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 biochemica...