Functional Sir2-RNA Interactions

Sir2-RNA 功能相互作用

• 批准号: 8511215
• 负责人: Jeffrey Scott Smith
• 金额: $ 23.7万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8511215
• 关键词: Affinity; Age; Aging; Agonist; Animal Model; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Caenorhabditis elegans; Caloric Restriction; Calorimetry; Catalysis; Cell physiology; Cells; ChIP-seq; Characteristics; Chromatin; Complement; Complementary DNA; Consensus; DNA; Data; Deacetylase; Dependence; Detection; Disease; Double-Stranded RNA; Drosophila genus; Environment; Enzymes; Eukaryota; Exploratory/Developmental Grant; Family; Family member; Fluorescence Anisotropy; Functional RNA; Future; Gene Targeting; Genes; Genetic Transcription; Goals; Health; High Pressure Liquid Chromatography; Histone Deacetylase; Human; Hybrids; In Vitro; Kinetics; Link; Longevity; Maintenance; Malignant Neoplasms; Mammals; Mating Types; Mediating; Metabolic; Methods; Neurodegenerative Disorders; Non-Insulin-Dependent Diabetes Mellitus; Nucleic Acid Binding; Nucleic Acids; Oligonucleotides; Open Reading Frames; Organism; Orthologous Gene; Pattern; Play; Population; Post-Translational Protein Processing; Proteins; RNA; RNA Binding; RNA Sequences; Reaction; Recombinant DNA; Recombinants; Regulation; Research Project Grants; Role; Saccharomyces cerevisiae; Saccharomycetales; Sir2-like Deacetylases; Sirtuins; Specificity; Structure; System; Techniques; Technology; Testing; Therapeutic; Titrations; Transfer RNA; Yeasts; Zinc; crosslink; deep sequencing; design; in vivo; inhibitor/antagonist; interest; mutant; novel; protein protein interaction; public health relevance; research study; small molecule; telomere; therapeutic target

DESCRIPTION (provided by applicant): The Sirtuins are a conserved family of NAD+-dependent protein deacetylases that function in a wide variety of cellular processes related to aging and age-associated disease. Due to their dependence on NAD+, sirtuin activity is closely linked to the metabolic and energy status of the cell, and therefore, has been proposed to help mediate the beneficial effects of caloric restriction. As a result, there is significant interest i characterizing additional mechanisms by which sirtuin activity is modulated, whether it is through protein-protein interactions, post-translational modifications, or via small molecule agonists and inhibitors. Sir2 from the budding yeast, Saccharomyces cerevisiae, is the founding family member and has been extensively studied for its role in transcriptional silencing at telomeres, the silent mating-type loci, and the rDNA. It has also served as an outstanding model system for interrogating Sir2 function in lifespan regulation. In preliminary experiments we have identified a novel RNA binding activity for yeast Sir2 that requires an intact zinc ribbon domain. The RNA interaction also stimulates the HDAC activity of Sir2 in vitro, suggesting that RNA or possibly other nucleic acid species may positively regulate Sir2 in vivo. Recent ChIP-Seq data has revealed that Sir2 surprisingly associates with active genes, in addition to the traditional silenced loci, thus supporting the hypothesis that Sir2 and RNA may functionally interact. In this exploratory project we propose two specific aims. The first is designed to identify RNA sequences that Sir2 interacts with in vivo, and to determine if there is any sequence specificity to the interaction. In the second specific aim, we propose to quantitatively characterize the binding affinity between Sir2 and RNA, and to determine the level of conservation with sirtuins from other species such as human SIRT1, the closest ortholog to yeast Sir2. To complement the binding experiments, the mechanism by which RNA stimulates HDAC activity will be investigated to determine whether RNA impacts catalysis (Vmax) or substrate binding (Km). These exploratory and developmental studies will define the basic mechanism and specificity of the functional Sir2-RNA interaction and set us up for more detailed functional and structure-function studies in the future.

描述（由申请人提供）：Sirtuins 是 NAD+ 依赖性蛋白脱乙酰酶的保守家族，其在与衰老和年龄相关疾病相关的多种细胞过程中发挥作用。由于它们对 NAD+ 的依赖性，sirtuin 活性与细胞的代谢和能量状态密切相关，因此，有人建议帮助调节热量限制的有益作用。因此，人们对表征调节沉默调节蛋白活性的其他机制非常感兴趣，无论是通过蛋白质-蛋白质相互作用、翻译后修饰，还是通过小分子激动剂和抑制剂。来自芽殖酵母（酿酒酵母）的 Sir2 是该家族的创始成员，因其在端粒、沉默交配型基因座和 rDNA 转录沉默中的作用而被广泛研究。它还作为询问 Sir2 在寿命调节中的功能的杰出模型系统。在初步实验中，我们发现了酵母 Sir2 的一种新的 RNA 结合活性，它需要完整的锌带结构域。 RNA 相互作用还在体外刺激 Sir2 的 HDAC 活性，这表明 RNA 或可能的其他核酸种类可能在体内正向调节 Sir2。最近的 ChIP-Seq 数据显示，除了传统的沉默位点之外，Sir2 还与活性基因出人意料地相关，从而支持了 Sir2 和 RNA 可能在功能上相互作用的假设。在这个探索性项目中，我们提出了两个具体目标。第一个旨在识别 Sir2 在体内相互作用的 RNA 序列，并确定这种相互作用是否存在任何序列特异性。在第二个具体目标中，我们建议定量表征 Sir2 和 RNA 之间的结合亲和力，并确定来自其他物种（例如人类 SIRT1）（与酵母 Sir2 最接近的直系同源）的去乙酰化酶的保守水平。为了补充结合实验，将研究 RNA 刺激 HDAC 活性的机制，以确定 RNA 是否影响催化 (Vmax) 或底物结合 (Km)。这些探索性和发展性研究将定义 Sir2-RNA 功能相互作用的基本机制和特异性，并为我们将来进行更详细的功能和结构功能研究奠定基础。