The Role of Limch1 Alternative Splicing in Skeletal Muscle Function

Limch1 选择性剪接在骨骼肌功能中的作用

• 批准号: 10449985
• 负责人: Matthew Penna
• 金额: $ 4.71万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-07 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10449985
• 关键词: Actin-Binding Protein; Actins; Adult; Affect; Age; Aging; Alternative Splicing; Amino Acids; Binding; Binding Proteins; Biological Assay; Birth; Body Weight Changes; CRISPR/Cas technology; Calcium; Characteristics; Defect; Development; Digitalis preparation; Disease; Epitopes; Event; Excision; Exons; Extensor; Fatigue; Fellowship; Female; Fiber; Fractionation; Generations; Genes; Genomic Segment; Goals; Hand Strength; Histologic; Homeostasis; Human; Immunoprecipitation; Label; Maintenance; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Mus; Muscle; Muscle Development; Muscle Weakness; Muscle function; Muscular Atrophy; Myopathy; Organelles; Outcome; Pattern; Phosphorylation Site; Physiological; Play; Process; Property; Protein Isoforms; Proteins; RNA Processing; RNA Splicing; Recovery; Regulation; Relaxation; Role; Skeletal Muscle; Soleus Muscle; Spatial Distribution; Specificity; Stress Fibers; Testing; Time; Tissues; base; calponin; cell motility; fetal; hydrophilicity; in vivo; insight; interest; male; muscle aging; muscle form; muscle physiology; postnatal; postnatal development; reduced muscle strength; transcriptome

Project Summary The goal of this project is to determine the functional consequences of developmentally regulated and conserved LIM and calponin homology domain 1 (Limch1) alternative splicing in skeletal muscle. Postnatal muscle development is a highly dynamic period associated with extensive transcriptome remodeling. A significant aspect of this process is widespread changes in alternative splicing, required for adaptation of tissues to adult function. The functional significance of many developmental and tissue specific alternative splicing transitions is unknown. These splicing events have significant implications since reversion of adult mRNA isoforms to fetal isoforms is observed in many skeletal muscle diseases. Limch1 is a putative actin-binding protein with LIM and calponin homology domains and several protein isoforms of unknown significance, generated by alternative splicing. The Limch1 gene expresses a ubiquitous protein isoform (uLimch1) in most tissues and a skeletal muscle specific isoform that predominates in adult skeletal muscle (mLimch1). mLimch1 contains an additional internal and in frame 454 amino acids encoded by six contiguous exons simultaneously included after birth. The developmental regulation and tissue specificity of this splicing transition is conserved in mouse and human. However, the significance of including these six exons of Limch1 in adult muscle is yet to be determined. To determine the physiologically relevant functions of mLimch1 and uLimch1 isoforms, CRISPR-Cas9 was used to delete the genomic segment containing the 6 alternatively spliced exons of Limch1 in vivo, thereby forcing the constitutive expression of the predominantly fetal isoform, uLimch1 in adult skeletal muscle (HOM Limch1 6exKO). Preliminary grip strength analysis showed that adult male and female mice from two independent homozygous (HOM) Limch1 6exKO founder lines had significant muscle weakness in vivo while maximum force and rate of relaxation is impacted ex vivo compared to wild-type (WT) age-matched controls. I will use the HOM Limch1 6exKO mice to determine the contribution of the muscle specific Limch1 isoform to skeletal muscle physiology and function. In the first aim, I will conduct extensive analysis of the HOM Limch1 6exKO mice through grip strength testing to determine extent of progression with aging, a thorough panel of ex vivo contractility assays to identify the predominant mechanism affecting force generation and skeletal muscle calcium analysis to identify the physiological defects resulting from removal of mLimch1, the Limch1 isoform normally present in adult muscle. In the second aim, functional characteristics of Limch1 protein will be delineated through in vivo localization studies and identification of interacting protein partners to determine the underlying mechanisms leading to strength loss in HOM Limch1 6exKO mice. By investigating the functional, spatial, and protein binding characteristics of mLimch1, the role of tissue specific regulation of Limch1 in skeletal muscle homeostasis will be uncovered. The results obtained from these studies will contribute to our understanding of the significance of RNA processing in skeletal muscle development and disease.

项目摘要 这个项目的目标是确定发育调节和保护的功能后果， 骨骼肌中LIM和钙调蛋白同源结构域1（Limch 1）的选择性剪接。生后肌 发育是与广泛的转录组重塑相关的高度动态的时期。一个重要方面 这一过程的一个重要特征是选择性剪接的广泛变化，这是组织适应成人功能所必需的。 许多发育和组织特异性选择性剪接转换的功能意义是未知的。 这些剪接事件具有重要意义，因为成体mRNA同种型向胎儿同种型的逆转是通过基因组的转录调控来实现的。 在许多骨骼肌疾病中观察到。Limch 1是一种与LIM和钙调蛋白结合的肌动蛋白 同源结构域和几种未知意义的蛋白质同种型，通过选择性剪接产生。的 Limch 1基因在大多数组织中表达普遍存在的蛋白质异构体（uLimch 1），并且在骨骼肌中特异性表达。 在成人骨骼肌中占优势的同种型（mLimch 1）。mLimch 1包含一个额外的内部和 框架454个氨基酸由出生后同时包括的六个连续外显子编码。发育 这种剪接转换的调节和组织特异性在小鼠和人中是保守的。但 在成年肌肉中包括Limch 1的这六个外显子的意义还有待确定。确定 为了确定mLimch 1和uLimch 1同种型的生理学相关功能，CRISPR-Cas9用于删除mLimch 1和uLimch 1同种型的生理学相关功能， 包含Limch 1的6个可变剪接外显子的基因组片段，从而迫使组成型 主要胎儿亚型uLimch 1在成人骨骼肌中的表达（HOM Limch 1 6 exKO）。 初步的握力分析表明，成年雄性和雌性小鼠来自两个独立的纯合子， (HOM)Limch 1 6 exKO建立者品系在体内具有显著的肌无力，而最大力和速率则在体内具有显著的肌无力。 与野生型（WT）年龄匹配的对照相比，离体松弛受到影响。我将使用HOM Limch 1 6 exKO小鼠，以确定肌肉特异性Limch 1同种型对骨骼肌生理学的贡献 和功能在第一个目标中，我将通过抓握对HOM Limch 1 6 exKO小鼠进行广泛的分析， 强度测试，以确定随着老化的进展程度，一个全面的小组离体收缩性测定， 确定影响力产生的主要机制和骨骼肌钙分析，以确定 mLimch 1（Limch 1同种型，通常存在于成年人中）的去除导致的生理缺陷 肌肉.第二个目标是通过体内实验研究Limch 1蛋白的功能特性， 定位研究和相互作用蛋白伴侣的鉴定，以确定潜在的机制 导致HOM Limch 1 6 exKO小鼠的力量损失。通过研究功能，空间和蛋白质结合， mLimch 1的特性，Limch 1在骨骼肌稳态中的组织特异性调节作用将 被发现。从这些研究中获得的结果将有助于我们理解 骨骼肌发育和疾病中的RNA加工。