Alternative Splicing Regulation and Mechanotransduction in Skeletal Muscle

项目总结/摘要 骨骼肌允许控制运动和微调协调整个生命的一个 单独的.在所有细胞中，对物理力的反应是至关重要的;然而，这在骨骼肌中尤其重要。 肌肉，以促进运动，收缩和力量的产生。机械转导允许细胞感知 并对外部环境做出反应。在肌肉细胞中，力通过Z盘传递， 散布在肌节和锚肌动蛋白丝之间。在许多肌肉疾病和肌病中， 机械传导被改变并导致力的损失、肌肉萎缩和增加的僵硬。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误的表达。 选择性剪接是一种RNA加工机制，允许单个基因编码多个 蛋白质异构体。骨骼肌的一个独特的特点是，它表现出最高水平的组织特异性 和进化上保守的选择性剪接。 在肌肉发育过程中，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，许多受剪接调控的基因编码的蛋白质参与了 并定位于肌节。其中一个基因编码加帽肌动蛋白 肌肉Z线亚单位β（CAPZB）蛋白。CAPZB除了在肌动蛋白帽化中发挥重要作用外， 在肌节组织中的非常规作用。令人惊讶的是，机械传导和替代 剪接在肌肉中相互连接尚未得到深入研究，可以揭示关于 肌肉疾病。在我的建议中，我假设选择性剪接调节有助于 骨骼肌力学性能的发展。我将在两个具体目标中检验这一假设。在 目的1，我将确定两个剪接调节剂的作用，多嘧啶束结合蛋白1（PTBP 1）和 震动蛋白（QK），通过拉伸细胞控制肌肉细胞的机械特性， 研究对机械敏感通路的影响。在目标2中，我将确定CAPZB及其剪接形式 通过使用力显微镜和功能研究有助于肌细胞的机械敏感性。 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 我将通过这个奖学金将促进我的成长，成为一个肌肉生物学家， RNA加工，以及扎实的指导、写作和教学技能。我的担保人和共同担保人都是专家 在肌肉生理学，细胞生物学，和选择性剪接，我已经招募了合作者和导师， 物理学和机械传导的专业知识，以促进多学科论文。都是 坚定地致力于指导和教育，并将支持我在我的博士学位。当我继续 我的职业生涯我最后，位于查佩尔山的北卡罗来纳州大学拥有强大的RNA生物学社区， 机械传导和膜运输，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.