Understanding the sorting, transport and fate of RNA cargo in Extracellular Vesicles at single molecule resolution

University Of Delaware, Newark DE

Investigators

Abstract

Extracellular vesicles (EVs) are a diverse group of lipid bilayer-bound vesicles of varying sizes and origins that are released out from all living cells. Initially thought of as cellular “dust” or “garbage bags”, EVs have now been found to play a role in a myriad of biological activities, ranging from maintaining homeostasis to cancer progression. EVs can carry a range of cargo, including proteins, lipids, and nucleic acids (such as DNA and RNA), that contribute to their diverse functions. While much research has focused on understanding the synthesis and release of EVs from the cell, little research has been conducted on how cargo gets sorted and packaged into EVs. This project focuses on the RNA component of EVs and seeks to understand the selection, packaging, and transport of linear and circular RNAs in EVs. Circular RNAs have a multitude of regulatory functions, and dysregulation of circular RNA expression has been linked to disease outcomes. Overall, this project will provide a new understanding of RNA transport via EVs. The project will also have educational impact through training of next-generation scientists through recruitment students at multiple stages, including K-12 students, first-generation college students, and undergraduates from low-income families. Students will gain multidisciplinary skills by incorporating studies of cell biology, biochemistry, imaging, bioinformatics, and computational tools. Extracellular vesicles (EVs) have been found to carry a wide variety of biomolecules including DNA, RNA, proteins, and lipids; however, their content varies based on the physiological state of the cell producing them. Moreover, EVs have been implicated in a wide spectrum of physiological and pathological conditions, and they can serve as important players in cell-cell communication. Leveraging the PI's previous expertise with single-molecule imaging systems for studying intracellular transport of RNAs from the nucleus to distant sites in cells, this project will address the long-standing question of how RNA cargo is sorted and packaged into EVs. There have been several theories proposed, but none of these are substantiated with direct visual evidence. Reporter constructs and genome-wide RNA sequencing of EV RNA cargo will be used to determine how and what factors govern the selective packaging of RNA cargo. The role of EV surface marker heterogeneity in RNA cargo content will also be evaluated, as well as its effect on uptake of EVs by recipient cells. The analysis of RNA sorting, tracking of RNA movement into EVs, and eventual transfer of RNAs into recipient cells will help to uncover the biological function of RNA cargo. Use of imaging at single-molecule resolution will provide the first visual validation of sorting and delivery processes and will serve as the basis of future advancements in our understanding of cell-cell communication, thereby enabling better engineering of EVs for therapy and diagnostic purposes. This project is jointly funded by the Division of Molecular and Cellular Biosciences (MCB) and the Established Program to Stimulate Competitive Research (EPSCoR). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.