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

项目概述RNA剪接调控是人类蛋白质组多样性和表型复杂性产生的主要机制。这种转录后调节机制在神经元细胞中尤为突出，越来越多的与剪接功能障碍相关的神经退行性疾病强调了其生物学相关性。对存活运动神经元（SMN）蛋白的研究提供了一个独特的机会来解决剪接调节的基本生物学和RNA功能障碍在人类疾病中的作用。SMN水平降低会导致脊髓性肌萎缩症（SMA）——一种常见的遗传性神经肌肉疾病，以运动神经元变性为特征。SMN在小核核糖核蛋白（snRNPs）的组装中具有良好的功能，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严重程度相关，并导致snRNP水平不均匀而非均匀下降，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的表型校正相吻合。尽管有这些进展，snRNP生物发生中SMN功能缺陷是如何选择性影响运动神经元的尚不清楚。该项目将研究我们的假设，即SMN的功能是赋予不同细胞类型独特的snRNP谱，以达到剪接调节的目的，并且SMN缺乏引发的这一过程的改变导致对运动神经元生物学至关重要的mrna的剪接缺陷。基于我们的初步研究结果，在Aim 1中，我们将分析SMN在不同细胞类型和发育过程中的小鼠组织中建立不同snRNP谱的作用。这些细胞类型特异性snRNP谱的剪接调节的相关性将在Aims 2和Aims 3中进行研究。在Aim 2中，我们将使用微阵列分析和SMN下调调控的细胞模型系统来研究SMN缺失对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中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缺乏影响的mrna。通过对其他类型的有丝分裂后神经元和来自SMA小鼠的初级运动神经元的比较分析，我们将定义一组mrna，其表达或选择性剪接在运动神经元中受到选择性影响。为了建立SMN控制snRNP生物发生与剪接调节之间的机制联系，我们将在Aim 3中分析snRNP谱中SMN依赖性改变与剪接变化之间的因果关系。最后，在Aim 4中，将在正常和缺SMN的ES细胞源性运动神经元中使用敲低和过表达实验，研究上述选定基因和备选剪接异构体的功能作用。这种方法应该识别出smn依赖性表达或选择性剪接对运动神经元存活和功能至关重要的mrna。

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.