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

SID-1和SID-2的遗传和生化分析

• 批准号: 7659988
• 负责人: CRAIG Patrick HUNTER
• 金额: $ 27万
• 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 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7659988
• 关键词: Affect; Animals; Antibodies; Behavior; Biochemical Genetics; Biological; Biological Assay; Body cavities; Caenorhabditis; Caenorhabditis elegans; Cell Line; Cell membrane; Cell physiology; Cells; Computer Systems Development; DNA; Defect; Development; Double-Stranded RNA; Drosophila genus; Electric Conductivity; Environment; Family; Gene Silencing; Genes; Genetic Screening; Growth; Homologous Gene; Human; Hyperplasia; Immune system; Integral Membrane Protein; Intestines; Laboratories; Learning; Liquid substance; Localized; Lymphocyte; Mammalian Cell; Measurement; Measures; Mediating; Molecular; Molecular Genetics; Movement; Mus; Nematoda; Neurons; Pattern; Phenotype; Physiological; Physiology; Process; Protein Binding; Proteins; Protocols documentation; RNA Interference; RNA Transport; Rana; Range; Refractory; Regulation; Reporter Genes; Research; Resistance; Resources; Role; Signal Transduction; Specificity; Substrate Specificity; System; T-Lymphocyte; Techniques; Testing; Therapeutic; Tissues; Transcript; Two-Hybrid System Techniques; Vertebrates; Work; blastomere structure; body cavity; extracellular; feeding; gain of function; human disease; insight; intercellular communication; invertebrate genome; loss of function; mutant; neuronal growth; protein function; targeted delivery; uptake

The use of short-interfering RNAs (siRNAs) to trigger RNA interference (RNAi) is actively being pursued as a therapeutic approach against many human diseases. However, the efficacy of RNAi as a gene-silencing therapy requires understanding how therapeutic siRNAs enter cells to gain access to the silencing machinery. In addition, we must rationally determine how to modify these dsRNAs molecules for more efficient delivery and targeting to select tissues and cells. Studies of dsRNA uptake and silencing in the nematode C. elegans have been instrumental in identifying key proteins involved in the process. Genetic screens for systemic RNAi defective (sid) mutants have identified two transmembrane proteins, SID-1 and SID-2 required for dsRNA uptake. SID-1 is an ancient protein with homologs detected in all sequenced vertebrate and many invertebrate genomes. A human homolog of SID-1 has recently been shown to be critical for cytoplasmic delivery of modified siRNAs, suggesting that dsRNA transport is a conserved function for this family of channel proteins. Current work in our laboratory has shown that knockdown of mouse SID-1 disrupts a wide range of processes, including animal growth, neuronal functions, and defects in immune system development. In contrast, SID-2 appears to be a rapidly evolving protein that is detectable by sequence similarity only in Caenorhabditis nematodes. However, the process that SID-2 enables, silencing by RNA uptake from the environment, is more widespread. Expression of either SID-1 or SID-2 in cells normally refractory to dsRNA, enables dsRNA import. Understanding the regulation and function of these proteins in the experimentally tractable nematode C. elegans and experimental vertebrate systems will provide valuable insights for the development of therapeutic RNAi 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 RNA transport mechanism of the dsRNA channel. 2) To characterize proteins that function with SID-1 and SID-2 and are necessary to allow dsRNA uptake using biochemical and genetic approaches. 3) To investigate the dsRNA channel in mouse immune-system development and function. 4). To isolate and characterize endogenous extracellular RNAs in C. elegans. This work may identify an ancient, gene-specific mechanism for intercellular communication in animals.

使用短干扰RNA（siRNA）来触发RNA干扰（RNAi）正被积极地研究。 作为治疗许多人类疾病的方法。然而，RNAi的功效 因为基因沉默疗法需要了解治疗性siRNA如何进入细胞， 进入消音器此外，我们必须理性地确定如何修改这些 dsRNA分子用于更有效地递送和靶向选择的组织和细胞。研究 线虫C.秀丽线虫在识别关键的 参与这个过程的蛋白质。系统性RNAi缺陷（sid）突变体的遗传筛选 鉴定了dsRNA摄取所需的两种跨膜蛋白SID-1和SID-2。SID-1是一个 在所有测序的脊椎动物和许多无脊椎动物中检测到同源物的古老蛋白质 基因组SID-1的人类同源物最近已被证明对细胞质中的细胞毒性至关重要。 修饰的siRNA的递送，表明双链RNA转运是其保守功能 通道蛋白家族。我们实验室目前的工作表明， SID-1破坏了广泛的过程，包括动物生长，神经元功能和缺陷 免疫系统的发展。相反，SID-2似乎是一种快速进化的蛋白质， 仅在小杆线虫中通过序列相似性可检测到。然而， SID-2使得通过从环境中摄取RNA而沉默更加普遍。表达 通常对dsRNA不敏感的细胞中的SID-1或SID-2使得dsRNA能够输入。理解 这些蛋白在实验上易处理的线虫C. elegans 和实验脊椎动物系统将提供有价值的见解， 治疗性RNAi 拟议研究的长期目标是了解 以及动物细胞内RNA转运的机制。为此， 本研究的主要目的是：1）研究dsRNA的特异性和RNA转运机制 频道2)为了表征与SID-1和SID-2一起发挥功能的蛋白质， 使用生物化学和遗传方法的dsRNA摄取。3)为了研究dsRNA通道， 小鼠免疫系统的发育和功能。4）。分离和表征内源性 胞外RNA在C.优雅的这项工作可能会确定一个古老的，基因特异性的机制， 动物的细胞间通讯。