Genetic analysis of FRG1 function in Drosophila

描述（由申请人提供）：面肩肱骨肌营养不良症（FSHD）是一种进行性衰弱综合征，是美国第三大最常见的营养不良类型，然而由于我们对其分子基础的了解不足，定向治疗的发展仍然受到阻碍。最近使用小鼠模型系统的进展表明，FRG1基因的过度表达足以引起许多FSHD症状。因此，减少FRG1的表达和/或功能是一种有希望的治疗干预途径。一些证据表明，FRG1与RNA剪接机制相关，当过表达时可能改变正常剪接，但FRG1的具体生化活性尚不清楚。在这个项目中，我们将利用果蝇中强大的遗传方法来确定FRG1发挥作用的分子途径。利用GAL4-UAS系统，我们将把FRG1的过表达特异性靶向成虫的间接飞行肌。这种肌肉的形态和功能很容易评估，因此它是遗传研究的理想目标。由于FSHD具有多种肌外作用，我们还将研究FRG1过表达对果蝇复眼的影响，复眼是一种高度有序的组织，经常被用于基因功能分析。利用这些组织中的过表达表型，我们将进行基因筛选，以确定修饰突变，逆转FRG1过度表达的有害影响。由于这种筛选是以无偏见的方式进行的，因此它们有可能识别出原本不会被考虑或调查的新的相互作用因素。由于之前没有特定的生化活性与FSHD相关，这些因子将为旨在治疗性抑制FRG1功能的策略提供新的靶点。在平行研究中，我们将利用已经在果蝇中开发的RNA剪接机制的广泛知识来询问FRG1是否是剪接体的必需组分。使用体外剪接系统，我们将确定剪接的具体步骤和剪接复合体的组装和FRG1起作用的功能。因此，这些研究将首次提供果蝇FRG1的遗传和生化分析。通过将果蝇作为FSHD的模型，这项工作将为未来的研究奠定基础，这些研究将详细了解FRG1的正常分子功能及其在肌肉中过表达的影响。因此，这些研究提供了揭示影响肌肉功能和疾病的新的生化途径的潜力。

DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is a progressively debilitating syndrome that is the nation's third most common type of dystrophy, yet the development of directed therapies remains hampered by our poor understanding of its molecular basis. Recent progress using a mouse model system suggests that overexpression of the FRG1 gene is sufficient to cause a number of FSHD symptoms. Thus a promising avenue for therapeutic intervention is the reduction of FRG1 expression and/or function. Several lines of evidence suggest that FRG1 associates with the RNA splicing machinery and may alter normal splicing when overexpressed, but the specific biochemical activity of FRG1 remains unknown. In this project we will utilize the powerful genetic approaches available in the fruitfly Drosophila melanogaster to identify molecular pathways through which FRG1 functions. Using the GAL4-UAS system we will target overexpression of FRG1 specifically to the indirect flight muscle of the adult fly. The morphology and function of this muscle are easily assessed and it therefore an ideal target for genetic studies. Because FSHD has a variety of extramuscular effects we will also examine the effects of FRG1 overexpression on the Drosophila compound eye, a highly ordered tissue that is frequently exploited for analysis of gene function. Using overexpression phenotypes in these tissues we will carry out genetic screens to identify modifier mutations that reverse the harmful effects of excess FRG1 expression. Because such screens are carried out in unbiased manner they offer the potential to identify novel interacting factors that would not otherwise have been considered or investigated. As no specific biochemical activity has previously been linked with FSHD, these factors will provide new targets for strategies aimed at therapeutic inhibition of FRG1 function. In parallel studies we will exploit the extensive knowledge of RNA splicing mechanisms already developed in Drosophila to ask whether FRG1 is a required component of the spliceosome. Using an in vitro splicing system we will identify the specific steps in splicing and splicing complex assembly and function where FRG1 acts. These studies will thus provide the first genetic and biochemical analysis of FRG1 in Drosophila. By developing Drosophila as a model for FSHD this work will form a basis for future studies that will elaborate a detailed understanding of both the normal molecular function of FRG1 and the effects of its overexpression in muscle. These studies thus offer the potential to uncover novel biochemical pathways that affect muscle function and disease.