Using Riboglow to Define RNP Interactions in Response to Environmental Stress

项目摘要/摘要 RNA的正确定位对于正确的RNA功能、下游蛋白质定位、细胞功能、 生物体发育和生物体健康。特别值得注意的是，RNA被适当地隔离到 核糖核蛋白(RNP)颗粒帮助细胞有效地应对压力，而持续的RNA定位 RNP颗粒可以作为与ALS、阿尔茨海默氏症和痴呆症有关的蛋白质的成核点。在.期间 应激，应激颗粒(SGS)在加工过程中从翻译机械中隔离RNA 降解核糖核酸。SGS和PBS在特定的压力条件下对接或相互作用，其中一个压力源已被证明 促进两种RNP颗粒间的信使核糖核酸转移。假设SG-PB对接和mRNA转移 在压力下作为一种额外的调节措施。假设信使核糖核酸的转移是由 与两种RNA结合蛋白TTP和CPEB1的特异性相互作用。然而，这两种假设在很大程度上仍然存在。 未经测试。为了确定在健康或应激状态和疾病状态下的RNA定位，科学家使用RNA- 成像工具。自生物学以来，使我们能够看到活细胞中的单分子RNA的工具具有特殊的价值 是令人难以置信的异质性，并对刺激做出实时反应。历史上用来监测mRNA的工具 RNP颗粒中的动力学使用许多荧光蛋白来显示RNA。由于RNP颗粒主要形成 由于蛋白质和RNA之间的非特异性相互作用，这个工具可能会损害我们的一些结论 关于结果数据。使用小分子荧光来可视化RNA的工具可能更适合于 调查这个问题。Riboglow是我的实验室、一个荧光工具实验室和 一个核糖核酸生物实验室。Riboglow是一种活细胞RNA成像工具，由两个主要部分组成：RNA 附加在目标RNA和小分子荧光探针上的适体。Riboglow表现优异 检测应激颗粒mRNA募集的金标准工具和初步数据显示 Riboglow可以实现活细胞中mRNA的单分子检测。然而，Riboglow仍然是一个新工具。 在这个方案中，我将1)优化和表征核辉光在活细胞中的单分子检测，2)量化 使用Riboglow标记RNA对基本RNA生物学的任何影响，3)使用Riboglow建立 导致SG-PB对接和信使核糖核酸转移的应激源，以及4)评估信使核糖核酸转移是 通过与TTP和CPEB1的特定相互作用而促进。我的研究将使科学受益于扩大单一的- 为RNA生物学家提供分子活细胞RNA成像工具箱，并为RNP提供基础知识 菲尔德。这将有助于进一步研究RNA对刺激反应的定位及其可能的关系 神经退行性疾病和环境应激源之间的关系。这笔奖学金将为我提供 生物化学工具开发和优化方面的高级培训，定量荧光显微镜 生物学研究，以及图像分析的数据科学。这套跨学科的技能将使我能够 实现我的职业目标，并对生物医学领域产生积极影响。

PROJECT SUMMARY/ABSTRACT Proper localization of RNA is critical for proper RNA function, downstream protein localization, cell function, organism development, and organism health. Of particular interest, proper sequestration of RNA into ribonucleoprotein (RNP) granules helps cells respond efficiently to stress, while persistent RNA localization to RNP granules may serve as nucleation points for proteins implicated in ALS, Alzheimer’s, and dementia. During stress, stress granules (SGs) sequester RNA from translational machinery while processing bodies (PBs) degrade RNA. SGs and PBs dock, or interact, in certain stress conditions and one stressor has been shown to promote transfer of mRNA between the two RNP granules. SG-PB docking and mRNA transfer is hypothesized to function as an additional regulatory measure during stress. mRNA transfer is hypothesized to be facilitated by specific interactions with two RNA-binding proteins, TTP and CPEB1. However, both hypotheses remain largely untested. In order to identify RNA localization in healthy or stressed and disease states, scientists use RNA- imaging tools. Tools that allow us to see single molecules of RNA in live cells are of particular value since biology is incredibly heterogenous and responds to stimuli in real time. The tool historically used to monitor mRNA dynamics in RNP granules uses many fluorescent proteins to visualize RNA. Since RNP granules form largely by nonspecific interactions between proteins and RNAs, this tool may compromise some of our conclusions about the resultant data. A tool that uses small molecule fluors to visualize RNA may be better suited to investigate this question. Riboglow was developed as a collaboration between my lab, a fluorescent tool lab, and an RNA-biology lab. Riboglow is a live-cell RNA-imaging tool that is comprised of two main parts: an RNA aptamer that is appended to an RNA of interest and a small-molecule fluorescent probe. Riboglow outperformed the gold-standard tool at detecting mRNA recruitment to stress granules and preliminary data shows that Riboglow can achieve single-molecule detection of mRNA in live cells. However, Riboglow remains a new tool. In this proposal, I will 1) optimize and characterize Riboglow for single-molecule detection in live cells, 2) quantify any effects that tagging an RNA with Riboglow has on basic RNA biology, 3) use Riboglow to establish a list of stressors that cause SG-PB docking and mRNA transfer, and 4) evaluate the hypothesis that mRNA transfer is facilitated by specific interactions with TTP and CPEB1. My research will benefit science by expanding the single- molecule live-cell RNA-imaging toolbox for RNA biologists and by yielding foundational knowledge for the RNP field. This will facilitate further studies on RNA localization in response to stimuli and the potential relationship between neurodegenerative diseases and environmental stressors. This fellowship will provide me with advanced training in biochemical tool development and optimization, quantitative fluorescence microscopy for biological studies, and data science for image analysis. This interdisciplinary set of skills will position me to achieve my career goals and positively impact the biomedical field.