Using Riboglow to Define RNP Interactions in Response to Environmental Stress

项目总结/摘要 RNA的适当定位对于RNA的适当功能、下游蛋白定位、细胞功能 有机体发育和有机体健康。特别令人感兴趣的是，将RNA适当隔离到 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ALS、阿尔茨海默氏症和痴呆症中涉及的蛋白质的成核点。期间 应激，应激颗粒（SG）在加工体（PB）时从翻译机器中隔离RNA 降解RNA。在某些应激条件下，SG和PB对接或相互作用，并且已经显示一种应激源 促进mRNA在两个RNP颗粒之间的转移。假设SG-PB对接和mRNA转移 作为压力下的额外调节措施。mRNA转移被假设为通过 与两种RNA结合蛋白TTP和CPEB 1的特异性相互作用。然而，这两种假设在很大程度上 未经测试为了确定健康或压力和疾病状态下的RNA定位，科学家使用RNA- 成像工具。让我们能够看到活细胞中单个RNA分子的工具具有特殊价值，因为生物学 是难以置信的异质性，并在真实的时间内对刺激做出反应。历史上用于监测mRNA的工具 RNP颗粒中的动力学使用许多荧光蛋白来可视化RNA。由于RNP颗粒主要形成于 通过蛋白质和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。使用小分子荧光剂来可视化RNA的工具可能更适合于 调查这个问题。Riboglow是由我的实验室，一个荧光工具实验室， RNA生物学实验室Riboglow是一种活细胞RNA成像工具，由两个主要部分组成： 附加到感兴趣的RNA上的适体和小分子荧光探针。Riboglow跑赢大盘 检测mRNA向应激颗粒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。 在这个提案中，我将1）优化和表征Riboglow用于活细胞中的单分子检测，2）定量 用Riboglow标记RNA对基本RNA生物学的任何影响，3）使用Riboglow建立一个列表， 引起SG-PB对接和mRNA转移的应激源，以及4）评估mRNA转移是 通过与TTP和CPEB 1的特定相互作用促进。我的研究将通过扩展单一的- 分子活细胞RNA成像工具箱的RNA生物学家和产生的基础知识的RNP 领域这将有助于进一步研究刺激反应中RNA的定位及其与细胞凋亡的关系 神经退行性疾病和环境压力之间的联系这个奖学金将为我提供 生化工具开发和优化高级培训，定量荧光显微镜， 生物学研究和用于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.