MFB: Characterization of the Biogenesis, Uptake, and Cellular Response to the Ribonucleoprotein Cargoes of Extracellular Vesicles using EV-CLASP

This research project focuses on Extracellular Vesicle-derived RNAs (EV-RNAs), a relatively understudied group of molecules involved in cell-to-cell communication. Found in bacteria, plants, parasites, and mammals, EV-RNAs play pivotal roles in gene regulation across various organisms. The Ascano and Weaver laboratories aim to elucidate the complex processes governing EV-RNA assembly and uptake in cells.

Recently, the Weaver laboratory identified a unique class of EVs enriched with a diverse set of RNAs. By utilizing the Ascano lab's innovative photochemical biology method to crosslink labeled RNAs to proteins, the team will explore how these RNAs are secreted out of the cell into EVs and then received by a different set of cells where they can influence the gene regulatory program.

The research will yield deeper understanding of cellular communication and introduce groundbreaking methodologies, shedding light on shared pathways between viruses and EVs. The broader impacts of this research extend beyond the scientific realm, with potential biotechnology applications for improving agricultural output, environmental monitoring capabilities, and human health.

Furthermore, the project will enable training of the next generation of scientists and generate new technologies and knowledge to benefit the scientific community and the general public.

This project aims to advance understanding of EV-RNAs and their role in intercellular communication. Aim 1 characterizes RNA-RBP complexes sorted into EVs using biotin-dependent proximity labeling (PL) and Extracellular Vesicle – CrossLinking and Solid-phase Purification (EV-CLASP). The role of the ER MCS linker protein VAP-A and the RBP Ago2 in RNP biogenesis and sorting into EVs will be studied to identify key RNP complexes and their fates.

Aim 2 defines pioneer interactions of EV-RNA unloading into recipient cells, utilizing EV-CLASP and proximity labeling assays for miRNA and mRNA. Expected outcomes encompass defining EV entry timeframe, identifying pioneer protein interactants, and understanding RNP complex exchange in EVs. The intellectual significance lies in introducing EV-CLASP, enabling precise isolation of 4SU-crosslinked RNAs and RBPs from donor and recipient cells.

This approach provides molecular granularity for tracking RNP exchanges. The interdisciplinary approach merges chemical and cell biology to understand RNA sorting into EVs at the ER-MCS, shedding light on the role of membraneless condensates. The outcomes are expected to define incoming EV-RNA interactions with recipient cell proteins, identifying key host determinants of EV RNA action, driving new directions in biotechnology for designer EVs in agriculture and human health.

This project is supported by the Genetic Mechanisms program/Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and by the Cellular and Biochemical Engineering program in the Directorate for Engineering.

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.