Alternative Splicing Regulation and Mechanotransduction in Skeletal Muscle

项目摘要/摘要 骨骼肌允许受控的运动和微调的协调贯穿整个生命 个人的。在所有细胞中，对物理力量的反应是至关重要的；然而，这在骨骼中尤其重要。 促进运动、收缩和力量产生的肌肉。机械转导使细胞能够感知 并对外部环境做出反应。在肌肉细胞中，力通过Z盘传递，这些力是 散布在肌节和锚定肌动蛋白细丝之间。在许多肌肉疾病和肌肉疾病中， 机械转导发生改变，导致力量丧失、肌肉萎缩和僵硬增加。在 在分子水平上，这些病理改变伴随着广泛的转录变化和错配。 对选择性剪接的调节，这是一种RNA处理机制，允许单个基因编码多个 蛋白质异构体。骨骼肌的一个独特特征是它表现出最高水平的组织特异性 以及进化上保守的选择性剪接。 在肌肉发育期间，会发生广泛的选择性剪接变化，以促进肌肉的成熟 组织。有趣的是，许多通过剪接发育调节的基因编码相关的蛋白质。 在膜运输中并定位于肌节。其中一个基因编码封闭式肌动蛋白。 肌肉Z线β亚基(CAPZB)蛋白。CAPZB除了在肌动蛋白封顶方面发挥关键作用外，还发挥着 在肌节组织中的非常规角色。令人惊讶的是，机械转导和替代 剪接在肌肉中是相互连接的还没有得到深入的研究，并可以揭示关于 肌肉疾病。在我的提案中，我假设替代拼接监管有助于 骨骼肌力学特性的发展。我将在两个具体目标上检验这一假设。在……里面 目的1，我将确定两个剪接调节因子，多嘧啶结合蛋白1(PTBP1)和 颤动蛋白(QK)，通过拉伸细胞和控制肌肉细胞的机械性能 研究对机械敏感通路的影响。在目标2中，我将确定CAPZB及其拼接的形成方式 通过使用力显微镜和功能研究来提高肌肉细胞的机械敏感性。 我的长期目标是成为一名独立的科学领导者，能够领导一个团队。因此，培训 我将通过这项奖学金获得帮助我成长为一名肌肉生物学家的专业知识 RNA处理，以及扎实的指导、写作和教学技能。我的赞助商和共同赞助商都是专家 在肌肉生理学、细胞生物学和选择性剪接方面，我与 物理学和机械传导学方面的专业知识，以促进一篇多学科的论文。他们都是 坚定不移地致力于指导和教育，并将在我获得博士学位期间和在我进入 我的事业。最后，北卡罗来纳大学教堂山分校拥有强大的RNA生物学社区， 机械转导和膜交易，这些都有助于我所在的协作环境。

PROJECT SUMMARY/ABSTRACT Skeletal muscle allows for controlled movement and fine-tuned coordination throughout the entire life of an individual. In all cells, the response to physical forces is critical; however, this is particularly important in skeletal muscles to facilitate movement, contraction, and force generation. Mechanotransduction allows cells to sense and respond to the external environment. In muscle cells, forces transmit through the Z-disc which are interspersed between sarcomeres and anchor actin filaments. In numerous muscular diseases and myopathies, mechanotransduction is altered and leads to loss of force, muscle wasting, and increased stiffness. At the molecular level, these pathological alterations are accompanied by extensive transcriptional changes and mis- regulation of alternative splicing, an RNA processing mechanism that allows single genes to code for multiple protein isoforms. A unique feature of skeletal muscle is that it exhibits one of the highest levels of tissue-specific and evolutionarily conserved alternative splicing. During muscle development, extensive alternative splicing changes occur to facilitate maturation of the tissue. Interestingly, numerous genes developmentally regulated by splicing encode proteins that are involved in membrane trafficking and localize to the sarcomere. One of these genes encodes the Capping Actin Protein of Muscle Z-Line Subunit Beta (CAPZB) protein. Besides its critical function in capping actin, CAPZB plays unconventional roles in sarcomere organization. Surprisingly, how mechanotransduction and alternative splicing are interconnected in muscles has not been deeply investigated and can reveal new insights about muscle diseases. In my proposal, I hypothesize that alternative splicing regulation contributes to the development of the mechanical properties of skeletal muscle. I will test this hypothesis in two specific aims. In aim 1, I will identify the role of two splicing regulators, the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking protein (QK), in controlling the mechanical properties of muscle cells by stretching cells and investigating effects on mechanosensitive pathways. In aim 2, I will determine how CAPZB and its splice forms contribute to the mechanosensitivity of muscle cells by using force microscopy and functional studies. My long-term goal is to be an independent scientific leader who can lead a team. Therefore, the training I will receive through this fellowship will facilitate my growth in becoming a muscle biologist with expertise in RNA processing, and solid skills in mentorship, writing and teaching. My sponsor and co-sponsor are experts in muscle physiology, cell biology, and, alternative splicing and I have recruited collaborators and mentors with expertise in physics and mechanotransduction to facilitate a multidisciplinary dissertation. All of them are strongly committed to mentoring and education and will support me during my Ph.D. and as I move forward in my career. Finally, the University of North Carolina at Chapel Hill has strong communities of RNA Biology, mechanotransduction, and membrane trafficking that contribute to the collaborative environment I am part of.