TITINmap · Towards Uncovering the Splicing Code of the Gigantic Gene Titin in Familial Dilated Cardiomyopathy

The sarcomeric protein titin (TTN) is mutated in 30% of patients with familial dilated cardiomyopathy (DCM), a common heart disease that is a global threat to the aging society. Aberrant alternative splicing of TTN is causally linked to DCM, therefore, to understand mechanisms of TTN splicing is crucial for developing therapeutic options, which so far do not exist for DCM. RBM20, a protein that regulates alternative splicing of TTN, is also mutated in many patients with an aggressive form of DCM. How RBM20 mediates alternative splicing of TTN is largely unknown. Due to its gigantic size, TTN mRNA is often precluded from transcriptome-wide single-cell analysis. Here, I aim to understand alternative splicing of TTN mediated by RBM20 on a single-cell level. To this end, I propose to develop a method for the detection of all transcript isoforms that can be produced by the 364 exons of TTN. Using this tool, termed TITIN-seq, together with complementary stem cell-based assays, I seek to analyze changes in the repertoire of TTN isoforms upon disease-relevant mutations in the RBM20 gene. Moreover, TITIN-seq is used to identify novel splice regulators of TTN, which, together with RBM20, could complete the picture of alternative splicing of TTN. The overarching goal is to construct a comprehensive map of TTN splicing by integrating data of all its isoforms in single cells and its regulatory proteins. I envision that knowledge of such a splice map can be exploited for developing therapeutic strategies to revert aberrant splicing of TTN in patients with DCM.