The Role of RNA in Defining FUS Specificity and Activity in Phase Separation and Splicing

项目总结 融合于肉瘤(Fus)是一种丰富的核rna结合蛋白，有助于调节几乎所有水平的 RNA处理。FUS的活性取决于它与rna特异性结合的能力，然而它仍然未知。 鉴于它对各种细胞RNA序列的亲和力，它如何实现特异性。一个引人注目的例子 FUS的特异性在于它自己的前-mRNA，它只在外显子7附近结合来抑制外显子跳跃， 导致了胡说八道的腐烂和FUS的自动调节。在神经系统疾病中肌萎缩侧索硬化 硬化症(ALS)和额颞部痴呆(FTD)，FUS错误定位于细胞质，合并到 应力颗粒，随后形成病理性包裹体。ALS/FTD中的FUS错误调节导致 广泛的基因表达中断，包括FUS自动调节，导致FUS和FUS的过度表达 病原聚集物的放大。尽管RNA结合在生理和病理过程中处于核心地位 FUS的活性、FUS：RNA相互作用及其功能结果仍然知之甚少。这项建议 旨在研究多个尺度上的FUS：RNA相互作用，以更好地了解FUS RNA结合 偏好以及RNA序列和结构在定义FUS活性中的作用。目标1将测试FUS绑定到 一组广泛的合成和生物RNA结构，包括来自FUS mRNA的外显子7。FUS：RNA 将使用EMSA、荧光各向异性、单分子FRET和核磁共振来表征相互作用 光谱学。这一目标还将测试ALS相关的FUS突变对RNA结合的影响。目标2将 根据FUS很容易将相分离为液体这一事实，在相分离分析中测试这些RNA 以依赖RNA的方式释放液滴。不同的RNA和FUS变体如何影响大小、数量 FUS液滴的形状和流动性，以及这与它们在AIM中确定的分子相互作用的对应关系 将遵守%1。最后，目标3将测试神经母细胞瘤中FUS剪接活性的RNA依赖性和 使用基于RT-PCR微基因报告分析和实时单分子成像的神经细胞。 由于FUS自动调节与ALS/FTD相关，因此将特别关注FUS自动调节。总而言之，这些目标 将有助于揭示FUS特异性和活性的机制。拟议的研究将主要进行 在王久明博士的支持和共同指导下，在Sua Myong博士的实验室里。明博士将提供 擅长RNA-蛋白质生物物理学和单分子方法，而王博士擅长于 神经生物学和神经退行性变，特别是ALS/FTD。赞助商、合作者和 约翰霍普金斯大学的科学界申请者将接受指导、技术培训和访问 用于完成拟议项目所需的资源和专业知识。此外，提案还概述了 许多有计划的职业发展活动，包括指导、教学和科学交流。 总体而言，这项培训计划旨在为申请者的独立研究生涯做好准备。

PROJECT SUMMARY Fused in sarcoma (FUS) is an abundant nuclear RNA binding protein that helps regulate nearly every level of RNA processing. FUS activity depends on its ability to specifically bind RNA, however it remains unknown how it achieves specificity given its affinity for a wide variety of cellular RNA sequences. A striking example of FUS specificity is on its own pre-mRNA where it binds exclusively near exon 7 to repress exon skipping, leading to nonsense mediated decay and FUS autoregulation. In neurological diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), FUS mislocalizes to the cytoplasm, incorporates into stress granules, and subsequently forms pathological inclusions. FUS misregulation in ALS/FTD causes widespread disruption of gene expression including FUS autoregulation, leading to overexpression of FUS and amplification of pathogenic aggregates. Although RNA binding is central to the physiological and pathological activities of FUS, the FUS:RNA interaction and its functional outcome remain poorly understood. This proposal seeks to investigate the FUS:RNA interaction across multiple scales to better understand FUS RNA-binding preferences and the role of RNA sequence and structure in defining FUS activity. Aim 1 will test FUS binding to a broad set of synthetic and biological RNA constructs including exon 7 from FUS mRNA. The FUS:RNA interaction will be characterized using EMSA, fluorescence anisotropy, single molecule FRET and NMR spectroscopy. This aim will also test the impact of ALS-associated FUS mutations on RNA binding. Aim 2 will test these RNAs in phase separation assays based on the fact that FUS readily phase separates into liquid droplets in an RNA-dependent manner. How different RNAs and FUS variants affect the size, number, shape, and fluidity of FUS droplets and how this corresponds to their molecular interactions determined in aim 1 will be observed. Finally, aim 3 will test the RNA dependence of FUS splicing activity in neuroblastoma and neuronal cells using an RT-PCR based minigene reporter assay and real-time single molecule imaging. Special attention will be given to FUS autoregulation due to its relevance to ALS/FTD. Together, these aims will help uncover the mechanism of FUS specificity and activity. The proposed research will primarily be carried out in the lab of Dr. Sua Myong with support and co-mentorship from Dr. Jiou Wang. Dr. Myong will provide expertise in RNA-protein biophysics and single molecule methods while Dr. Wang has expertise in neurobiology and neurodegeneration, specifically ALS/FTD. Between the sponsors, collaborators, and scientific community at Johns Hopkins the applicant will receive the mentorship, technical training, and access to resources and expertise necessary to accomplish the proposed project. Additionally, the proposal outlines many planned activities for career development including mentorship, teaching, and scientific communication. Overall, this training plan has been designed to prepare the applicant for an independent research career.