Elucidating the Mechanism in the Regulation of RNA-binding Protein Phase Separation

摘要： RNA结合蛋白(RBP)的液-液分离是一种最近被认可的细胞内分离方法 区划是各种无膜细胞器的生物发生的基础。尽管有明确的生物学意义 实用、失调的RNA结合蛋白(RBP)相分离导致蛋白质聚集和纤维形成 形成，这是许多神经退行性疾病的关键病理特征。中心假说 我的工作和我的实验室的驱动力是，逆转反常相变和聚集是一种 与这些致命疾病作斗争的潜在途径。事实上，我们和其他人已经确定，核进口 KAPβ2等受体可逆转蛋白异常相变和上调KAPβ2的表达 可以抑制疾病相关的RNA结合蛋白引起的神经退行性变。因此，NIR和其他 蛋白相变的调节剂可以在治疗上被用来恢复正常的功能 无膜细胞器和减轻由异常相变引起的蛋白毒性。然而，有几个关键 必须填补有关相变调节的知识空白，为发展这方面的工作奠定基础 治疗策略。因此，我的研究计划的长期目标是实现更全面的 了解蛋白质相分离的调节机制，并利用我们对 发展具有治疗潜力的策略以逆转异常引起的蛋白毒性的阶段调节 相变。我试图阐明蛋白质相分离调节的分子机制，两者 在健康和疾病条件下。 我们建议在未来五年重点关注三个主题：(1)定义KAPβ2的机制 逆转蛋白质的异常相变。(2)界定KAPβ2和其他核进口的范围和专用性 受体在调节活细胞的相分离中的作用。(3)根据我们的初步结果， 具有特定序列的RNA寡核苷酸具有不同的调节RNA结合蛋白相的活性 分离，我们将确定RNA在调节RBP相分离和发育中的序列空间 基于单分子荧光的创新生物物理方法研究RNA的作用机制 功能。我们的工作将极大地促进我们对阶段调控机制的理解 细胞中的分离，以及这一调节的破坏如何导致疾病状况。此外，这项工作 将为制定战略以增强KAPβ2的活性以及基于RNA2奠定基础 可以缓解毒性异常相变的寡核苷酸，这两者都为开发 恢复细胞内健康蛋白质相的创新疗法。

Abstract: Liquid–liquid phase separation of RNA-binding proteins (RBP) is a recently appreciated means of intracellular compartmentalization underpins the biogenesis of diverse membraneless organelles. Despite clear biological utility, dysregulated phase separation of RNA-binding protein (RBP) leads to protein aggregation and fibrils formation, which are key pathological features of numerous neurodegenerative diseases. The central hypothesis of my work, and the driving force of my lab, is that reversing aberrant phase transition and aggregation is a potential avenue to combat these fatal diseases. In fact, we and others have established that nuclear import receptors (NIRs) such as Kapβ2 can reverse protein aberrant phase transition and elevated Kapβ2 expression can suppress neurodegeneration caused by disease-linked RNA-binding proteins. Thus, NIRs and other regulators of protein phase transition could be leveraged therapeutically to restore normal function of membraneless organelles and mitigate proteotoxicity caused by aberrant phase transition. However, several key knowledge gaps regarding the regulation of phase transition must be filled, to lay the foundation to develop such therapeutic strategies. Thus the long-term goal of my research program is to achieve a more comprehensive understanding of the regulatory mechanism of protein phase separation and leverage our understanding of phase regulation to develop strategies with therapeutic potential to reverse proteotoxicity induced by aberrant phase transition. I seek to elucidate the molecular mechanism of the regulation of protein phase separation, both in health and disease conditions. We propose to focus on three main themes over the next five years: (1) Define the mechanism of how Kapβ2 reverses protein aberrant phase transition. (2) Define the scope and specificity of Kapβ2 and other nuclear import receptors in their function in regulating phase separation in live cells. (3) Based on our preliminary results that RNA oligonucleotides with specific sequences have diverse activities in regulating RNA-binding protein phase separation, we will identify the sequence space of RNA in regulating RBP phase separation and develop innovative single-molecule fluorescence-based biophysical methods to understand the mechanism of RNA’s function. Our work will significantly contribute to our understanding of the regulatory mechanism of phase separation in cells and how the breakdown of this regulation leads to disease conditions. Furthermore, this work will set the stage for developing strategies to enhance the activity of Kapβ2, as well as RNA-based oligonucleotides that can mitigate toxic aberrant phase transition, both of which provide the basis for developing innovative therapeutics to restore the healthy protein phase in cells.