Molecular Genetic Analysis of Extracellular RNAs in C. elegans

描述（由申请人提供）：秀丽隐杆线虫中RNA干扰（RNAi）的一个显著特性是它与细胞间RNA转运途径的关联。这种联系调动了dsrna沉默信号，并使沉默从起始位点传播到整个动物和后代。这种现象被称为系统性RNAi，在许多多细胞生物中是一个保守的过程。通过遗传分析，我们分离出了系统性RNAi缺陷突变体（sid），并鉴定出了相应的蛋白（sid）。SID-1是一个广泛保守的dsRNA通道，它选择性地和特异性地将dsRNA转运到细胞中，是全身性RNAi所必需的。哺乳动物的SID-1同源物对于修饰sirna的细胞质递送至关重要，这表明dsRNA转运是该通道蛋白家族的保守功能。SID-2是一种推测的dsRNA受体，仅在肠腔膜上表达和定位。SID-2通过肠上皮将摄入的dsRNA转运到动物体内以触发RNAi。这种序列特异性基因沉默以响应环境遇到的dsRNA的过程，被称为环境RNAi，在自然界中广泛存在，包括在哺乳动物中。

DESCRIPTION (provided by applicant): A remarkable property of RNA interference (RNAi) in C. elegans is its association with intercellular RNA transport pathways. This linkage mobilizes dsRNA-silencing signals and enables silencing to spread from the site of initiation throughout the animal and to the progeny. This phenomenon, known as systemic RNAi, is a conserved process among many multicellular organisms. Through genetic analysis, we have isolated systemic RNAi defective mutants (sid) and have identified the corresponding proteins (SID). SID-1 is a widely conserved dsRNA channel that selectively and specifically transports dsRNA into cells and is essential for systemic RNAi. A mammalian SID-1 homolog is critical for cytoplasmic delivery of modified siRNAs, suggesting that dsRNA transport is a conserved function for this family of channel proteins. SID-2 is a putative dsRNA receptor that is expressed and localized exclusively to the luminal membrane of the intestine. SID-2 transports ingested dsRNA across the intestinal epithelium into the animal to trigger RNAi. This process of sequence-specific gene silencing in response to environmentally-encountered dsRNA, known as environmental RNAi, is widespread throughout nature, including in mammals. The development of RNAi-based drugs enables targeting of previously "undruggable" disease related genes and has exciting therapeutic potential. However, the efficacy and safety of RNAi as a gene-silencing therapy requires understanding how therapeutic dsRNAs enter cells to gain access to the silencing machinery. In addition, we must rationally determine how to modify these dsRNA molecules for more efficient delivery and targeting to select tissues and cells. Our analysis of dsRNA transport through the SID-1 channel indicates that even minor modifications, such as removing an oxygen atom from the ribose sugar, can block dsRNA transport. Thus, understanding the regulation and function of these proteins in the experimentally tractable nematode C. elegans will provide valuable insights for the advancement of RNAi-based drugs as a novel class of therapeutic agents in humans. The long-term objective of the proposed research is to understand the physiological importance and mechanism of intercellular RNA transport in animals. Towards this end, the specific aims of this proposal are: 1) To characterize the specificity and regulation of the SID-1 dsRNA channel; 2) To characterize ingested dsRNA uptake mechanism in environmental RNAi; 3) To characterize extracellular dsRNA transport pathways; and 4) To isolate and characterize endogenous extracellular RNAs in C. elegans. These aims address, through a combination of genetic, biochemical and biophysical approaches, questions at the leading edge of the recently discovered field of intercellular RNA transport and further explore the possibility that extracellular RNA molecules underlie a novel means of signaling in multicellular organisms. Remarkably, this process, which was unknown 10 years ago, now has immediate clinical relevance. PUBLIC HEALTH RELEVANCE: The specificity and potency of dsRNA-based gene silencing lends tremendous hope for the treatment of a wide range of human diseases including cancer. Although clinical trials for several RNAi-based drugs are already underway, the mechanisms underlying dsRNA transport and uptake are poorly understood. Understanding how dsRNA crosses cell membranes to trigger RNAi will lead to improvements in targeting and delivery efficiency, and will limit potential deleterious side effects of this exciting therapeutic strategy.