PROJECT SUMMARY This project aims to understand how nucleic acids prevent protein aggregation and aid protein folding. Protein misfolding and aggregation lead to many debilitating diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In addition to many other roles in the cell, we recently discovered that nucleic acids are powerful molecular chaperones. Chaperones are responsible for maintaining proteome stability by preventing toxic protein aggregation and enabling protein folding. Given the preponderance of nucleic acids in the cell, their role in stress granules, and their potent chaperone activity, it is highly likely that they play a major role in protein homeostasis. At the current stage of investigation, we have virtually no knowledge of how nucleic acids prevent protein aggregation, how they interact with other chaperones, or what role they play in protein folding. This project will elucidate how nucleic acids work as molecular chaperones, a previously unrecognized property of these important molecules. The work described in the K99 phase of this grant will provide me with the necessary skills to successfully complete the R00 phase as an independent scientist and will lay the foundation for the R00 phase research. In the K99 phase, I will learn new in vitro and in vivo techniques for characterizing chaperone function geared towards nucleic acid chaperones. Using these techniques, I will examine the sequence specificity of nucleic acid chaperones using a genetic screen (Aim 1A). I will then generate the foundation for the first model of how nucleic acids prevent aggregation and participate in protein folding by studying how these sequences aid in protein folding and interact with other chaperones (Aim 1B). I will also develop structural biology methods using both NMR spectroscopy and X-ray crystallography to analyze complexes of chaperone-active nucleotide chains and partially folded protein substrates (Aim 2). In the R00 phase of this project, I will continue to expand our studies of nucleic acid chaperone function to include the larger structural vocabulary of RNA (Aim 1). I will also apply the structural biology techniques developed in the K99 phase toward understanding the relationship between nucleic acid chaperone structure and function, and elucidating how nucleic acid chaperones affect the folding of protein substrates (Aim 2). Together, these data will allow us to construct a comprehensive model describing the chaperone function of RNA and DNA. In the future, this model may form the basis for designing nucleic acid-based therapeutics for currently untreatable protein misfolding diseases.