Alternative splicing regulation of CLTC in the heart

心脏中 CLTC 的选择性剪接调节

基本信息

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
项目类别：
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&apos -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

项目摘要

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.

抽象的选择性剪接在控制蛋白质功能、细胞发育和选择性剪接是一种共转录和转录后 RNA 加工机制，可实现一个基因产生多个转录本，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存在于人类 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，并且这些剪接模式的重新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选择性地剪接在组织特异性的结构中。然而，这些事件是如何调节的以及这些亚型的功能作用心肌细胞仍然难以捉摸，该提案将研究一种特定的膜运输剪接。典型地发生在编码网格蛋白重链 (CLTC) 蛋白的基因中。 Cltc 外显子在网格蛋白介导的内吞作用中发挥作用，但也发挥非常规的结构作用。 31选择性剪接发生在心脏中，我们之前观察到Cltc外显子31的缺失在小鼠身上使用 CRISPR 编辑可阻止压力后肥厚和心力衰竭的进展我认为心脏中 Cltc 外显子 31 剪接的调节调节内吞和我将讨论网格蛋白的结构功能，它会影响心肌细胞的生长。在目标 1 中，我将通过评估确定 Cltc 剪接在心脏中的生理后果。 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）。我将证明心脏中 Cltc 外显子 31 剪接的调节机制。 (QKI) 和聚嘧啶束结合蛋白 1 (PTBP1) RNA 结合蛋白控制 Cltc 外显子 31 剪接并且这些 RBP 以高亲和力与位于 Cltc pre-mRNA 转录本内的共有基序结合（蛋白质敲低、体外重建和小基因报告基因表达与基序删除配对）。我的长期目标是从事研究密集型职业并成为该领域的独立领导者我在奖学金期间接受的培训将促进我的科学、通过提供扩展我的实验工具包和网络的机会来实现专业和个人成长与受人尊敬的研究人员合作，增强我作为一名强大科学家的信心。北卡罗来纳州教堂山分校提供丰富的 RNA、心血管、遗传和分子细胞生物学家，我已经招募了在这些领域具有专业知识的导师和合作者他们所有人都致力于在我的奖学金期间教育和支持我，这将奠定我的基础。我为我的研究计划和未来的科学事业取得成功做好了准备。