Functional Sir2-RNA Interactions

描述（由申请人提供）：Sirtuins是保守的NAD+依赖性蛋白脱乙酰酶家族，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，沉默调节蛋白的活性与细胞的代谢和能量状态密切相关，因此，已被提议帮助介导热量限制的有益效果。因此，人们对表征调节沉默调节蛋白活性的其他机制有很大兴趣，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。Sir 2来自芽殖酵母，酿酒酵母，是创始家族成员，并已被广泛研究其在端粒，沉默交配型基因座和rDNA的转录沉默的作用。它也是一个出色的模型系统，用于询问Sir 2在寿命调节中的功能。在初步实验中，我们已经确定了一种新的酵母Sir 2的RNA结合活性，需要一个完整的锌带结构域。RNA的相互作用也刺激了体外Sir 2的HDAC活性，这表明RNA或其他可能的核酸物质可以在体内正向调节Sir 2。最近的ChIP-Seq数据显示，除了传统的沉默位点之外，Sir 2还与活性基因相关联，从而支持Sir 2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。第一个是设计来鉴定Sir 2在体内相互作用的RNA序列，并确定是否存在相互作用的任何序列特异性。在第二个具体的目标，我们建议定量表征Sir 2和RNA之间的结合亲和力，并确定与其他物种，如人SIRT 1，最接近的直系同源酵母Sir 2的sirtuins的保护水平。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的研究将确定的基本机制和特异性的功能Sir 2-RNA相互作用，并设置我们在未来更详细的功能和结构-功能的研究。

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.