SMN Control of snRNP Biogenesis: Role in RNA Splicing and Motor Neuron Survival

描述（由申请人提供）：项目概要RNA剪接调控是产生人类蛋白质组多样性和表型复杂性的主要机制。这种转录后调节机制在神经元细胞中特别突出，并且与剪接功能障碍相关的神经退行性疾病的数量越来越多，强调了其生物学相关性。运动神经元存活蛋白的研究为阐明剪接调控的基础生物学和RNA功能障碍在人类疾病中的作用提供了一个独特的机会。SMN水平降低导致脊髓性肌萎缩症（SMA）-一种常见的遗传性神经肌肉疾病，其特征在于运动神经元变性。SMN在小核核糖核蛋白（snRNP）的组装中具有公认的功能，snRNP是剪接机制的重要组成部分。在SMA小鼠中，snRNP组装受损的程度与疾病的严重程度相关，并导致snRNP水平的不均匀而非均匀降低，从而导致组织snRNP谱的改变。此外，恢复正常snRNP水平与疾病动物模型中的表型校正一致。尽管有这些进展，但snRNP生物发生中SMN功能缺陷如何选择性影响运动神经元尚不清楚。该项目将研究我们的假设，即SMN的功能赋予不同的细胞类型以独特的snRNP轮廓，用于剪接调节的目的，并且SMN缺陷引发的这一过程中的改变会导致运动神经元生物学关键mRNA的剪接缺陷。在我们初步研究结果的基础上，在目标1中，我们将分析SMN在不同细胞类型以及发育过程中小鼠组织中建立不同snRNP谱的作用。将在目的2和3中研究这些细胞类型特异性snRNP谱的剪接调节的相关性。在目标2中，我们将使用微阵列分析和细胞模型系统与SMN的调控敲低研究SMN耗竭对RNA剪接的后果。基于我们产生大量从具有正常和降低水平的SMN的小鼠胚胎干（ES）细胞分化的运动神经元的能力，我们将鉴定与SMA相关的细胞类型中受SMN缺乏影响的mRNA。通过使用其他类型的有丝分裂后神经元以及SMA小鼠的初级运动神经元的比较分析，我们将定义其表达或选择性剪接在运动神经元中选择性受影响的mRNA的集合。为了建立SMN控制snRNP生物发生和剪接调控之间的机制联系，将在目的3中分析snRNP谱中SMN依赖性改变和剪接变化之间的因果关系。最后，在目的4中，将在正常和SMN缺陷型ES细胞衍生的运动神经元中使用敲低和过表达实验来研究上述鉴定的所选基因和选择性剪接同种型的功能作用。这种方法应该确定的mRNA的SMN依赖性表达或选择性剪接是运动神经元的生存和功能的关键。 公共卫生相关性：了解RNA加工的机制和调控的生物医学相关性通过与RNA代谢缺陷相关的人类遗传性疾病的日益增长的列表而突出。该项目旨在确定脊髓性肌萎缩症（SMA）蛋白在基因表达的转录后控制中的正常作用，并确定其表达改变可能导致SMN缺陷运动神经元变性的基因，SMA中选择性影响的神经元细胞。这些研究应该提供深入了解RNA调控的基本机制和SMA发病机制的分子缺陷，并有可能为治疗开发确定新的候选靶点。

DESCRIPTION (provided by applicant): Project Summary Regulation of RNA splicing is the primary mechanism responsible for generating the proteome diversity and phenotypic complexity of humans. This post-transcriptional regulatory mechanism is particularly prominent in neuronal cells and the increasing number of neurodegenerative disorders that are associated with splicing dysfunction underscores its biological relevance. The study of the survival motor neuron (SMN) protein provides a unique opportunity to address the basic biology of splicing regulation and the role of RNA dysfunction in human disease. Reduced SMN levels cause spinal muscular atrophy (SMA)-a common inherited neuromuscular disorder characterized by motor neuron degeneration. SMN has a well-established function in the assembly of small nuclear ribonucleoproteins (snRNPs), which are the essential components of the splicing machinery. In SMA mice, the degree of snRNP assembly impairment correlates with disease severity and causes an uneven rather than uniform decrease in the levels of snRNPs, resulting in the alteration of the snRNP profile of tissues. Moreover, restoration of normal snRNP levels coincides with phenotypic correction in animal models of disease. Despite these advances, how defective SMN function in snRNP biogenesis selectively affects motor neurons is unknown. This project will investigate our hypothesis that SMN functions to endow distinct cell types with unique snRNP profiles for the purpose of splicing regulation and that alterations in this process triggered by SMN deficiency cause splicing defects in mRNAs critical for motor neuron biology. Building on the results of our preliminary studies, in Aim 1 we will analyze SMN role in establishing distinct snRNP profiles in different cell types as well as mouse tissues during development. The relevance for splicing regulation of these cell type-specific snRNP profiles will be studied in Aims 2 and 3. In Aim 2, we will investigate the consequences of SMN depletion on RNA splicing using microarray analyses and cellular model systems with regulated knockdown of SMN. Based on our ability to generate large numbers of motor neurons differentiated from mouse embryonic stem (ES) cells with normal and reduced levels of SMN, we will identify mRNAs affected by SMN deficiency in the cell type relevant to SMA. Through comparative analyses using other types of post-mitotic neurons as well as primary motor neurons from SMA mice, we will define the set of mRNAs whose expression or alternative splicing is selectively affected in motor neurons. In order to establish a mechanistic link between SMN control of snRNP biogenesis and splicing regulation, the cause-effect relationship between SMN-dependent alterations in the snRNP profile and splicing changes will be analyzed in Aim 3. Finally, in Aim 4, the functional role of selected genes and alternative splicing isoforms identified above will be studied using knockdown and over-expression experiments in both normal and SMN- deficient ES cell-derived motor neurons. This approach should identify mRNAs whose SMN-dependent expression or alternative splicing is critical for motor neuron survival and function. PUBLIC HEALTH RELEVANCE: Project Narrative The biomedical relevance of understanding the mechanisms and regulation of RNA processing is highlighted by the growing list of human genetic disorders associated with defects in RNA metabolism. This project is designed to define the normal role of the spinal muscular atrophy (SMA) protein in the post-transcriptional control of gene expression as well as to identify genes whose altered expression may contribute to degeneration of SMN-deficient motor neurons, the neuronal cells selectively affected in SMA. These studies should provide insights into the basic mechanisms of RNA regulation and the molecular defects underlying SMA pathogenesis with the potential of identifying new candidate targets for therapeutic development.