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

这项研究的重点是胞外囊泡衍生的RNAs(EV-RNAs)，这是一组相对研究较少的参与细胞间通讯的分子。在细菌、植物、寄生虫和哺乳动物中发现的EV-RNAs在各种生物的基因调控中发挥着关键作用。Ascano和Weaver实验室旨在阐明控制EV-RNA在细胞中组装和摄取的复杂过程。最近，韦弗实验室发现了一类独特的电动汽车，富含不同的RNA。通过利用Ascano实验室的创新光化学生物学方法将标记的RNA与蛋白质交联，该团队将探索这些RNA如何从细胞分泌到EVS，然后被另一组细胞接收，在那里它们可以影响基因调控程序。这项研究将加深对蜂窝通信的理解，并引入开创性的方法，揭示病毒和电动汽车之间的共同路径。这项研究的更广泛影响超出了科学领域，潜在的生物技术应用于提高农业产量、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。该项目旨在增进对EV-RNAs及其在细胞间通信中的作用的了解。目的1利用生物素依赖的邻近标记(PL)和胞外囊泡交联固相纯化(EV-CLAP)技术对分离到EVS中的RNA-RBP复合体进行表征。将研究ER MCS连接蛋白VAP-A和RBP Ago2在RNP生物发生和分选EVS中的作用，以确定关键的RNP复合体及其命运。目的2利用EV-CLASP和邻近标记分析miRNA和mRNA，定义EV-RNA卸载到受体细胞的先驱相互作用。预期结果包括确定EV进入的时间框架，确定先驱蛋白质相互作用物，以及了解EVS中的RNP复合体交换。其智力意义在于引入EV-CLAP，使4SU-交联型RNA和限制性商业惯例能够从供体和受体细胞中精确分离。这种方法为跟踪RNP交换提供了分子粒度。这种跨学科的方法融合了化学和细胞生物学，以了解在ER-MCS中将RNA分类到EV中，揭示了无膜冷凝物的作用。预计结果将确定EV-RNA与受体细胞蛋白质的相互作用，确定EV RNA作用的关键宿主决定因素，推动农业和人类健康中设计EV的生物技术新方向。该项目由生物科学局的遗传机制计划/分子和细胞生物科学部以及工程局的细胞和生化工程计划支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.