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

RNA 在定义 FUS 特异性以及相分离和剪接活性中的作用

• 批准号: 10400579
• 负责人: Laura R. Ganser
• 金额: $ 2.51万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2022-11-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10400579
• 关键词: Affect; Affinity; Alternative Splicing; Amyotrophic Lateral Sclerosis; Attention; Binding; Binding Sites; Biological; Biological Assay; Biophysics; Cell Line; Cells; Communication; Communities; Complex; Cytoplasm; Cytoplasmic Granules; Dependence; Development; Disease; EMSA; Educational process of instructing; Exons; Fluorescence Anisotropy; Gene Expression; Genetic Transcription; Homeostasis; Human; Link; Liquid substance; Mentorship; Messenger RNA; Methods; MicroRNAs; Molecular; Molecular Conformation; Motor Neurons; Mus; Mutation; NMR Spectroscopy; Nerve Degeneration; Neurobiology; Neuroblastoma; Neurodegenerative Disorders; Neurons; Nonsense-Mediated Decay; Nuclear RNA; Pathogenicity; Pathologic; Phase; Physiological; Polyadenylation; Positioning Attribute; Process; Property; Proteins; RNA; RNA Binding; RNA Folding; RNA Probes; RNA Processing; RNA Recognition Motif; RNA Sequences; RNA Splicing; RNA Transport; RNA-Binding Proteins; Regulation; Reporter; Reporting; Research; Resources; Reverse Transcriptase Polymerase Chain Reaction; Role; Shapes; Specificity; Structure; Structure-Activity Relationship; System; Testing; Time; Training; Variant; Work; base; career; career development; design; exon skipping; experimental study; fluidity; frontotemporal lobar dementia-amyotrophic lateral sclerosis; functional outcomes; fused in sarcoma; in vivo; induced pluripotent stem cell; mRNA Precursor; molecular imaging; mutant; nervous system disorder; neuroblastoma cell; overexpression; predictive modeling; predictive test; preference; recruit; single molecule; single-molecule FRET; stem cells; stoichiometry; stress granule

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.

项目摘要 融合在肉瘤（FUS）是一种丰富的核RNA结合蛋白，有助于调节几乎每一个水平的肿瘤， RNA加工FUS的活性取决于其特异性结合RNA的能力，但它仍然未知 它是如何达到特异性的，因为它对各种各样的细胞RNA序列都有亲和力。一个突出的例子 FUS特异性的一部分是在其自身的前体mRNA上，其中它仅在外显子7附近结合以抑制外显子跳跃， 导致无义介导的衰变和FUS自动调节。在神经系统疾病肌萎缩侧索硬化 硬化症（ALS）和额颞叶痴呆（FTD）中，FUS错误定位于细胞质，并入细胞质中， 应力颗粒，随后形成病理性内含物。ALS/FTD病因中的FUS失调 基因表达的广泛破坏，包括FUS自身调节，导致FUS的过度表达， 致病性聚集体的扩增。虽然RNA结合是生理和病理的核心， 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FUS：RNA在多个尺度上的相互作用，以更好地了解FUS RNA结合 偏好和RNA序列和结构在定义FUS活性中的作用。目标1将测试FUS与 一组广泛的合成和生物RNA构建体，包括来自FUS mRNA的外显子7。标签：RNA 将使用EMSA、荧光各向异性、单分子FRET和NMR来表征相互作用 谱该目的还将测试ALS相关FUS突变对RNA结合的影响。目标2将 基于FUS容易相分离成液体的事实，在相分离测定中测试这些RNA 以RNA依赖的方式。不同的RNA和FUS变体如何影响细胞的大小，数量， FUS液滴的形状和流动性，以及这如何对应于目标中确定的分子相互作用 1将被观察。最后，目标3将测试成神经细胞瘤中FUS剪接活性的RNA依赖性， 使用基于RT-PCR的小基因报告基因测定和实时单分子成像来检测神经元细胞。 由于FUS与ALS/FTD的相关性，将特别关注FUS自动调节。总之，这些目标 将有助于揭示FUS特异性和活性的机制。该研究将主要用于 在Sua Myong博士的实验室里，在Jiou Wang博士的支持和共同指导下，明医生会提供 在RNA-蛋白质生物物理学和单分子方法方面的专业知识，而王博士在 神经生物学和神经变性，特别是ALS/FTD。在赞助商、合作者和 科学界在约翰霍普金斯，申请人将获得指导，技术培训，并获得 为完成拟议项目所需的资源和专业知识。此外，该提案还概述了 许多计划的职业发展活动，包括指导，教学和科学交流。 总体而言，该培训计划旨在为申请人的独立研究生涯做好准备。