Molecular Genetic Analysis of Extracellular RNAs in C. elegans

描述（由申请人提供）：在体液（血清、唾液、母乳等）中发现的细胞外rna正在作为疾病的敏感序列特异性标记物和可能的细胞间信使进行研究。了解特定rna是如何被选择和输出的，以及它们是否以及如何被输入到靶细胞中，对于理解它们作为生物标志物、细胞功能介质和潜在治疗药物的意义是不可或缺的。模型系统秀丽隐杆线虫中输出和输入双链RNA （dsRNA）沉默信号的RNA转运体的发现和表征为这一新兴领域提供了分子和遗传学的见解。在秀丽隐杆线虫中，引入的dsRNA诱导RNA干扰（RNAi），这种干扰可以在细胞和组织之间传播。系统性RNAi缺陷（Sid）突变体的遗传分析不仅确定了RNA转运体，还确定了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，以支持细胞间RNA转运。SID-1是一个dsRNA门控的dsRNA特异性通道，赋予细胞关键的dsRNA输入和输出活性。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的核酸转运对dsRNA具有特异性，并且对dsRNA的微小化学修饰敏感。类似的实验表明，鱼类、小鼠和人类的SID-1同源物也能运输dsRNA。如果人类同源物对dsRNA具有同样的特异性，并且不耐受修饰，那么设计用于稳定血清中治疗性沉默RNA并降低其免疫原性的无数RNA修饰可能会导致将这些RNA递送到细胞中的困难。在AIM 1中，我们将确定常见的天然和非天然RNA修饰对秀丽隐杆线虫和培养细胞中dsRNA运输的影响。这些结果将直接适用于设计有效的人类治疗性sirna。哺乳动物细胞外rna通常在外泌体中发现，外泌体是由晚期内小体/多泡体（LE/MVB）分泌的囊泡。SID-5是一种新的LE/MVB相关蛋白，是有效输出RNA沉默信号所必需的，这表明外泌体可能在秀丽隐杆线虫中运输RNA沉默信号。在AIM 2中，我们建议表征新的SID蛋白的活性、定位和功能，包括SID-1和SID-5相互作用蛋白，以深入了解RNA输出的重要生物学过程。这些结果将为外泌体生物学和细胞外rna的新兴领域提供遗传基础和机制理解。在AIM 3中，我们建议利用Hunter实验室开发的实验专业知识和资源直接分离内源性细胞外rna，然后使用秀丽隐杆线虫中成熟的分子遗传学工具来表征它们的表达、运输和功能。了解细胞外RNA信号如何促进动物生理是理解其作为生物标志物和治疗靶点在癌症治疗和其他生物过程中的重要性的关键。

DESCRIPTION (provided by applicant): 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.