RNA interference: Does R2D2 witness Dicing?

RNA干扰：R2D2见证了Dicing吗？

• 批准号: 8370576
• 负责人: Tracey Ann Lincoln
• 金额: $ 5.39万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8370576
• 关键词: Amino Acids; Binding; Biochemical; Biochemical Genetics; Biological Assay; Cleaved cell; Complex; Congenital Abnormality; Coupled; Development; Dicer Enzyme; Disease; Double-Stranded RNA; Double-Stranded RNA Binding Domain; Drosophila genus; Drosophila melanogaster; Drosophila melanogaster Proteins; Ensure; Enzymes; Eukaryotic Cell; Genetic; Germ Lines; Goals; Human; Huntington Disease; In Vitro; Lead; Learning; Life; Malignant Neoplasms; Mammals; Maps; Messenger RNA; Modeling; Molecular; Monitor; Multienzyme Complexes; Mutation; Nucleotides; Parasites; Parkinson Disease; Pharmacologic Substance; Population; Process; Production; Proteins; RNA Interference; RNA Interference Pathway; Repetitive Sequence; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease III; Ribonucleases; Role; Site-Directed Mutagenesis; Small Interfering RNA; Small RNA; System; Techniques; Testing; Therapeutic; Transgenic Organisms; Virus Diseases; Work; Yeasts; base; design; fly; genetic element; high throughput screening; human DICER1 protein; improved; in vivo; mutant; neuronal cell body; tool; yeast two hybrid system

DESCRIPTION (provided by applicant): RNA interference (RNAi) is an evolutionarily conserved cellular defense against foreign or parasitic genetic elements. This regulatory system ensures genetic stability. Loss of RNAi in the soma may lead to cancer; loss of RNAi in the germ line may cause birth defects. In Drosophila melanogaster, the proteins Dicer-2 and R2D2 are essential for the RNA interference (RNAi) pathway. These proteins form a stable heterodimer in vitro, and without Dicer-2, R2D2 is unstable in vivo. Yet in vitro analysis indicates that Dicer-2 can carry out one of its functions-cleaving dsRNA-in the absence of R2D2. The goal of this study is to identify the amino acids required for the binding of these two proteins to each other, so as to design mutant flies in which I can test whether R2D2 must be bound to Dicer-2 during dicing or, rather, must only be present at subsequent steps in the production of an active RNAi enzyme complex. To this end, I propose to use a reverse yeast two- hybrid screen to select mutants of Dicer-2 that are unable to bind R2D2. I will carry out quantitative biochemical tests to identify a mutant that retains its ability to cleave long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs), the guides that direct RNAi. I will generate transgenic flies that have two forms of Dicer-2: the mutant form of Dicer-2 that no longer binds R2D2 but is able to cleave dsRNA, as well as an existing mutant Dicer-2 that binds R2D2 but is diminished in its ability to cleave dsRNA. Using established techniques, I will examine these flies to determine whether RNAi can occur in the living fly when the processing dsRNA into siRNAs and the loading of the siRNAs into functional complexes are decoupled. I will also use molecular tools, including quantitative RT-PCR and tiling microarrays, to monitor the levels of genetic elements that are normally silenced, and high throughput sequencing of the small RNA populations in these flies to determine the in vivo consequences of separating these processes.

描述（由申请人提供）：RNA干扰（RNAi）是一种进化上保守的细胞防御外来或寄生遗传因子。这种调控系统确保了遗传稳定性。索马中RNAi的缺失可能导致癌症;生殖系中RNAi的缺失可能导致出生缺陷。在黑腹果蝇中，蛋白质Dicer-2和R2D2是RNA干扰（RNAi）途径所必需的。这些蛋白质在体外形成稳定的异源二聚体，并且在没有Dicer-2的情况下，R2D2在体内不稳定。然而，体外分析表明，Dicer-2可以在不存在R2D2的情况下执行其功能之一-切割dsRNA。这项研究的目的是确定这两种蛋白质相互结合所需的氨基酸，以便设计突变果蝇，我可以在其中测试R2D2是否必须在切割过程中与Dicer-2结合，或者更确切地说，必须仅存在于活性RNAi酶复合物生产的后续步骤中。为此，我建议使用反向酵母双杂交筛选来选择不能结合R2D2的Dicer-2突变体。我将进行定量生化测试，以确定一种突变体，该突变体保留了将长双链RNA（dsRNA）切割成小干扰RNA（siRNA）的能力，siRNA是指导RNAi的向导。我将产生具有两种形式的Dicer-2的转基因果蝇：不再结合R2D2但能够切割dsRNA的Dicer-2的突变形式，以及结合R2D2但切割dsRNA的能力减弱的现有突变Dicer-2。使用已建立的技术，我将检查这些苍蝇，以确定是否RNAi可以发生在活的苍蝇时，处理dsRNA到siRNA和加载的siRNA到功能复合物是解耦的。我还将使用分子工具，包括定量RT-PCR和平铺微阵列，来监测通常沉默的遗传元件的水平，并对这些果蝇中的小RNA群体进行高通量测序，以确定分离这些过程的体内后果。