Molecular Genetic Analysis of Extracellular RNAs in C. elegans

在体液（血清、唾液、母乳等）中发现的细胞外RNA正在被调查为敏感的 疾病的序列特异性标记物，以及可能的细胞间信使。了解如何具体 RNA被选择和输出，它们是否以及如何被输入到靶细胞中是不可或缺的。 了解它们作为生物标志物、细胞功能介质和潜在的 治疗学RNA转运蛋白的发现和表征 模型系统中的双链RNA（dsRNA）沉默信号C.线虫提供了分子和 对这一新兴领域的遗传学见解。 In C.在线虫中，引入的dsRNA诱导RNA干扰（RNAi），其可以在细胞之间传播， 组织中系统性RNAi缺陷（Sid）突变体的遗传分析不仅鉴定了RNA转运蛋白， 但在普遍的细胞生物学过程中起作用的调节和生物发生因子， RNA运输。SID-1是一种dsRNA门控的dsRNA特异性通道，它赋予了关键的dsRNA输入和输出功能。 活性细胞。对表达野生型和突变型SID-1蛋白的培养细胞的分析表明， SID-1的酸转运对dsRNA是特异性的，并且对dsRNA的甚至微小的化学修饰敏感。 类似的试验表明，鱼、小鼠和人SID-1同源物也转运dsRNA。如果人类 同源物对dsRNA同样具有特异性，并且对修饰不耐受， 设计用于稳定血清中的治疗性沉默RNA并降低其免疫原性可能有助于 很难将这些RNA输送到细胞中。在AIM 1中，我们将确定常见的天然和 非天然RNA修饰对两种C.线虫和培养细胞。这些结果将 直接适用于设计有效的人治疗性siRNA。哺乳动物细胞外RNA是 通常在外来体中发现，分泌囊泡来源于晚期内体/多泡体（LE/MVB）。 SID-5是一种新的LE/MVB相关蛋白，是RNA沉默信号有效输出所必需的，表明 外泌体可能在C.优雅在AIM 2中，我们建议描述 新SID蛋白的活性、定位和功能，包括SID-1和SID-5相互作用蛋白， 深入了解RNA输出的重要生物过程。这些结果将提供一个遗传基础 以及对外来体生物学和细胞外RNA新兴领域的机制理解。在AIM 3中 我们建议利用亨特实验室开发的实验专业知识和资源， 内源性胞外RNA，然后使用C. elegans来表征它们的表达、运输和功能。了解细胞外RNA信号 对动物生理学的贡献是理解它们作为生物标志物和治疗靶点的重要性的关键 用于癌症治疗和其他生物过程。!

Extracellular RNAs found in body fluids (serum, saliva, breast milk, etc.) are being investigated as sensitive sequence-specific markers for disease and as possible intercellular messengers. Understanding how specific RNAs are selected and exported and whether and how they are imported into target cells is integral to understanding their significance as biomarkers, as mediators of cellular functions, and as potential therapeutics. The discovery and characterization of RNA transporters that function to export and import double-stranded RNA (dsRNA) silencing signals in the model system C. elegans is providing molecular and genetic insights into this emerging field. In C. elegans, introduced dsRNA induces RNA interference (RNAi) that can spread between cells and tissues. Genetic analysis of systemic RNAi defective (Sid) mutants has identified not only RNA transporters, but regulatory and biogenesis factors that function in universal cell biological processes to support intercellular RNA transport. SID-1, a dsRNA-gated dsRNA-specific channel, confers the key dsRNA import and export activity to cells. Analysis of cultured cells expressing wild-type and mutant SID-1 proteins shows that nucleic acid transport by SID-1 is specific for dsRNA and sensitive to even minor chemical modifications to the dsRNA. Similar assays indicate that fish, mouse, and human SID-1 homologs also transport dsRNA. If the human homologs are equally specific for dsRNA and intolerant to modification, then the myriad of RNA modifications designed to stabilize therapeutic silencing RNAs in sera and reduce their immunogenicity may contribute to the difficulty of delivering these RNAs into cells. In AIM 1 we will determine the effect of common natural and unnatural RNA modifications on dsRNA transport in both C. elegans and cultured cells. These results will be directly applicable to the design of effective human therapeutic siRNAs. Mammalian extracellular RNAs are often found within exosomes, secreted vesicles derived from late endosome/multivesicular bodies (LE/MVB). SID-5 is a novel LE/MVB associated protein required for efficient export of RNA silencing signals, indicating that exosomes may transport RNA silencing signals in C. elegans. In AIM 2 we propose to characterize the activity, localization, and function of new SID proteins, including SID-1 and SID-5 interacting proteins to gain insight into the biological processes important for RNA export. These results will provide a genetic foundation and mechanistic understanding for the emerging fields of exosome biology and extracellular RNAs. In AIM 3 we propose to exploit the experimental expertise and resources developed in the Hunter lab to directly isolate endogenous extracellular RNAs and then to use the well established molecular genetic tools available in C. elegans to characterize their expression, transport, and function. Understanding how extracellular RNA signals contribute to animal physiology is key to understanding their significance as biomarkers and therapeutic targets for cancer therapy and other biological processes. !