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

细胞外囊泡（EVs）是一组不同大小和来源的脂质双分子层结合囊泡，从所有活细胞中释放出来。最初被认为是细胞的“灰尘”或“垃圾袋”，现在已经发现电动汽车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。电动汽车可以携带一系列货物，包括蛋白质、脂质和核酸（如DNA和RNA），这有助于它们的多种功能。虽然很多研究都集中在了解电池中电动汽车的合成和释放，但关于货物如何分类和包装成电动汽车的研究却很少。本项目重点关注电动汽车的RNA成分，并试图了解电动汽车中线性和圆形RNA的选择，包装和运输。环状RNA具有多种调节功能，而环状RNA表达的失调与疾病结果有关。总的来说，该项目将为RNA通过电动汽车的转运提供新的认识。该项目还将通过招收K-12学生、第一代大学生、低收入家庭本科生等不同阶段的学生，培养下一代科学家，并产生教育影响。学生将通过结合细胞生物学、生物化学、成像、生物信息学和计算工具的研究获得多学科技能。细胞外囊泡（EVs）被发现携带多种生物分子，包括DNA、RNA、蛋白质和脂质；然而，它们的含量根据产生它们的细胞的生理状态而变化。此外，电动汽车涉及广泛的生理和病理条件，它们可以在细胞-细胞通讯中发挥重要作用。利用PI之前在单分子成像系统方面的专业知识，研究RNA从细胞核到细胞中遥远部位的细胞内运输，该项目将解决长期存在的RNA货物如何分类和包装成ev的问题。有几个理论被提出，但没有一个是由直接的视觉证据证实的。报告构建和EV RNA货物的全基因组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表面标记物异质性在RNA载货量中的作用，以及其对受体细胞摄取EV的影响。分析RNA分选，跟踪RNA进入ev的运动，并最终将RNA转移到受体细胞将有助于揭示RNA货物的生物学功能。单分子分辨率成像的使用将首次提供分类和递送过程的视觉验证，并将作为我们对细胞-细胞通信理解的未来进步的基础，从而使电动汽车的治疗和诊断目的的更好的工程。该项目由分子和细胞生物科学部（MCB）和促进竞争性研究的既定计划（EPSCoR）共同资助。该奖项反映了美国国家科学基金会的法定使命，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.