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

细胞外囊泡（EV）是从所有活细胞释放出的具有不同大小和来源的脂质双层结合囊泡的不同群组。最初被认为是细胞的“灰尘”或“垃圾袋”，现在已经发现EV在无数的生物活动中发挥作用，从维持体内平衡到癌症进展。EV可以携带一系列货物，包括蛋白质，脂质和核酸（如DNA和RNA），这些货物有助于其多样化的功能。虽然许多研究都集中在了解电动汽车从细胞中的合成和释放，但很少有研究涉及货物如何被分类和包装成电动汽车。该项目的重点是EV的RNA组分，并试图了解EV中线性和环状RNA的选择，包装和运输。环状RNA具有多种调节功能，环状RNA表达的失调与疾病结果有关。总的来说，该项目将提供通过EV的RNA运输的新理解。该项目还将通过在多个阶段招募学生，包括K-12学生，第一代大学生和来自低收入家庭的本科生，培养下一代科学家，从而产生教育影响。细胞外囊泡（extracellular vesicle，EV）是一种生物大分子，它可以携带DNA、RNA、蛋白质和脂质等多种生物分子，但其含量因细胞的生理状态而异。此外，EV与广泛的生理和病理条件有关，它们可以作为细胞间通讯的重要参与者。利用PI先前在单分子成像系统方面的专业知识，研究RNA从细胞核到细胞中远端位点的细胞内转运，该项目将解决长期存在的问题，即RNA货物如何被分选和包装到EV中。已经提出了几种理论，但没有一种是用直接的视觉证据证实的。EV RNA货物的报告构建体和全基因组RNA测序将用于确定如何以及哪些因素控制RNA货物的选择性包装。还将评估EV表面标志物异质性在RNA货物含量中的作用，以及其对受体细胞摄取EV的影响。对RNA分选的分析，RNA进入EV的运动的跟踪，以及RNA最终转移到受体细胞中将有助于揭示RNA货物的生物学功能。使用单分子分辨率的成像将提供分选和递送过程的第一次视觉验证，并将作为我们对细胞间通讯理解的未来进步的基础，从而能够更好地设计用于治疗和诊断目的的EV。该项目由分子和细胞生物科学部（MCB）和刺激竞争性研究的既定计划（EPSCoR）共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.