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

描述(申请人提供)：线虫中的RNAi自然是系统性的；基因沉默在单个细胞或组织中开始传播，在整个动物及其后代中沉默该基因。线虫的一种蛋白质SID-1已被鉴定，它是系统传递沉默信息所必需的。SID-1被预测包含11个完整的膜结构域和一个大的胞外结构域，这表明它可能是摄取或运输dsRNA的通道或受体。此外，已鉴定出两个哺乳动物基因在跨膜区具有相似的总体预测结构和广泛的同源性。线虫的第二个完整膜蛋白SID-2也是吸收沉默信息所必需的。该方案的目的是研究SID-1和SID-2的亚细胞定位、结构、活性和调控，并开始鉴定哺乳动物的SID-1同源物。为了确定Sid蛋白是否作为通道或受体，并鉴定和表征它们与dsRNA的可能相互作用，我们将使用来自野生型和突变胚胎的线虫原代细胞系来研究它们的转运活性。为了证实这些发现，我们将检测在异源系统中表达的SID-1和SID-2的转运活性。小鼠SID-1同源物将在线虫和昆虫细胞系中表达，以确定它们是否可以补充SID-1突变或执行SID-1的任何功能。小鼠胚胎干细胞将以表达SID同源物和dsRNA摄取活性以及沉默信息的细胞间传播为特征。最后，我们将开始对小鼠ES细胞和嵌合体胚胎中的同源物进行功能分析。这些研究可能会对人类遗传病和病毒感染的治疗产生直接影响。RNAi已被证明可以有效地抑制艾滋病毒和脊髓灰质炎感染，并使癌基因失效，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将dsRNA输送到培养和体内的人类细胞中。

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.