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

描述(由申请人提供)：项目摘要：RNA剪接的调节是产生人类蛋白质组多样性和表型复杂性的主要机制。这种转录后调控机制在神经细胞中尤为突出，与剪接功能障碍相关的神经退行性疾病的数量不断增加，突显了它的生物学相关性。对存活运动神经元(SMN)蛋白的研究为解决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独特的机会。SMN水平降低会导致脊髓性肌萎缩症(SMA)--一种常见的以运动神经元变性为特征的遗传性神经肌肉疾病。SMN在组装小核核糖核蛋白(SnRNPs)方面具有成熟的功能，而SnRNPs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水平不均匀而不是均匀的下降，从而导致组织的SnRNP轮廓的改变。此外，在疾病的动物模型中，恢复正常的SnRNP水平与表型纠正是一致的。尽管有这些进展，但SNRNP生物发生中SMN功能缺陷如何选择性地影响运动神经元尚不清楚。本项目将调查我们的假设，即SMN功能是为了剪接调控目的而赋予不同类型的细胞独特的SnRNP图谱，以及SMN缺乏引发的这一过程中的变化会导致对运动神经元生物学至关重要的mRNAs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的SnRNP谱中的作用。在目标2和目标3中，我们将研究这些细胞类型特异的SnRNP谱与剪接调控的相关性。在目标2中，我们将使用微阵列分析和具有受调控的SMN下调的细胞模型系统来研究SMN耗尽对RNA剪接的影响。基于我们从SMN水平正常和降低的小鼠胚胎干细胞分化出大量运动神经元的能力，我们将在与SMA相关的细胞类型中识别受SMN缺陷影响的mRNAs。通过使用其他类型的有丝分裂后神经元以及来自SMA小鼠的初级运动神经元的比较分析，我们将定义其在运动神经元中的表达或选择性剪接受到选择性影响的一组mRNAs。为了建立SMN对SnRNP生物发生的调控和剪接调控之间的机制联系，目标3将分析依赖SMN的SnRNP谱变化和剪接变化之间的因果关系。最后，在目标4中，将通过敲除和过度表达实验研究上述选定基因和选择性剪接异构体在正常和SMN缺陷ES细胞来源运动神经元中的功能作用。这种方法应该确定其SMN依赖的表达或选择性剪接对运动神经元的生存和功能至关重要的mRNAs。

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.