Liquid?liquid phase separation of RNA-binding proteins (RBP) is a recently appreciated means of intracellular compartmentalization underpins the biogenesis of diverse membraneless organelles. Despite clear biological utility, dysregulated phase separation of RNA-binding protein (RBP) leads to protein aggregation and fibrils formation, which are key pathological features of numerous neurodegenerative diseases. The central hypothesis of my work, and the driving force of my lab, is that reversing aberrant phase transition and aggregation is a potential avenue to combat these fatal diseases. In fact, we and others have established that nuclear import receptors (NIRs) such as Kap?2 can reverse protein aberrant phase transition and elevated Kap?2 expression can suppress neurodegeneration caused by disease-linked RNA-binding proteins. Thus, NIRs and other regulators of protein phase transition could be leveraged therapeutically to restore normal function of membraneless organelles and mitigate proteotoxicity caused by aberrant phase transition. However, several key knowledge gaps regarding the regulation of phase transition must be filled, to lay the foundation to develop such therapeutic strategies. Thus the long-term goal of my research program is to achieve a more comprehensive understanding of the regulatory mechanism of protein phase separation and leverage our understanding of phase regulation to develop strategies with therapeutic potential to reverse proteotoxicity induced by aberrant phase transition. I seek to elucidate the molecular mechanism of the regulation of protein phase separation, both in health and disease conditions. We propose to focus on three main themes over the next five years: (1) Define the mechanism of how Kap?2 reverses protein aberrant phase transition. (2) Define the scope and specificity of Kap?2 and other nuclear import receptors in their function in regulating phase separation in live cells. (3) Based on our preliminary results that RNA oligonucleotides with specific sequences have diverse activities in regulating RNA-binding protein phase separation, we will identify the sequence space of RNA in regulating RBP phase separation and develop innovative single-molecule fluorescence-based biophysical methods to understand the mechanism of RNA?s function. Our work will significantly contribute to our understanding of the regulatory mechanism of phase separation in cells and how the breakdown of this regulation leads to disease conditions. Furthermore, this work will set the stage for developing strategies to enhance the activity of Kap?2, as well as RNA-based oligonucleotides that can mitigate toxic aberrant phase transition, both of which provide the basis for developing innovative therapeutics to restore the healthy protein phase in cells.

Public Health Relevance

Liquid?liquid phase separation of proteins and nucleic acids drives the biogenesis of diverse membraneless organelles with important biological functions. Dysregulated phase separation of RNA-binding proteins leads to the formation of toxic fibrils, which is a pathological hallmark in multiple fatal neurodegenerative diseases. Elucidating molecular mechanisms of phase separation regulation and developing strategies to reverse aberrant phase transition and fibrillization will lay the foundation for advances in novel therapeutic strategies to combat these fatal diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZRG1)
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Sakalian, Michael
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Thomas Jefferson University
Schools of Medicine
United States
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