The role of the non-coding RNAs Malat1 and 7SK in motoneuron disease

非编码 RNA Malat1 和 7SK 在运动神经元疾病中的作用

• 批准号: 255331586
• 负责人: Dr. Michael Briese
• 金额: --
• 依托单位: Institut für Klinische Neurobiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/255331586?language=en
• 关键词: role; non; coding; RNAs; Malat1

Non-coding RNAs are emerging as key regulators of cellular functions. Whilst the number of identified non-coding RNAs has increased substantially over the last years their individual role in specialized cells has remained relatively elusive. Highly polarized cells such as neurons might utilize non-coding RNAs to establish and maintain their often extensive axonal processes. Additionally, the role of non-coding RNAs in neurodegenerative diseases is not well-understood. In the proposed project we would like to investigate the roles of the non-coding RNAs 7SK and Malat1 in spinal muscular atrophy (SMA), one of the most common genetic causes of infant lethality. In SMA, deficiency of the Survival Motor Neuron (SMN) protein causes degeneration of spinal motoneurons leading to atrophy of associated muscles. We previously identified the RNA-binding protein hnRNP R as an interactor of SMN. During the first SPP 1738 funding period we characterized the RNA interactome of hnRNP R in motoneurons by iCLIP and found the non-coding RNA 7SK as the top candidate. We detected hnRNP R/7SK complexes in the cytosol of motoneurons including their axons and found that knockdown of either hnRNP R or 7SK reduced axon outgrowth accompanied by similar transcriptome alterations in the axons and somata. In the proposed project we would like to expand upon these findings and investigate the functions of 7SK and hnRNP R in vivo. For this purpose we would like to generate a 7SK knockout mouse and characterize it using cell biology techniques and transcriptome analyses. Additionally, we would like to characterize a newly generated hnRNP R knockout mouse and determine whether similar defects occur. In addition to 7SK, hnRNP R also interacts with Malat1, an abundant nuclear long non-coding RNA. Our preliminary data show that Malat1 is upregulated in Smn-deficient motoneurons and that nuclear hnRNP R levels are increased and cytosolic levels are decreased in these cells. These data suggest that subcellular hnRNP R/7SK complexes might be disturbed in SMA in a Malat1-dependent manner. Therefore, we would like to investigate the composition of cytosolic hnRNP R/7SK complexes by proteome analysis and determine whether these complexes are altered in SMA motoneurons. Finally, we will determine whether upregulation of Malat1 as seen in SMA causes such alterations of hnRNP R/7SK complexes and whether reduction of Malat1 can restore these defects. We anticipate that the techniques used in this project, such as primary motoneuron culture, and functional and histopathological analyses of mouse models of SMA will be of interest to other members of the SPP 1738. Taken together, the results generated in this project will help to further understand the functions of 7SK, Malat1 and hnRNP R in motoneurons and their roles in the pathomechanism of SMA.

非编码rna正在成为细胞功能的关键调节因子。虽然鉴定的非编码rna的数量在过去几年中大幅增加，但它们在特化细胞中的个体作用仍然相对难以捉摸。高度极化的细胞如神经元可能利用非编码rna来建立和维持其广泛的轴突过程。此外，非编码rna在神经退行性疾病中的作用尚不清楚。在这个项目中，我们希望研究非编码rna 7SK和Malat1在脊髓性肌萎缩症（SMA）中的作用，这是婴儿死亡最常见的遗传原因之一。在SMA中，存活运动神经元（SMN）蛋白的缺乏导致脊髓运动神经元变性，导致相关肌肉萎缩。我们之前发现rna结合蛋白hnRNP R是SMN的相互作用物。在spp1738资助的第一个时期，我们用iCLIP表征了hnRNP R在运动神经元中的RNA相互作用，并发现非编码RNA 7SK是首选候选。我们在包括轴突在内的运动神经元的细胞质中检测了hnRNP R/7SK复合物，发现hnRNP R或7SK的敲低都会减少轴突的生长，并伴随着轴突和体细胞中类似的转录组改变。在拟议的项目中，我们希望扩展这些发现，并研究7SK和hnRNP R在体内的功能。为此，我们希望产生一个7SK基因敲除小鼠，并利用细胞生物学技术和转录组分析对其进行表征。此外，我们想表征新产生的hnRNP基因敲除小鼠，并确定是否发生类似的缺陷。除了7SK外，hnRNP R还与Malat1相互作用，Malat1是一种丰富的核长链非编码RNA。我们的初步数据显示，在缺smn的运动神经元中，Malat1表达上调，细胞核hnRNP水平升高，胞浆水平降低。这些数据表明亚细胞hnRNP R/7SK复合物可能以malat1依赖的方式在SMA中受到干扰。因此，我们希望通过蛋白质组学分析来研究胞质hnRNP R/7SK复合物的组成，并确定这些复合物在SMA运动神经元中是否发生改变。最后，我们将确定SMA中Malat1的上调是否会导致hnRNP R/7SK复合物的改变，以及Malat1的减少是否可以恢复这些缺陷。我们期望在这个项目中使用的技术，如原代运动神经元培养，以及小鼠SMA模型的功能和组织病理学分析，将对spp1738的其他成员感兴趣。综上所述，本项目的研究结果将有助于进一步了解7SK、Malat1和hnRNP R在运动神经元中的功能及其在SMA发病机制中的作用。