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

项目摘要/摘要 每个细胞的核是由DNA、RNA和蛋白质组成的复杂排列，这种排列是动态组织的 进入各种核机构和隔间，通常围绕共享的功能和调节 角色。然而，尽管这些核弹舱中的许多在几十年前首次被发现，但一个主要的 表征这些隔室的挑战是，目前还没有生物化学方法来 隔离单独的核隔室。重要的是，许多核机构的标记和维护 核内保留的长非编码RNA(LncRNAs)。在这里，我们的目标是开发几项新技术来 提纯核区的分子成分及其形成的必要性和充分性 车厢。这些技术结合在一起将使我们能够系统地解决以下问题： 目标1：特异地、准确地提纯特定蛋白的最佳生化条件是什么？ 以确定它们的DNA、RNA和蛋白质成分？一位批评者 生物化学净化的一个方面是在识别直接 分子间相互作用，而不是可能导致间接相互作用的过度交联化。使用已知的核 我们将优化RAP以分离核仁、核斑点和副斑点等隔间 DNA-RNA、RNA-RNA、RNA-蛋白质相互作用。我们的目标是确定普遍适用的净化 确定核和其他亚细胞结构内分子相互作用的条件。 目的2：核间隔中涉及的DNA、RNA和蛋白质因子是什么？尽管最著名的是 核体以lncRNAs为特征，其他RNA、DNA和蛋白质因子仍不是很清楚。 在这里，我们将开发技术来对组成各种核体的分子因素进行分类。我们 将通过在核域内对这些组件进行共存来进一步验证这些组件 使用可视化方法(例如，RNA-DNA联合FISH)。总的来说，我们的目标是将新技术应用于 核体中的RNA、DNA和蛋白质的系统和全面目录。 目标3：lncRNAs和蛋白质对于核分区是必要的和/或足够的吗？在这里，我们将 开发一种名为CRISPR-Display的新技术平台，允许添加长RNA货物 并由CRISPR-CAS9系统传递到基因组中的所需位置。我们将开发这项技术来 测试lncRNA分子是否足以驱动核组织。我们还将使用CRISPR-DISPLAY 复合几个可用于招募蛋白质的RNA适配子(具有相互结合的蛋白质表位 RNA适配子)，并测试它们是否足以形成核隔室。同时，我们将执行损失- 建立核结构域需要功能方法来识别蛋白质和/或RNA组分。 总的来说，我们的建议旨在开发强大的、多方面的技术来对分子进行分类 亚核结构的组成部分，验证这些相互作用，并测试它们的生物学重要性。

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.