In vivo models of small RNP biogenesis and Spinal Muscular Atrophy

 描述（由申请人提供）：小核糖核蛋白（RNP）是生命三个王国中必不可少的细胞成分。事实上，真核基因的表达需要一个名副其实的星座的小非编码RNP参与生物体功能的多个方面。长期目标是了解控制小RNP的生物发生和功能的分子机制。作为剪接体的关键元件，Sm类小核（sn）RNP在转录后基因调控中是必不可少的。Sm类RNP颗粒的组装被认为是由运动神经元（SMN）蛋白复合物介导的，SMN蛋白复合物将Sm蛋白加载到snRNA上，形成核心RNP。了解这一过程对人类健康至关重要，因为人类SMN1突变会导致一种称为脊髓性肌萎缩症（SMA）的遗传疾病。每五十个无关的个体中就有一个是SMA的携带者，使这种疾病成为严重的健康问题。不幸的是，大多数SMA患者通常在幼儿期死亡。SMA是由SMN蛋白水平降低引起的，而SMN表达的完全丧失导致产前致死。虽然SMN1已被确定为SMA的突变基因，但该疾病的下游触发因素仍然是一个谜。新出现的证据表明，SMN具有额外的组织特异性功能，特别是在肌肉和神经元中。然而，对SMN如何执行其各种功能的分子理解缺失。因此，详细了解SMN复合物在小RNP代谢和神经肌肉发育中所起的作用是必不可少的。因此，本申请的主要目的是确定SMN和其他snRNP生物发生因子突变对动物体内生存力和发育的影响。为了实现这一目标，我们开发了果蝇作为模型系统。我们产生了一个等位基因系列的苍蝇表达SMN错义突变来自人类SMA患者。使用这个遗传平台，我们希望识别功能分离突变，这些突变将SMN的假定管家和组织特异性功能分离，使我们能够独立研究它们。我们将采用全基因组技术与分子遗传学和生物化学一起，以确定细胞途径和蛋白质结合伙伴，被破坏的SMA引起的点突变。由于已知参与snRNP生物发生的其他基因的突变可能表现为SMN功能障碍的方面，因此还提出了实验来鉴定由SMN丧失引起的剪接和基因表达中的snRNP依赖性与snRNP独立性变化。综合数据将阐明亚型SMN突变的分子，细胞和发育后果，并导致更好地了解脊髓性肌萎缩症。

 DESCRIPTION (provided by applicant): Small ribonucleoproteins (RNPs) are essential cellular components in all three kingdoms of life. Indeed, eukaryotic gene expression requires a veritable constellation of small non-coding RNPs that participate in multiple aspects of organismal function. The long-term goal is to understand the molecular mechanisms that govern the biogenesis and function of small RNPs. As key elements of the spliceosome, the Sm-class small nuclear (sn)RNPs are essential for post-transcriptional gene regulation. Assembly of Sm-class RNP particles is thought to be mediated by the Survival Motor Neuron (SMN) protein complex, which loads Sm proteins onto snRNAs, forming the core RNP. Understanding this process is important for human health, as mutations in human SMN1 result in a genetic disorder called Spinal Muscular Atrophy (SMA). One in fifty unrelated individuals is a carrier for SMA, making this disease a serious health concern. Unfortunately, most people with SMA typically die in early childhood. SMA is caused by reduced levels of SMN protein, whereas complete loss of SMN expression results in prenatal lethality. Although SMN1 has been identified as the mutant gene in SMA, the downstream trigger of the disease remains a mystery. Emerging evidence suggests that SMN has additional tissue-specific functions, especially in muscles and neurons. However, a molecular understanding of how SMN carries out its various functions is missing. Hence, detailed knowledge of the roles played by the SMN complex in small RNP metabolism and neuromuscular development is essential. Therefore, the major objective of this application is to determine the consequences of mutations in SMN and other snRNP biogenesis factors to animal viability and development in vivo. To address this objective we have developed Drosophila as a model system. We generated an allelic series of flies expressing SMN missense mutations derived from human SMA patients. Using this genetic platform, we expect to identify separation-of-function mutations that uncouple the putative housekeeping and tissue-specific functions of SMN, enabling us to study them independently. We will employ genome-wide techniques together with molecular genetics and biochemistry to identify cellular pathways and protein binding partners that are disrupted by SMA-causing point mutations. Because mutations in other genes known to be involved in snRNP biogenesis may phenocopy aspects of SMN dysfunction, experiments are also proposed to identify snRNP-dependent versus snRNP-independent changes in splicing and gene expression that result from loss of SMN. The combined data will elucidate the molecular, cellular and developmental consequences of hypomorphic SMN mutations, and lead to a better understanding of Spinal Muscular Atrophy.