Transcriptome processing networks in skeletal muscle: mechanisms and functions

项目摘要 该项目的长期目标是确定范围、监管机制和功能 骨骼肌中受调控的前信使核糖核酸加工的后果。大量基因表达Pre-Pre 经过选择性剪接以产生保守的肌肉特异性蛋白的mRNAs具有不同的功能 其中有哪些是未知的。这些异构体通常出现在胎儿晚期或出生后早期。颠覆 选择性剪接的缺失是影响骨骼肌的疾病的共同特征，通常涉及回复到 胎儿的异构体，但对这些变化在发病机制中的作用知之甚少。一个潜在的 这一建议的假设是，重点关注具有保守的胎儿和成人蛋白亚型的基因，包括 肌肉特异的成体亚型不仅会发现以前未知的亚型特有的功能，而且 此外，先前未知的基因功能对成人肌肉的动态平衡至关重要。确定…的功能 体内的选择性剪接事件我们正在使用CRISPR介导的外显子移除经历肌肉- 特定的和/或出生后受监管的纳入。在第一个目标中，我们将确定条纹的功能 肌肉特异性微管结合蛋白的亚型，其中肌肉特异性外显子缺失 显著破坏骨骼肌肌纤维的微管结构并影响肌力 一代。在第二个目的中，我们将确定骨骼肌特异性Limch1亚型的功能， 它的缺失也会降低活体骨骼肌的功能。在第三个目标中，我们将使用CRISPR 将表位标签介导性导入内源基因以监测基因的时间和空间细节 体内单个细胞水平的选择性剪接转变和成体肌肉特异性亚型。这 这项研究有望确定以前未知的基因功能，增加对成人骨骼的了解 肌肉动态平衡及其破坏在疾病中的影响。

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.