The Role of Limch1 Alternative Splicing in Skeletal Muscle Function

项目摘要 该项目的目标是确定发育调节和保护的功能后果。 LIM和Calponin同源域1(Limch1)在骨骼肌中的选择性剪接。产后肌肉 发育是一个高度动态的时期，伴随着广泛的转录组重塑。一个重要的方面 在这个过程中，选择性剪接发生了广泛的变化，这是组织适应成年功能所必需的。 许多发育和组织特异性选择性剪接转变的功能意义尚不清楚。 这些剪接事件具有重要的意义，因为成人mRNA亚型逆转为胎儿亚型 在许多骨骼肌疾病中观察到。Limch1是一个与LIM和Calponin结合的肌动蛋白结合蛋白 同源结构域和几种未知意义的蛋白质亚型，由选择性剪接产生。这个 Limch1基因在大多数组织中表达一种普遍存在的蛋白亚型(ULimch1)，并在骨骼肌中表达一种特异的 主要存在于成人骨骼肌中的亚型(MLimch1)。MLimch1包含一个额外的内部和 出生后由6个相邻外显子同时包含的454个氨基酸组成的框架。发展中的 这种剪接转换的调节和组织特异性在小鼠和人类中是保守的。然而， 在成人肌肉中包括Limch1的这六个外显子的意义尚未确定。要确定 MLimch1和uLimch1亚型的生理相关功能，使用CRISPR-Cas9删除 包含Limch1的6个选择性剪接外显子的基因组片段在体内，从而迫使构成 胎儿为主的异构体uLimch1在成人骨骼肌中的表达(Hom Limch1 6exKO)。 初步握力分析表明，成年雄性和雌性小鼠分别来自两个独立的纯合子 (Hom)Limch1 6exKO建立系在活体内有明显的肌肉无力，而最大力量和速度 与野生型(WT)年龄匹配的对照组相比，松弛在体外受到影响。我会用Hom Limch1 6exKO小鼠测定肌肉特异性Limch1亚型对骨骼肌生理的贡献 和功能。在第一个目标中，我将通过GRIP对Hom Limch1 6exKO小鼠进行广泛的分析 强度测试以确定随年龄增长的进展程度，一组全面的体外收缩分析可 确定影响力量产生的主要机制，并进行骨骼肌钙分析以确定 由于去除mLimch1引起的生理缺陷，mLimch1亚型通常存在于成人中 肌肉。在第二个目标中，将通过体内实验描述Limch1蛋白的功能特性 相互作用蛋白质伙伴的定位研究和鉴定以确定潜在的机制 导致Hom Limch1 6exKO小鼠力量丧失。通过研究功能、空间和蛋白质结合 MLimch1的特性、组织特异性调节在骨骼肌内稳态中的作用 被揭开。这些研究的结果将有助于我们理解 骨骼肌发育和疾病中的RNA加工。

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.