The role of RNA editing in organizing the cytoplasm.

. Introduction: RNA editing introduces nucleotide base substitutions post- transcriptionally, expanding the transcriptome and proteome without the need for additional gene programs. RNA editing relies on precise, spatiotemporal interactions between RNA-editing enzymes and RNA substrates, yet the features of RNA that guide these interactions, and the impact of editing events on protein-RNA interactions, remain poorly understood. In this proposal, I will examine how RNA sequence and structure establish RNA editing sites, and how RNA editing changes RNA sequence and structure to tune RNA-protein interactions within the cell. I hypothesize that RNA editing tailors RNA-protein interactions to drive local protein synthesis. Research: In Aim 1, I will identify novel sites of RNA editing across the transcriptome of the giant, single cell, Physarum polycephalum. I will examine the RNA structures and sequences associated with these sites of modification and implement a machine learning approach to predict new sites of RNA editing that will have general applications beyond Physarum. In Aim 2, I will quantitatively assess the spatial organization of RNA editing, and how these events alter the composition and function of protein-RNA assemblies to drive changes in local protein synthesis. The overall outcome will be a mechanistic understanding of how cells govern RNA editing, and the role of RNA editing in organizing the cytoplasm. Training: I will complete my training with Prof. Amy Gladfelter at Duke University. During the training period, I will work with innovative collaborators to acquire new skills that will enable me to identify RNA editing across the genome using bioinformatics, probe and manipulate RNA structure, and implement machine learning approaches for the prediction of novel editing sites. These skillsets will accelerate discovery during the remainder of my training and form the core expertise of my independent lab. Specifically, I will learn powerful strategies to (1) identify signatures of RNA within sequencing data with Fred Dietrich at Duke University: (2) map RNA structure with Alain Laederach at UNC Chapel Hill; and (3) develop machine learning approaches for novel RNA editing site prediction with Rohit Singh at Duke University. Environment: Prof. Gladfelter is a supportive and inspiring mentor who fosters creativity and collaboration. Duke university, and the greater Research Triangle in which it is a part, is a hub for world-class RNA biology and will provide valuable opportunities to learn from experienced scientists. This K99/R00 award will enable me to pursue exciting new research directions beyond my core skillsets, form strong collaborations with leading labs, and immerse myself in new disciplines through a variety of courses, seminars, workshops, and conferences. Impact on Public Health: RNA-based technologies have gained attention as emerging genetic therapies due to their ability to modify the transcriptome without altering the genome. My work aims to uncover fundamental mechanisms of RNA editing and identify novel editing strategies. Through my work, I hope to aid in the development of RNA editing therapies and clinical interventions for genetic disease that result from Single-Nucleotide-Polymorphisms. Project Number: 1K99GM164668-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Zach Geisterfer | Institution: DUKE UNIVERSITY, DURHAM, NC | Award Amount: $125,000 | Activity Code: K99 | Study Section: Special Emphasis Panel[ZRG1 MGG-M (80)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11350208