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

破译lncRNA介导的核区室组织的功能和机制

• 批准号: 9767760
• 负责人: Mitchell Guttman
• 金额: $ 74.96万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9767760
• 关键词: Address; Antisense RNA; Binding; Biochemical; Biogenesis; Biological; CRISPR/Cas technology; Catalogs; Cell Nucleolus; Cell Nucleus; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Detection; Development; Epitopes; Equilibrium; Gene Expression Regulation; Genes; Genome; Genomics; Health; Human; Image Analysis; Imagery; Individual; Lamins; Link; Location; MALAT1 gene; Maps; Measures; Mediating; Messenger RNA; Methods; Molecular; Monitor; Nuclear; Nuclear Structure; Polycomb; Post-Transcriptional Regulation; Property; Proteins; Proteomics; RNA; RNA Binding; RNA Processing; RNA Sequences; RNA Splicing; RNA-Protein Interaction; Regulation; Research; Ribosomal RNA; Role; Site; Structure; Subcellular structure; System; Technology; Testing; Untranslated RNA; Work; aptamer; biochemical tools; crosslink; gene interaction; human disease; insight; loss of function; molecular imaging; new technology; novel; nuclear imaging; public health relevance; recruit; single molecule; tool

 DESCRIPTION (provided by applicant): 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. Usin 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.

 描述(申请人提供)：每个细胞的核是DNA、RNA和蛋白质的复杂排列，动态地组织成各种核体和隔间，通常围绕共同的功能和调节角色排列。然而，虽然许多核隔室是在几十年前首次发现的，但确定这些核隔室的一个主要挑战是，目前还没有分离单个核隔室的生化方法。重要的是，许多核体是由核保留的长非编码RNA(LncRNAs)标记和维持的。在这里，我们的目标是开发几种新的技术来纯化核结构域的分子成分，以及它们形成这些间隔的必要性和充分性。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：在什么最佳生化条件下专门和准确地提纯特定的核体和隔室，以便确定它们的DNA、RNA和蛋白质成分？生化净化的一个关键方面是在识别直接连接的交联条件之间的精细平衡 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。使用已知的核隔室，如核仁、核斑点和核小球，我们将优化RAP来分离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纯化条件，以确定核和其他亚细胞结构中的分子相互作用。目的2：核间隔中涉及的DNA、RNA和蛋白质因子是什么？虽然大多数已知的核体具有lncRNAs的特征，但其他RNA、DNA和蛋白质因子仍然没有得到很好的定义。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。我们将使用可视化方法(例如RNA-DNA Co-FISH)通过在核域中共定位这些组件来进一步验证这些组件。总体而言，我们的目标是将新技术应用于系统和全面的核体RNA、DNA和蛋白质目录。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，它允许CRISPR-Cas9系统将长RNA货物附加并运送到基因组中所需的位置。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多路传输几个RNA适配子，这些RNA适配子可用于招募蛋白质(与RNA适配子结合的相互蛋白质表位)，并测试它们是否足以形成核间隔。同时，我们将执行功能丧失方法来确定建立核结构域所需的蛋白质和/或RNA组分。总的来说，我们的建议旨在开发强大和多方面的技术，以编目亚核结构的分子成分，验证这些相互作用，并测试它们的生物学重要性。