Functional Sir2-RNA Interactions

描述(由申请人提供)：sirtuins是一个保守的NAD+依赖的蛋白去乙酰酶家族，在与衰老和年龄相关疾病相关的各种细胞过程中发挥功能。由于对NAD+的依赖，sirtuin的活性与细胞的代谢和能量状态密切相关，因此，已被认为有助于调节热量限制的有益效果。因此，人们对调节sirtuin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。Sir2是发芽酵母的创始家族成员，因其在端粒转录沉默、沉默交配型基因和rDNA中的作用而被广泛研究。它也是询问Sir2在寿命调节中的功能的一个杰出的模式系统。在初步实验中，我们已经确定了酵母Sir2的一种新的RNA结合活性，它需要一个完整的锌带结构域。RNA的相互作用在体外也能刺激Sir2的HDAC活性，提示在体内，RNA或其他核酸物种可能对Sir2有正向调节作用。最近的芯片序列数据显示，除了传统的沉默基因外，Sir2还出人意料地与活跃的基因相关联，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。第一个目的是识别Sir2在体内与之相互作用的RNA序列，并确定这种相互作用是否存在序列特异性。在第二个特定目标中，我们建议定量地表征Sir2与RNA之间的结合亲和力，并确定与其他物种的sirtuins的保守水平，例如人类SIRT1，与酵母Sir2的最接近的同源物。为了补充结合实验，将研究RNA刺激HDAC活性的机制，以确定RNA是影响催化(Vmax)还是影响底物结合(Km)。这些探索性和发育性的研究将确定功能Sir2-RNA相互作用的基本机制和特异性，并为未来更详细的功能和结构-功能研究奠定基础。

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.