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: 10314946
• 负责人: Matthew Penna
• 金额: $ 4.64万
• 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/10314946
• 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 该项目的目的是确定发育受监管和保守的功能后果 LIM和CALPONIN同源域1（Limch1）骨骼肌中的替代剪接。产后肌肉 开发是与广泛的转录组重塑相关的高度动态时期。一个重要的方面 此过程的替代剪接发生了广泛的变化，这是将组织适应成人功能所必需的。 许多发育和组织特异性替代剪接跃迁的功能意义尚不清楚。 这些剪接事件具有重大影响，因为成年mRNA同工型对胎儿同工型是 在许多骨骼肌疾病中观察到。 limch1是一种假定的肌动蛋白结合蛋白，lim和calponin 同源域和几种具有未知意义的蛋白质同工型，由替代剪接产生。这 Limch1基因在大多数组织中表达无处不在的蛋白质同工型（ULIMCH1）和骨骼肌肉特异性 在成年骨骼肌中占主导的同工型（Mlimch1）。 Mlimch1包含一个额外的内部和 框架454氨基酸由六个连续外显子编码，同时包括出生后。发展 该剪接过渡的调节和组织特异性在小鼠和人类中是保守的。但是， 在成年肌肉中包括这六个limch1外显子的意义尚待确定。确定 Mlimch1和Ulimch1同工型的生理相关功能，CRISPR-CAS9用于删除 基因组段包含6个limch1的6个剪接外显子，从而迫使本构 成年骨骼肌中主要是胎儿同工型的表达（HOM LIMCH1 6EXKO）。 初步握力强度分析表明，来自两个独立纯合的成年男性和雌性小鼠 （HOM）LIMCH1 6EXKO创始人线在体内具有明显的肌肉无力，而最大力和速率 与野生型（WT）年龄匹配的对照组相比，放松受到了体内影响。我将使用hom limch1 6Exko小鼠确定肌肉特异性limch1同工型对骨骼肌生理的贡献 和功能。在第一个目标中，我将通过握把对HOM LIMCH1 6EXKO小鼠进行广泛的分析 强度测试以确定衰老的进展程度，这是一个彻底的体内收缩力测定面板 确定影响力产生和骨骼肌肉钙分析的主要机制，以识别 由于去除Mlimch1而导致的生理缺陷，通常存在于成人中的Limch1同工型 肌肉。在第二个目标中，将通过体内划定Limch1蛋白的功能特性 定位研究和相互作用蛋白伴侣的鉴定，以确定基本机制 导致HOM LIMCH1 6EXKO小鼠的力量损失。通过研究功能，空间和蛋白质结合 Mlimch1的特征，组织特异性调节在骨骼肌稳态中的作用将 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处理。