Alternative splicing regulation of CLTC in the heart

心脏中 CLTC 的选择性剪接调节

• 批准号: 10749474
• 负责人: Gabrielle Marie Gentile
• 金额: $ 3.88万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749474
• 关键词: 5' -AMP-activated protein kinase; Actins; Affinity; Alternative Splicing; Animals; Architecture; Atrial Natriuretic Factor; Attenuated; Binding; Binding Proteins; Biological; Biological Assay; Biology; Brain; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular Pathology; Cardiovascular Physiology; Cardiovascular system; Cell membrane; Cell physiology; Cells; Cellular Structures; Clathrin; Clathrin Heavy Chains; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Confocal Microscopy; Consensus; Cytoskeletal Modeling; Cytoskeleton; Deceleration; Development; EFRAC; Education; Endocytosis; Equilibrium; Event; Exhibits; Exons; Faculty; Fellowship; Foundations; Future; Gene Expression Regulation; Generations; Genes; Genetic; Goals; Growth; Heart; Heart Diseases; Heart failure; Human; Hypertrophy; In Vitro; Mediating; Membrane; Mentors; Microtubules; Molecular; Mus; Muscle Fibers; Natriuretic Peptides; North Carolina; Operative Surgical Procedures; Output; Pattern; Peptide Receptor; Personal Growth; Phosphorylation; Phosphotransferases; Physiological; Physiology; Play; Polypyrimidine Tract-Binding Protein; Post-Transcriptional RNA Processing; Protein Biosynthesis; Protein Isoforms; Proteins; Proteome; Proteomics; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Regulation; Report (document); Reporter; Research; Research Personnel; Research Proposals; Role; Scientist; Shortening Fraction; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Skeletal Muscle; Spliced Genes; Stress; Survival Rate; Tissues; Training; Trans-Activators; Transcript; Universities; Western Blotting; Wild Type Mouse; aorta constriction; career; cell growth; experience; experimental study; heart function; in vivo; insight; knock-down; mRNA Precursor; mouse model; multidisciplinary; overexpression; pressure; prevent; programs; protein function; protein protein interaction; protein transport; reconstitution; recruit; response; success; therapeutic target; trafficking

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.

抽象的 替代剪接在控制蛋白质功能，细胞发育和 组织身份。替代剪接是一种共同和后的RNA处理机制，可实现 一个基因生成多个转录本，从而扩大了细胞蛋白质组的多样性。 这种无处不在的基因调节机制发生在人类中超过95％的多焦虑基因中。 心脏表现出最具体和高度组成的替代剪接程序之一， 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替代在组织特异性中剪接 方式;但是，如何调节这些事件以及这些同工型的功能作用 心肌细胞仍然难以捉摸。该建议将调查一个特定的膜贩运剪接 发生在编码网格蛋白重链（CLTC）蛋白的基因中。 CLTC经典 网格蛋白介导的内吞作用期间的功能也起着非常规的结构作用。 CLTC外显子 31替代剪接发生在心脏中，我们先前观察到CLTC外显子的缺失31 在小鼠中使用CRISPR编辑阻碍了压力后肥大和心力衰竭的进展 超载。我假设CLTC外显子31在心脏中的调节调节内吞和 网格蛋白的结构功能，影响心肌细胞生长。我将通过 两个目标。在AIM 1中，我将通过评估确定心脏中CLTC剪接的物理后果 CLTC同工型表达对心肌细胞信号传导，细胞骨架结构和 蛋白质蛋白相互作用（免疫印迹，共聚焦显微镜和蛋白质组学）。在AIM 2中，我将确定 控制CLTC外显子31拼接的调节机制。我将证明震颤 （QKI）和息肉酰亚胺氨结合蛋白1（PTBP1）RNA结合蛋白控制CLTC外显子31剪接 并且这些RBP与位于CLTC前MRNA转录本内的共识基序具有高亲和力结合 （蛋白质敲低，体外重组和与基序缺失配对的微型报告）。 我的长期目标是拥有研究密集型职业，并成为 心血管生物学。我在此奖学金期间将接受的培训将有助于我的科学培训 通过提供扩展我的实验工具包的机会，专业和个人成长 有了受人尊敬的研究人员，并提高了我作为强大科学家的信心。重要的是，大学 北卡罗来纳州教堂山（Chapel Hill）提供了丰富的RNA，心血管，遗传和 分子细胞生物学家，我曾在每种方面招募具有专业知识的导师和合作者 字段。他们所有人都致力于在我的整个奖学金中教育和支持我，这将设定 我的研究建议和未来的科学生涯都取得了成功。