Transcriptome processing networks in skeletal muscle: mechanisms and functions

骨骼肌转录组处理网络：机制和功能

• 批准号: 10359820
• 负责人: Thomas A Cooper
• 金额: $ 45.16万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359820
• 关键词: Actin-Binding Protein; Actins; Adult; Affect; Age; Alleles; Alternative Splicing; Amino Acids; Animals; Antibodies; Antisense Oligonucleotides; Architecture; Binding Proteins; CRISPR/Cas technology; Cells; Cellular Structures; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Data; Development; Disease; Environment; Epitopes; Event; Excision; Exons; Failure; Funding; Generations; Genes; Genomics; Goals; Homeostasis; Immunofluorescence Microscopy; Individual; Injury; Knowledge; Label; Length; MAP4; Mediating; Microscopic; Microscopy; Microtubules; Modification; Molecular Analysis; Monitor; Mus; Muscle; Muscle Development; Muscle Weakness; Muscle function; Neuromuscular Junction; Nuclear; Organelles; Pathogenesis; Physiological; Protein Isoforms; Proteins; Publishing; RNA Splicing; Resolution; Role; Skeletal Muscle; Specificity; Stress; Striated Muscles; Structure; Tissues; Transport Process; Variant; Work; alpha Tubulin; c-myc Genes; calponin; density; fetal; flexor digitorum brevis; gene function; in vivo; insight; mRNA Precursor; postnatal; postnatal development; repaired; response; skeletal muscle differentiation; skeletal muscle weakness; transcriptome; zygote

Project Summary The long-term goal of this project is to determine the extent, regulatory mechanisms, and functional consequences of regulated pre-mRNA processing in skeletal muscle. A large number of genes express pre- mRNAs that undergo alternative splicing to produce conserved muscle-specific protein isoforms the functions of which are unknown. These isoforms often appear during late fetal or early postnatal development. Disruption of alternative splicing is a common feature of diseases affecting skeletal muscle, often involving reversion to fetal isoforms, yet little is known about the contributions of these changes to pathogenesis. An underlying hypothesis of this proposal is that a focus on genes with conserved fetal and adult protein isoforms, including adult isoforms that are muscle-specific, will discover not only previously unknown isoform-specific functions but also previously unknown gene functions critical for adult muscle homeostasis. To identify the functions of alternative splicing events in vivo we are using CRISPR-mediated removal of exons that undergo muscle- specific and/or postnatally regulated inclusion. In the first aim, we will determine the functions of a striated muscle-specific isoform of the Map4 microtubule-binding protein in which deletion of the muscle-specific exon significantly disrupts microtubule architecture in skeletal muscle myofibers and impacts muscle force generation. In the second aim we will determine the function of the skeletal muscle-specific Limch1 isoform, the absence of which also decreases skeletal muscle function in vivo. In the third aim we will use CRISPR- mediated introduction of epitope tags into endogenous genes to monitor the temporal and spatial details of the alternative splicing transitions and adult muscle-specific isoforms at the level of individual cells in vivo. This study is expected to identify previously unknown gene functions, increase understanding of adult skeletal muscle homeostasis and the impact of its disruption in disease.

项目摘要 该项目的长期目标是确定范围，监管机制和功能 骨骼肌中受调节前MRNA加工的后果。大量基因表达 经过替代剪接以产生保守的肌肉特异性蛋白质同工型的mRNA 其中未知。这些同工型通常在胎儿后期或早期产后发育中出现。破坏 替代剪接是影响骨骼肌的疾病的共同特征，通常涉及回归到 胎儿同工型，但对这些变化对发病机理的贡献知之甚少。基础 该提议的假设是关注具有保守胎儿和成人蛋白质同工型的基因，包括 具有特异性肌肉特异性的成年同工型，不仅会发现以前未知的同工型特异性功能，还会发现 以前未知的基因对成人肌肉稳态至关重要。确定功能 在体内替代剪接事件我们正在使用CRISPR介导的外显子去除肌肉 - 特定和/或产后调节的纳入。在第一个目标中，我们将确定横纹的功能 MAP4微管结合蛋白的肌肉特异性同工型，其中肌肉特异性外显子缺失 显着破坏骨骼肌肌纤维中的微管结构并影响肌肉力 一代。在第二个目标中，我们将确定骨骼肌肉特异性的Limch1同工型的功能， 缺失也降低了体内骨骼肌功能。在第三个目标中，我们将使用crispr- 介导的表位标签引入内源基因，以监测该基因的时间和空间细节 在体内单个细胞水平上的替代剪接跃迁和成年肌肉特异性同工型。这 预计研究将识别以前未知的基因功能，增加对成人骨骼的理解 肌肉稳态及其对疾病的破坏的影响。