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

该研究项目的重点是细胞外囊泡衍生的RNA（EV-RNA），这是一组参与细胞间通讯的分子。EV-RNA存在于细菌、植物、寄生虫和哺乳动物中，在各种生物体的基因调控中发挥着关键作用。Ascano和Weaver实验室的目标是阐明控制EV-RNA组装和细胞摄取的复杂过程。最近，Weaver实验室发现了一类独特的富含多种RNA的EV。通过利用Ascano实验室创新的光化学生物学方法将标记的RNA与蛋白质交联，研究小组将探索这些RNA如何从细胞中分泌到EV中，然后被一组不同的细胞接收，在那里它们可以影响基因调控程序。这项研究将加深对细胞通信的理解，并引入开创性的方法，揭示病毒和EV之间的共享途径。这项研究的更广泛影响超出了科学领域，具有潜在的生物技术应用，可提高农业产量、环境监测能力和人类健康。此外，该项目将能够培训下一代科学家，并产生新的技术和知识，使科学界和公众受益。该项目旨在促进对EV-RNA及其在细胞间通讯中的作用的理解。目的1利用生物素依赖性邻近标记（PL）和细胞外囊泡-交联和固相纯化（EV-CLASP）表征RNA-RBP复合物分选成EV。将研究ER MCS接头蛋白VAP-A和RBP Ago 2在RNP生物发生和分选成EV中的作用，以鉴定关键的RNP复合物及其命运。目的2利用EV-CLASP和miRNA和mRNA的邻近标记测定，定义EV-RNA卸载到受体细胞中的先锋相互作用。预期成果包括定义EV进入时间表，识别先锋蛋白质相互作用物，并了解EV中的RNP复合物交换。知识意义在于引入EV-CLASP，使得能够从供体和受体细胞中精确分离4SU交联的RNA和RBP。该方法提供了用于跟踪RNP交换的分子粒度。跨学科的方法融合了化学和细胞生物学，以了解RNA在ER-MCS中分选到EV中，从而揭示了无膜浓缩物的作用。这些结果预计将定义EV-RNA与受体细胞蛋白的相互作用，确定EV RNA作用的关键宿主决定因素，为农业和人类健康领域的电动汽车设计师推动生物技术的新方向。该项目由遗传机制计划/生物科学理事会的分子和细胞生物科学部以及生物科学理事会的细胞和生物化学工程计划工程。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.