Structure-Function Mapping of the Nuclear Pore Complex-Renewal

摘要 核孔复合体(Npc)是一种巨大的大分子集合体，它是 胞核和胞质之间的双向运输。我们已经确定了组成要素，建筑， 并详细介绍了原型酵母NPC的高精度结构。然而，尽管我们不断增加的结构性 关于NPC的信息，我们仍然缺乏对其众多功能的机制的基本了解。 有了我们的详细地图，我们第一次处于一个独特的位置来绘制和揭示结构 与功能状态相关的变化，揭示了NPC关键方面的潜在机制 功能。我们的假设是，尽管有一些重叠，但离散和不同的结构阶段和状态是 与NPC的不同功能相关联。因此，我们建立了一条强大的渠道来分析NPC 以及它们在结构和功能上处于限定的官能态的邻位缔合物 我们绘制定量的表型信息。这些信息将使我们能够从NPC的静态模型 到运转中的机器的工作模型，为我们的NPC地图注入生命，并剖析出 功能在结构层面得到机械支持。我们关注两个这样的功能，它们是 核功能的中心在两个相关的水平上：第一，作为运输的调节者，npc控制着mrna的包装。 并输出到细胞质以调节和调节基因的表达；第二，npc直接控制 通过结合染色质及其调节剂来改变表观遗传表达状态的基因。这两个过程都是 在分子水平上不完全了解，并对细胞功能有深远的影响，这一点得到了证明 与这些功能相关的鼻咽癌相关蛋白的破坏导致许多人类 疾病。对于目标1，我们将通过研究确定NPC介导的mRNP输出的分子机制 在mRNP出口的规定中间阶段，国家方案实际上是“冻结”的。对于目标2，我们将确定 鼻咽癌介导的染色质组织的分子机制，特别是亚端粒基因 沉默。利用我们已建立的渠道，我们将确定和结构性地描述这些NPC阶段和 各州及其周边地区之间的关系。实现这些目标将生成NPC结构-功能图 史无前例的细节，这将对现场了解mRNP如何输出和 染色质重塑机器与NPC的不同部分协同工作，以实现其功能 并将阐明与这些过程中的功能障碍相关的许多障碍的性质。这个 由此产生的结构-功能NPC图有望为挖掘NPC的巨大潜力奠定基础 一种针对多种人类疾病的药物靶点，从癌症到传染病，再到发育和 神经紊乱。 1

ABSTRACT Nuclear pore complexes (NPCs) are huge macromolecular assemblies that serve as the only conduit for bidirectional transport between the nucleus and cytoplasm. We have determined the constituents, architecture, and detailed high precision structure of the archetypal yeast NPC. However, despite our increasing structural information on NPCs, we still lack a fundamental understanding of the mechanics of numerous of its functions. With our detailed maps in hand, we are, for the first time, in a unique position to map and reveal the structural changes associated with functional states that throw light on mechanisms underlying critical aspects of NPC function. Our hypothesis is that, despite some overlap, discrete and distinct structural stages and states are associated with NPCs’ varied functions. We have therefore established a powerful pipeline for analyzing NPCs and their vicinal associated complexes both structurally and functionally in defined functional states onto which we map quantitative phenotypic information. This information will allow us to move from static models of NPCs to working models of the machine in action, breathing life into our NPC maps and dissecting out how particular functionalities are mechanistically supported at the structural level. We focus on two such functionalities that are central to nuclear function at two related levels: first, as a regulator of transport, NPCs control mRNA packaging and export to the cytoplasm to both mediate and regulate gene expression; and second, NPCs directly control genes by binding chromatin and its regulators to alter expression states epigenetically. Both processes are incompletely understood at the molecular level, and have profound effects on cellular function as evidenced by the fact that disruptions of NPC-associated proteins associated with these functions lead to many human diseases. For Aim 1, we will determine the molecular machinery of NPC-mediated mRNP export by studying NPCs effectively “frozen” in defined intermediate stages of mRNP export. For Aim 2, we will determine the molecular machinery of NPC-mediated chromatin organization, specifically focusing on subtelomeric gene silencing. Using our established pipeline, we will identify and structurally characterize these NPC stages and states and their vicinal interactomes. Realizing these Aims will generate NPC structure-function maps in unprecedented detail, which will be of great use to the field to understanding how the mRNP export and chromatin remodeling machineries act in concert with different parts of the NPC to enable their functionalities and will shed light on the nature of numerous disorders associated with dysfunction in these processes. The resulting structure-function NPC maps promise to set the stage for tapping the NPC’s tremendous potential as a drug target for many human conditions ranging from cancers to infectious diseases to developmental and neurological disorders. 1