splice regulation. We will conditionally silence the expression of putative binding partners. We will use CRISPRi to modulate the expression of each gene, to determine if each protein is a potential therapeutic target. With the completion of these aims, we will directly test our hypothesis that would explain the pattern of human RBM20 mutations, and provide a molecular mechanism for pathological splicing. We will have defined the molecular targets and protein partners of RBM20. Drugs that alter RBM20 activity could enhance cardiac health and repair, via a novel mechanism. These studies will provide a foundation for developing a human iPS-CM-based platform to develop new therapeutics.
We are building a model system for studying dilated cardiomyopathy (DCM) a life threatening disease. We are using human induced pluripotent stem (iPS) cells to functionally assess putative disease mutations in a gene called RBM20, which causes ~3% of all DCM. Our study is important because DCM is the most common indication for cardiac transplantation in the Western world. With the completion of these aims, we will develop rapidly evolving technologies will provide a new paradigm to study human genetics and disease.
|Mandegar, Mohammad A; Huebsch, Nathaniel; Frolov, Ekaterina B et al. (2016) CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Cell Stem Cell 18:541-53|