Mechanisms of RNA Packaging into Exosomes

背景： 质膜为细胞质物质的传播设置了一道屏障。这使得对于复杂的多细胞生物体来说至关重要的细胞特化成为可能。在这个模型中，细胞不应该交换细胞质或核材料，因为这将损害致力于建立和维持细胞特性的重大努力。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展和功能。令人惊讶的是，在某些情况下，由细胞释放的称为外体的胞外小泡可以克服质膜屏障，将一个细胞产生的细胞质内容输送到另一个细胞的细胞质中。这就提出了一些根本性的问题，即如何控制外切体对RNA细胞间通讯的生理影响，以确保维持细胞的身份和功能。细胞用来控制外切体介导的RNA传递的一个主要机制是通过选择性地将大量仅特定的RNA包装到外切体中(许多其他RNA可能在外切体中可检测到，但通常不够丰富，不足以与生理相关)。了解将RNA包装成外切体的机制可以产生对RNA生物学和外切体的生理功能的基本生物学见解。 总体目标和理论基础： 最近的文献表明，在严格的条件下，通过将RNA与蛋白质共价交联来分离RNA-蛋白质复合体，可以系统地鉴定数百种新型的RNA结合蛋白质。我们已经建立了类似的方法来从外切体中纯化所有的RNA-蛋白质复合体，以及与前-miR-451结合的RNA结合蛋白，我们的初步数据表明，这在外切体中高度丰富。我们建议在外体中生成一个新的RNA结合蛋白的目录，并分析它们在将特定RNA包装到外体中的作用。最后，我们将在细胞或小鼠模型中测试外体中新的RNA结合蛋白和RNA的生物学影响。 假设：蛋白质结合特定的RNA序列和结构以将其招募到外切体中 具体目标 1.从细胞和外体中纯化前-miR-451复合体，鉴定和表征蛋白质对将前-miR-451包装到外体中的影响。 2.从多种细胞类型的外体中纯化所有的RNA-蛋白质复合体，并通过其中的一个子集来鉴定包装到外体中的RNA。 3.检测新的RNA结合蛋白和外切体中的RNA对小鼠的生物学影响。 重要意义： 在外切体中，mRNAs只占RNA的一小部分。外切体中最丰富的RNA是人们知之甚少的非编码RNA。使用公正的方法，这项研究将定义几种较少研究的类型的RNA是如何选择性地包装到外体中的，并旨在了解这些RNA通过外体进行细胞间转移的影响。

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 another14. 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.