Alternative splicing regulation of CLTC in the heart

摘要 选择性剪接在控制蛋白质功能、细胞发育和 组织身份选择性剪接是一种共转录和转录后RNA加工机制， 单个基因产生一个以上的转录本，从而扩大细胞蛋白质组的多样性。 这种普遍存在的基因调控机制发生在人类95%以上的多外显子基因中。 心脏表现出最具组织特异性和高度保守的选择性剪接程序之一， 这些剪接模式的重编程是心血管疾病的标志。 在心脏中，编码膜运输蛋白的基因以组织特异性的方式选择性剪接， 方式;然而，这些事件是如何调节的，以及这些亚型在细胞内的功能作用。 心肌细胞仍然难以捉摸。本提案将研究一种特定的膜运输剪接 在编码网格蛋白重链（CLTC）蛋白的基因中发生的事件。CLTC经典 在网格蛋白介导的内吞作用中起作用，但也起非常规的结构作用。Cltc外显子 31选择性剪接发生在心脏，我们以前观察到Cltc外显子31的缺失， 在小鼠中使用CRISPR编辑阻止了压力后肥大和心力衰竭的进展 超载。我推测，心脏中Cltc外显子31剪接的调节调节了内吞和内分泌， 网格蛋白的结构功能，影响心肌细胞生长。我将用下面的话来说明这个假设： 两个目标。在目标1中，我将通过评估心脏中Cltc剪接的生理后果， CLTC亚型表达对心肌细胞信号传导、细胞骨架结构和 蛋白质-蛋白质相互作用（免疫印迹、共聚焦显微镜和蛋白质组学）。在目标2中，我将确定 控制心脏中Cltc外显子31剪接的调节机制。我会证明震动 (QKI)和多聚嘧啶束结合蛋白1（PTBP 1）RNA结合蛋白控制Cltc外显子31剪接 并且这些RBP以高亲和力结合位于Cltc前mRNA转录物内的共有基序 （蛋白质敲除、体外重建和与基序缺失配对的小基因报告基因表达）。 我的长期目标是拥有一个研究密集型的职业生涯，并成为一个独立的领导者， 心血管生物学我在研究期间接受的培训将有助于我的科学， 通过提供机会来扩大我的实验工具包，网络， 与受人尊敬的研究人员，并增加我作为一个强大的科学家的信心。重要的是， 位于查佩尔山的北卡罗来纳州提供了丰富的RNA、心血管、遗传和 分子细胞生物学家，我已经招募了在每一个领域都有专业知识的导师和合作者， 领域的他们都致力于教育和支持我在我的奖学金，这将设置 我希望我的研究计划和未来的科学事业都能成功。

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.