Alternative splicing regulation of CLTC in the heart

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ABSTRACT Alternative splicing wields extraordinary power in controlling protein function, cell development, and tissue identity. Alternative splicing is a co- and post-transcriptional RNA processing mechanism that enables a single gene to generate more than one transcript, thereby expanding the diversity of a cell’s proteome. This ubiquitous mechanism of gene regulation occurs in more than 95% of multiexonic genes in humans. The heart exhibits one of the most tissue specific and highly conserved alternative splicing programs, and the reprogramming of these splicing patterns is a hallmark of cardiovascular diseases. In the heart, genes encoding membrane trafficking proteins are alternatively spliced in a tissue-specific manner; however, how these events are regulated and the functional roles of these isoforms within cardiomyocytes remains elusive. This proposal will investigate one specific membrane trafficking splicing event that takes place in the gene encoding the clathrin heavy chain (CLTC) protein. CLTC classically functions during clathrin-mediated endocytosis but plays unconventional structural roles as well. Cltc exon 31 alternative splicing occurs in the heart, and we have previously observed that deletion of Cltc exon 31 using CRISPR editing in mice impedes the progression of hypertrophy and heart failure after pressure overload. I hypothesize that the regulation of Cltc exon 31 splicing in the heart modulates the endocytic and structural functions of clathrin, which impacts cardiomyocyte cell growth. I will address this hypothesis with two aims. In Aim 1, I will determine the physiological consequences of Cltc splicing in the heart by assessing the impact of CLTC isoform expression on cardiomyocyte cell signaling, cytoskeleton architecture, and protein-protein interactions (immunoblotting, confocal microscopy, and proteomics). In Aim 2, I will identify the regulatory mechanism governing Cltc exon 31 splicing in the heart. I will demonstrate that the quaking (QKI) and polypyrimidine tract binding protein 1 (PTBP1) RNA-binding proteins control Cltc exon 31 splicing and that these RBPs bind with high affinity to consensus motifs located within the Cltc pre-mRNA transcript (protein knockdowns, in vitro reconstitutions, and minigene reporter expression paired with motif deletions). My long-term goal is to have a research-intensive career and become an independent leader in cardiovascular biology. The training that I will receive during this fellowship period will facilitate my scientific, professional, and personal growth by providing opportunities to expand my experimental toolkit, network with esteemed researchers, and increase my confidence as a strong scientist. Importantly, the University of North Carolina at Chapel Hill offers a rich research community of RNA, cardiovascular, genetic, and molecular cell biologists, and I have recruited mentors and collaborators with expertise in each of these fields. All of them are committed to educating and supporting me throughout my fellowship, which will set me up for success with both my research proposal and my future scientific career.