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Monitoring RNA restructuring and translation in real time using a human cell-free extract system

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Here I adapted a fluorescent-based helicase assay to monitor RNA restructuring in a high-fidelity translation system using HeLa cell free extracts. In Chapter 1, I describe the method of how to successfully make large quantities of robust translation competent cell-free extracts from HeLa S3 cells, a mammalian suspension cell line. These extracts form the foundation for the remaining work outlined in this dissertation. In Chapter 2 I address how I redesigned the method to demonstrate the physiological implications of these RNA restructuring events. I discuss both engineering of the dual-assay mRNA reporter, and optimization of assay conditions for recapitulating faithful translational regulation and RNA restructuring in the cell free extracts. Moreover, I explore the contribution of the eIF4F complex to the unwinding activity, greatly extending previous work from our lab using a purified reconstituted system. Chapter 3 outlines the method by which I generated factor-dependent stable cell lines, using Invitrogen’s Flp-in™ T-REx™ system, and adapt these cell lines to liquid suspension culture to produce large scale factor-dependent extracts. I applied this tool to modulate the amount of eIF4F complex components and associated factors to aim to understand the residual unwinding activity observed in my RNA restructuring assay. The work outlined in this dissertation underscores the feasibility of monitoring in vitro real-time RNA restructuring dynamics. Additionally, I believe my system could serve to screen potential therapeutic agents to identify inhibitors of RNA restructuring.

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This item is under embargo until May 11, 2024.