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

SID-1 和 SID-2 的遗传和生化分析

• 批准号: 6838163
• 负责人: CRAIG Patrick HUNTER
• 金额: $ 29.52万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6838163
• 关键词: Caenorhabditis elegans; Drosophilidae; RNA interference; antisense nucleic acid; cell line; embryonic stem cell; genetically modified animals; immunofluorescence technique; intermolecular interaction; intracellular transport; laboratory mouse; membrane proteins; molecular genetics; protein localization; protein structure function; reporter genes; yeast two hybrid system

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.

描述（由申请人提供）：C.线虫是天然系统性的;在单个细胞或组织中开始的基因沉默扩散，使该基因在整个动物及其后代中沉默。梭已经鉴定了线虫蛋白SID-1，其是沉默信息的系统传递所需的。预测SID- 1含有11个完整的膜结构域和一个大的胞外结构域，这表明它可能充当摄取或转运dsRNA的通道或受体。此外，两个哺乳动物基因已被确定具有相似的整体预测结构和广泛的跨膜结构域的同源性。第二个C。线虫整合膜蛋白SID-2也是摄取沉默信息所必需的。本计划的目标是研究SID-1和SID-2的亚细胞定位、结构、活性和调节，并开始表征哺乳动物SID-1同源物。为了确定SID蛋白是否充当通道或受体，并鉴定和表征它们与dsRNA的假定相互作用，将使用来源于野生型和突变体胚胎的线虫原代细胞系研究它们的转运活性。为了证实这些发现，将测定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。小鼠胚胎干细胞将表征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。最后，我们将开始对ES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已被证明可以有效抑制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，但主要障碍仍然是在培养和体内将dsRNA递送到人类细胞中。