Mechanisms of RNA Packaging into Exosomes

背景：质膜对细胞质物质的扩散起屏障作用。这使得细胞特化成为可能，这对于复杂的多细胞生物来说是必不可少的。在这个模型中，细胞不应该交换细胞质或核物质，因为这将损害致力于建立和维持细胞身份的重大努力。细胞已经进化出相互沟通的方法，以允许复杂器官分子在其表面或释放到细胞外空间的发育和功能。令人惊讶的是，在某些情况下，细胞释放的胞外囊泡（称为外泌体）可以克服质膜屏障，将一个细胞产生的细胞质内容物运送到另一个细胞的细胞质中1-4。这就提出了一个基本问题，即如何控制外泌体对RNA细胞间通讯的生理影响，以确保细胞身份和功能的维持。细胞用来控制外泌体介导的rna递送的一个主要机制是选择性地将大量特异性rna包装到外泌体中（许多其他rna可能在外泌体中检测到，但通常不够丰富，不足以与生理相关）。了解将RNA打包到外泌体的机制可以为RNA生物学和外泌体的生理功能提供基本的生物学见解。总体目标和基本原理：最近的出版物表明，在严格的条件下，通过共价交联RNA与蛋白质分离RNA-蛋白质复合物，可以系统地鉴定数百种新的RNA结合蛋白31,32。我们已经建立了类似的方法，从外泌体中纯化所有rna -蛋白复合物，以及与pre-miR-451结合的rna结合蛋白，我们的初步数据表明该蛋白在外泌体中高度富集。我们建议在外泌体中生成一个新的RNA结合蛋白目录，并剖析它们在将特定RNA包装到外泌体中的作用。最后，我们将在细胞或小鼠模型中测试新的rna结合蛋白和rna外泌体的生物学影响。假设：蛋白质结合特定的RNA序列和结构，将它们招募到外泌体中。从细胞和外泌体中纯化pre-miR-451复合物，以鉴定和表征蛋白质对将pre-miR-451包装到外泌体的影响。2. 从多种细胞类型的外泌体中纯化所有RNA-蛋白复合物，并通过其中的一个子集鉴定包装到外泌体中的RNA。3. 测试新rna结合蛋白和外泌体rna对小鼠的生物学影响。意义：mrna只代表外泌体中rna的一小部分。外泌体中最丰富的rna是人们知之甚少的非编码rna。使用无偏倚的方法，本研究将定义几种很少研究类型的RNA如何被选择性地包装到外泌体中，并旨在了解外泌体对这些RNA的细胞间转移的影响。

Background: The plasma membrane erects a barrier to the spread of cytoplasmic materials. This enables cellular specialization that is essential for complex multicellular organisms. In this model cells should NOT exchange cytoplasmic or nuclear materials, as this would compromise the significant efforts devoted to establishing and maintaining cellular identity. Cells have evolved methods to communicate amongst themselves to allow the development and function of complex organs molecules on their surface or released into the extracellular space. Surprisingly, in some circumstances extracellular vesicles released by cells and called exosomes can overcome the plasma membrane barrier and deliver cytoplasmic contents produced in one cell into the cytoplasm of another1-4. This raises fundamental questions about how the physiological impact of intercellular communication of RNA by exosomes is controlled to ensure the maintenance of cellular identity and function. One principal mechanism that cells use to control exosome-mediated delivery of RNAs is by selectively packaging large amounts of only specific RNAs into exosomes (many other RNAs may be detectable in exosomes, but are often not abundant enough to be physiologically relevant). Understanding the mechanisms which package RNAs into exosomes can generate fundamental biological insights into RNA biology and physiological functions of exosomes. Over-Arching Aims and Rationale: Recent publications demonstrated that hundreds of novel RNA-binding proteins can be systematically identified by covalently cross-linking RNA to proteins to isolate RNA-protein complexes in stringent conditions31,32. We have established similar methods to purify all RNA-protein complexes from exosomes, and RNA-binding proteins bound to a pre-miR-451 which our preliminary data demonstrates is highly enriched in exosomes. We propose to generate a catalogue of new RNA binding proteins in exosomes and dissect their roles in packaging specific RNAs into exosomes. Finally, we will test the biological impact of new RNA-binding proteins and RNAs in exosomes in cell or mouse models. Hypothesis: Proteins bind specific RNA sequences and structures to recruit these into exosomes Specific Aims 1. Purify pre-miR-451 complexes from cells and exosomes to identify and characterize the impact of proteins on packaging pre-miR-451 into exosomes. 2. Purify all RNA-protein complexes from exosomes of multiple cell types and identify RNA packaged into exosomes by a subset of these. 3. Test the biological impact in mice of new RNA-binding proteins and RNAs in exosomes. Significance: mRNAs represent a tiny fraction of the RNAs in exosomes. The most abundant RNAs in exosomes are poorly understood non-coding RNAs. Using unbiased approaches this research will define how RNA of several little studied types is selectively packaged into exosomes and aims to understand the impact of intercellular transfer of these RNAs by exosomes.