Methylation of Sm proteins - Roles in snRNP Biogenesis and germline specification

描述(申请人提供)：SM类小核糖核蛋白(SnRNPs)是剪接体的核心成分，是体内剪接所必需的。此外，Sm蛋白似乎在生殖系的指定中起着更原始的作用。该项目的总体目标是了解Sm蛋白组装成SnRNPs以进行剪接或组装成特定的核糖核蛋白(RNPs)的机制，这些核糖核蛋白是决定生殖细胞命运所需的。在这两条看似不同的途径的结合点上，是PRMT5复合体。SnRNPs的生物发生是高度协调的，需要几个基本的辅助因素。特别是，细胞质SnRNP的组装依赖于两个异构体复合体，PRMT5复合体和SMN复合体。重要的是，降低SMN蛋白水平的突变与一种称为脊髓肌萎缩症(SMA)的神经退行性疾病相关。患有这种疾病的患者通常在儿童时期很早就去世了。SMA的分子病因尚不清楚，然而，SNRNP生物发生的扰动被认为是一个主要的致病因素。对SnRNP组装机制的详细了解将有助于揭示疾病过程。PRMT5复合体负责对称地对Sm蛋白质进行二甲基化，并通过这一活性被认为与SMN复合体合作，介导有效的SnRNP组装。然而，目前还缺乏对这一过程的体内检查。此外，PRMT5与BLIMP1相关，是小鼠生殖细胞发育所必需的。因此，PRMT5活性的中断可能会影响两条不同但可分离的途径--SNRNP的生物发生和生殖细胞的发育。为了从机制上深入了解这些过程，我们开发了利用果蝇作为模型系统的体内方案以及哺乳动物细胞培养技术。目的1探讨Sm蛋白甲基化对SnRNP生物发生的要求。由于SMN与甲基化的Sm蛋白具有更高的亲和力，这种修饰可能作为SMA表型的修饰物。具体地说，哺乳动物中缺乏Sm蛋白甲基化可能是SMA的表型。或者，增加甲基化可能会减轻疾病的严重程度。目的2研究生殖细胞规范中对DART的要求，并说明Sm蛋白在这一过程中所起的特殊作用。摘要：脊髓性肌萎缩症是一种常见的遗传性疾病，会导致儿童早期死亡。虽然导致这种疾病的基因已经确定，但最终导致疾病病理的机制尚不清楚。为了开发有效的治疗方法，对疾病基因产物的分子理解是必不可少的。

DESCRIPTION (provided by applicant): Sm class small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome and are required for splicing in vivo. In addition, Sm proteins appear to have a more ancestral role in specification of the germline. The overall goal of this project is to understand the mechanism by which Sm proteins are assembled into snRNPs for the purpose of splicing or into specialized ribonucleoproteins (RNPs) required to establish germ cell fate. At the nexus of these two seemingly disparate pathways is the PRMT5 complex. The biogenesis of snRNPs is highly orchestrated, requiring several essential co-factors. In particular, cytoplasmic snRNP assembly relies on two heteromeric complexes, the PRMT5 complex and the SMN complex. Importantly, mutations that reduce the level of SMN protein are correlated with a neurodegenerative disease known as Spinal Muscular Atrophy (SMA). Patients with the disease often die very early in childhood. The molecular etiology of SMA is unknown, however, perturbation of snRNP biogenesis is thought to be a major contributing factor. A detailed understanding of the mechanism of snRNP assembly will shed light on the disease process. The PRMT5 complex is responsible for symmetrically dimethylating Sm proteins, and by virtue of this activity, is thought to cooperate with the SMN complex in mediating efficient snRNP assembly. However, an in vivo examination of this process is currently lacking. Furthermore, PRMT5 associates with BLIMP1 and is required for germ cell development in the mouse. Disruption of PRMT5 activity could thus impinge on two distinct but separable pathways - snRNP biogenesis and germ cell development. To gain mechanistic insight into these processes we have developed in vivo protocols making use of Drosophila as a model system as well as mammalian cell culture techniques. Aim 1 explores the requirement for Sm protein methylation in snRNP biogenesis. Since SMN binds with a higher affinity to methylated Sm proteins, this modification may act as a modifier of the SMA phenotype. Specifically, lack of Sm protein methylation in mammals may phenocopy SMA. Alternatively, increased methylation may ameliorate the disease severity. Aim 2 examines the requirement for DartS, the fly ortholog of PRMT5, in germ cell specification and addresses the specific role played by Sm proteins in this process. Lay summary: Spinal Muscular Atrophy, a common genetic disease, results in very early childhood mortality. Whereas the gene responsible for the disease has been identified, the mechanism that ultimately results in the disease pathology is unknown. In order to develop effective therapies, a molecular understanding of the disease gene product is essential.