Genetic and Biochemical Analysis of SID-1 and SID-2

描述（由申请人提供）：秀丽隐杆线虫的RNAi具有天然的系统性；基因沉默始于单个细胞或组织的传播，使该基因在整个动物及其后代中沉默。已经鉴定出秀丽隐杆线虫的一种蛋白质SID-1，它是系统传递沉默信息所必需的。预计SID- 1包含11个完整的膜结构域和一个大的细胞外结构域，这表明它可能作为dsRNA摄取或运输的通道或受体。此外，两个哺乳动物基因在跨膜结构域具有相似的总体预测结构和广泛的同源性。另一种秀丽隐杆线虫整体膜蛋白SID-2也需要吸收沉默信息。本提案的目标是研究SID-1和SID-2的亚细胞定位、结构、活性和调控，并开始表征哺乳动物SID-1同源物。为了确定SID蛋白是作为通道还是受体，并鉴定和表征它们与dsRNA的推定相互作用，将使用来自野生型和突变胚胎的线虫原代细胞系研究它们的运输活性。为了证实这些发现，将对异种系统中表达的SID-1和SID-2的转运活性进行分析。小鼠SID-1同源物将在线虫和昆虫细胞系中表达，以确定它们是否能补充SID-1突变或执行SID-1的任何功能。小鼠胚胎干细胞将以SID同源物的表达和dsRNA摄取活性以及沉默信息的细胞间传播为特征。最后，我们将开始对胚胎干细胞和嵌合体胚胎中的小鼠同源物进行功能分析。这些研究可能对人类遗传疾病和病毒感染的治疗产生直接影响。RNAi已被证明能有效抑制HIV和脊髓灰质炎感染，并使癌基因失活，使细胞恢复正常生长行为。尽管RNAi已被证明是有效的，但一个主要的障碍仍然是在培养和体内将dsRNA输送到人类细胞中。

DESCRIPTION (provided by applicant): RNAi in C. elegans is naturally systemic; gene silencing initiated in a single cell or tissue spreads, silencing that gene throughout the animal and its progeny. A C. elegans protein, SID-1, has been identified that is required for the systemic transmission of silencing information. SID- 1 is predicted to contain 11 integral membrane domains and a large extracellular domain suggesting that it may act as a channel or receptor for the uptake or transport of dsRNA. Furthermore, two mammalian genes have been identified with a similar overall predicted structure and extensive homology in the transmembrane domains. A second C. elegans integral membrane protein SID-2 is also required for uptake of silencing information. The goals of this proposal are to investigate the subcellular localization, structure, activity, and regulation of SID-1 and SID-2 and to begin to characterize mammalian SID-1 homologs. To determine whether either SID protein acts as a channel or receptor and to identify and characterize their presumed interactions with dsRNA, their transport activity will be investigated using nematode primary cell lines derived from wild-type and mutant embryos. To corroborate these findings, the transport activity of SID-1 and SID-2 expressed in heterologous systems will be assayed. The mouse SID-1 homologs will be expressed in nematodes and insect cell lines to determine whether they can complement sid-1 mutations or perform any of the functions of SID-1. Mouse embryonic stem cells will be characterized for expression of SID homologs and dsRNA uptake activity as well as cell-tocell spreading of silencing information. Finally, we will begin a functional analysis of the mouse homologs in ES cells and chimera embryos. These studies may have a direct impact on the treatment of human genetic disease and viral infection. RNAi has been shown to effectively inhibit HIV and polio infection and to disable oncogenes, returning cells to normal growth behavior. Although RNAi has been shown to be effective, a major obstacle remains delivery of dsRNA into human cells both in culture and in vivo.