Deciphering the function and mechanisms of lncRNA-mediated organization of nuclear compartments

项目概要/摘要 每个细胞的细胞核都是由DNA、RNA和蛋白质组成的复杂结构， 分成不同的核体和隔室，这些核体和隔室通常围绕着共同的功能和调节 角色然而，虽然许多这些核隔间在几十年前就被首次发现， 表征这些区室的挑战是目前没有生物化学方法来表征这些区室。 隔离出各个核隔间重要的是，许多核体是由 核内长链非编码RNA（lncRNA）。在这里，我们的目标是开发几种新的技术， 纯化核结构域的分子成分以及它们在形成这些核结构域中的必要性和充分性。 隔间这些技术将使我们能够系统地解决以下问题： 目的1：特异性和准确纯化特异性的最佳生化条件是什么 核体和隔室，以确定它们的DNA，RNA和蛋白质成分？一个关键 生物化学纯化的一个方面是识别直接生物化学性质的交联条件之间的精细平衡。 分子相互作用，而不是过度交联，这可能导致间接相互作用。使用已知的核 隔室，如核仁，核斑点，paraspeckles，我们将优化RAP分离 DNA-RNA，RNA-RNA，RNA-protein interactions.我们的目标是确定普遍适用的净化 条件，以确定核和其他亚细胞结构内的分子相互作用。 目的2：什么是DNA，RNA和蛋白质因子参与核隔室？虽然大多数人都知道 核体的特征在于lncRNA，其它RNA、DNA和蛋白质因子仍然不太明确。 在这里，我们将开发技术来分类组成各种核体的分子因子。我们 将通过核域中这些组件的共定位来进一步验证这些组件 使用可视化方法（例如RNA-DNA Co-FISH）。总的来说，我们的目标是将新技术应用于 核体中RNA、DNA和蛋白质系统和全面目录。 目的3：lncRNA和蛋白质是否是核区室化所必需和/或足够的？这里我们将 开发一种新的技术平台，称为CRISPR-Display，它允许将长RNA货物附加到 并通过CRISPR-Cas9系统递送至基因组中的期望位点。我们将开发这项技术， 测试lncRNA分子是否足以驱动核组织。我们还将使用CRISPR-Display 多路复用几种RNA适体，其可用于募集蛋白质（具有结合蛋白质表位的互补蛋白质表位 RNA适体），并测试它们是否足以形成核隔室。同时，我们将执行损失- 需要鉴定蛋白质和/或RNA组分的功能方法来建立核结构域。 总的来说，我们的建议旨在开发强大的和多方面的技术来分类分子 亚核结构的组成部分，验证这些相互作用，并测试其生物重要性。

Project Summary/Abstract The nucleus of each cell is a complex arrangement of DNA, RNA, and protein that is dynamically organized into various nuclear bodies and compartments that are often arranged around shared functional and regulatory roles. Yet, while many of these nuclear compartments were first identified several decades ago a major challenge with characterizing these compartments is that there are currently no biochemical methods for isolating individual nuclear compartments. Importantly, many nuclear bodies are marked and maintained by nuclear retained long non-coding RNAs (lncRNAs). Here we aim to develop several novel technologies to purify the molecular constituents of nuclear domains and their necessity and sufficiency in forming these compartments. Together these technologies will allow us to systematically address the following questions: Aim 1: What are the optimal biochemical conditions in which to specifically and accurately purify specific nuclear bodies and compartments in order to identify their DNA, RNA and protein components? A critical aspect of biochemical purifications is the fine balance between cross-linking conditions that identify direct molecular interactions while not over-crosslinking that could result in indirect interactions. Using known nuclear compartments such as the nucleolus, nuclear speckles, and paraspeckles we will optimize RAP for isolating DNA-RNA, RNA-RNA, RNA-protein interactions. We aim to identify universally applicable purification conditions to identify molecular interactions within nuclear and other subcellular structures. Aim 2: What are the DNA, RNA and protein factors involved in nuclear compartments? Although most known nuclear bodies are characterized by lncRNAs the other RNA, DNA and protein factors remain less well defined. Here we will develop technologies to catalog the molecular factors that comprise various nuclear bodies. We will further validate these components through colocalization of these components within a nuclear domain using visualization approaches (e.g. RNA-DNA Co-FISH). Overall we aim to apply new technologies to systematic and comprehensive catalog of RNA, DNA and Proteins in nuclear bodies. Aim 3: Are lncRNAs and proteins necessary and/or sufficient for nuclear compartmentalization? Here we will develop a novel technology platform, termed CRISPR-Display, which allows long RNA cargos to be appended and delivered by CRISPR-Cas9 systems to a desired site in the genome. We will develop this technology to test if lncRNA molecules are sufficient to drive nuclear organization. We will also use CRISPR-Display to multiplex several RNA aptamers that can be used to recruit proteins (with reciprocal protein epitopes that bind RNA aptamers) and test if they are sufficient to form nuclear compartments. In parallel we will perform loss-of- function approaches to identify protein and or RNA components are required for establishing nuclear domains. Collectively our proposal aims to develop powerful and multifaceted technologies to catalog the molecular components of subnuclear structures, validate these interactions and test their biological importance.