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

 描述（由申请人提供）：每个细胞的细胞核是DNA、RNA和蛋白质的复杂排列，动态组织成各种核体和隔室，这些核体和隔室通常围绕共同的功能和调节作用排列。然而，虽然许多这些核隔室是几十年前首次确定的，但表征这些隔室的一个主要挑战是，目前还没有分离单个核隔室的生物化学方法。重要的是，许多核体由核保留长非编码RNA（lncRNA）标记和维持。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，以及它们在形成这些隔室中的必要性和充分性。这些技术将使我们能够系统地解决以下问题：目标1：什么是最佳的生化条件，在其中具体和准确地纯化特定的核体和车厢，以确定其DNA，RNA和蛋白质成分？生物化学纯化的一个关键方面是识别直接化学反应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。利用已知的核区室，如核仁，核斑点，和paraspeckles，我们将优化RAP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？尽管大多数已知的核体的特征在于lncRNA，其他RNA，DNA和蛋白质因子仍然不太清楚。在这里，我们将开发技术来分类组成各种核体的分子因子。我们将通过使用可视化方法（例如RNA-DNA Co-FISH）在核域内共定位这些组件来进一步验证这些组件。总的来说，我们的目标是将新技术应用于核体中RNA，DNA和蛋白质的系统和全面的目录。目的3：lncRNA和蛋白质是否是核区室化所必需和/或足够的？在这里，我们将开发一种新的技术平台，称为CRISPR-Display，它允许通过CRISPR-Cas9系统将长RNA货物附加并递送到基因组中的所需位点。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来多路复用几种RNA适体，这些适体可用于招募蛋白质（具有结合RNA适体的相互蛋白质表位），并测试它们是否足以形成核隔室。与此同时，我们将进行功能丧失的方法，以确定蛋白质和/或RNA组件所需的建立核域。总的来说，我们的提案旨在开发强大而多方面的技术，对亚核结构的分子组成进行编目，验证这些相互作用并测试其生物重要性。

 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.