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

基本信息

批准号：
10649476
负责人：
Matthew Penna
金额：
$ 3.17万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-07 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10649476
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 Fetal Skeleton 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 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 calponin cell motility constitutive expression fetal hydrophilicity in vivo insight interest male muscle aging muscle form muscle physiology postnatal postnatal development reduced muscle strength transcriptome

项目摘要

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.

项目概要该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 和钙调蛋白同源域 1 (Limch1) 的选择性剪接。产后肌肉发育是一个高度动态的时期，与广泛的转录组重塑相关。一个重要的方面这一过程的关键是选择性剪接的广泛变化，这是组织适应成体功能所必需的。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意义，因为成人 mRNA 异构体向胎儿异构体的逆转是很容易的。在许多骨骼肌疾病中观察到。 Limch1 是一种推定的肌动蛋白结合蛋白，具有 LIM 和钙调蛋白同源结构域和几种意义不明的蛋白质亚型，由选择性剪接产生。这 Limch1 基因在大多数组织中表达普遍存在的蛋白质亚型 (uLimch1) 和骨骼肌特异性在成人骨骼肌中占主导地位的亚型 (mLimch1)。 mLimch1 包含一个额外的内部和框架 454 个氨基酸由出生后同时包含的六个连续外显子编码。发展性的这种剪接转变的调节和组织特异性在小鼠和人类中是保守的。然而， Limch1 的这 6 个外显子在成人肌肉中的重要性尚未确定。确定 mLimch1 和 uLimch1 亚型的生理相关功能，使用 CRISPR-Cas9 删除体内含有 Limch1 的 6 个选择性剪接外显子的基因组片段，从而迫使组成型主要胎儿亚型 uLimch1 在成人骨骼肌中的表达 (HOM Limch1 6exKO)。初步握力分析显示，成年雄性和雌性小鼠来自两个独立的纯合子 (HOM) Limch1 6exKO 创始人系在体内具有显着的肌肉无力，而最大力和速率与年龄匹配的野生型（WT）对照相比，放松受到离体影响。我将使用 HOM Limch1 6exKO 小鼠确定肌肉特异性 Limch1 亚型对骨骼肌生理学的贡献和功能。在第一个目标中，我将通过握力对 HOM Limch1 6exKO 小鼠进行广泛分析力量测试以确定衰老进展的程度，一系列全面的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去除 mLimch1（通常存在于成人中的 Limch1 亚型）导致的生理缺陷肌肉。第二个目标是通过体内研究描绘Limch1蛋白的功能特征。定位研究和识别相互作用的蛋白质伙伴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 的特征，Limch1 的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 加工。