Mechanisms of large non-coding RNA regulation of gene expression

大非编码RNA基因表达调控机制

• 批准号: 9165109
• 负责人: Colleen Adare McHugh
• 金额: $ 9万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9165109
• 关键词: Address; Affect; Amino Acids; Antisense RNA; Binding; Biological Assay; Cell Nucleus; Cell physiology; Complex; Crystallization; Development; Disease; Dissection; Female; Foundations; Gene Expression; Gene Expression Regulation; Goals; Human; In Vitro; Individual; Knowledge; Lead; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Mentors; Messenger RNA; Methods; Molecular; Molecular Structure; Nucleotides; Outcome; Pathway interactions; Phase; Play; Positioning Attribute; Process; Proteins; RNA Binding; RNA Splicing; RNA, Ribosomal, 18S; Research; Resolution; Ribosomal RNA; Roentgen Rays; Role; Site; Structural Models; Structure; System; Testing; Training; U1 small nuclear RNA; Untranslated RNA; Work; X Chromosome; cell growth; cell growth regulation; human disease; improved; insight; mutant; protein complex; protein function; research study; scaffold; tumor progression

PROJECT SUMMARY Large non-coding RNAs (lncRNAs) comprise a newly recognized class of thousands of functional RNA molecules that play important roles in a wide variety of cellular pathways. These lncRNAs can have significant effects on gene expression, and are involved in controlling regulatory networks critical for cellular growth and differentiation. Many lncRNAs are important players in human diseases, especially human cancers. An emerging hypothesis is that lncRNAs regulate gene expression and cellular function by binding functional protein complexes to organize and coordinate their activities. However, a main challenge in understanding the mechanisms of lncRNA-mediated regulation of gene expression and cellular function has been a lack of knowledge of their protein interaction partners. To address this research bottleneck, I have developed a method to comprehensively define the proteins that directly interact with a lncRNA, termed RNA Antisense Purification with Mass Spectrometry (RAP-MS). This method has so far been highly successful in identifying direct and specific interaction partners of several cellular RNAs, including Xist lncRNA, 18S ribosomal RNA, U1 small nuclear RNA, and 45S pre-ribosomal RNA. In the proposed work I will use RAP-MS to identify the functional protein interactors of three lncRNAs (Tsix, Neat1, and Malat1) then identify the interacting regions of lncRNAs and proteins and create detailed high resolution structural models of lncRNA and protein complexes. Dissection of the sequence determinants of lncRNA-protein interactions at the level of nucleotide and amino acid interactions will lead to an improved understanding of the mechanisms of action of lncRNAs in controlling gene expression and regulating cellular function at a molecular level. The goals of this study are to identify direct and specific lncRNA-interacting proteins using RAP-MS (Aim 1), determine the functions of lncRNA-interacting proteins (Aim 2), and dissect the molecular structure of lncRNA- protein complexes (Aim 3). Taken together, the results of these experiments will reveal how lncRNAs bind and coordinate proteins into higher-order complexes to achieve their roles in regulating gene expression and cellular function. An understanding of functional lncRNA-protein interactions at the level of amino acid and nucleotide interactions will provide insight into the mechanisms of action of lncRNAs and reveal molecular principles guiding lncRNA-protein assembly. In addition, identification of the underlying principles of lncRNA- protein function at a detailed molecular level will lead to new opportunities to control and manipulate the action of non-coding RNAs in mammalian cells during the initiation and progression of cancer and other disease processes.

项目摘要 大型非编码RNA（largenon-codingRNA，lncRNA）是近年来发现的一类功能性RNA 在多种细胞途径中发挥重要作用的分子。这些lncRNA可以具有显著的 对基因表达的影响，并参与控制对细胞生长至关重要的调控网络， 分化许多lncRNA是人类疾病，特别是人类癌症的重要参与者。一个 一种新的假说是lncRNA通过结合蛋白质来调节基因表达和细胞功能， 功能性蛋白质复合物来组织和协调它们的活动。然而，一个主要挑战是， 了解lncRNA介导的基因表达和细胞功能调节机制， 缺乏对蛋白质相互作用伙伴的了解。为了解决这个研究瓶颈，我 开发了一种方法来全面定义直接与lncRNA（称为RNA）相互作用的蛋白质 质谱法反义纯化（RAP-MS）。到目前为止，这种方法非常成功， 鉴定几种细胞RNA的直接和特异性相互作用伴侣，包括Xist lncRNA、18 S 核糖体RNA、U1小核RNA和45 S前核糖体RNA。在拟议的工作中，我将使用RAP-MS 为了鉴定三种lncRNA（Tsix，Neat 1和Malat 1）的功能性蛋白质相互作用物， lncRNA和蛋白质的相互作用区域，并创建详细的lncRNA的高分辨率结构模型 和蛋白质复合物。在蛋白质水平上剖析lncRNA-蛋白质相互作用的序列决定簇 核苷酸和氨基酸的相互作用将导致更好地理解的作用机制， lncRNA在分子水平上控制基因表达和调节细胞功能。这个的目标 研究是使用RAP-MS鉴定直接和特异性lncRNA相互作用蛋白（目的1），确定 lncRNA相互作用蛋白的功能（目的2），并剖析lncRNA的分子结构， 蛋白质复合物（Aim 3）。总之，这些实验的结果将揭示lncRNA如何结合 并将蛋白质协调成更高级的复合物，以实现其在调节基因表达中的作用， 细胞功能在氨基酸水平上理解功能性lncRNA-蛋白质相互作用， 核苷酸相互作用将提供对lncRNA作用机制的深入了解，并揭示分子生物学机制。 指导lncRNA-蛋白质组装的原理。此外，lncRNA的基本原理的鉴定- 在详细的分子水平上研究蛋白质的功能，将为控制和操纵蛋白质的作用提供新的机会。 在癌症和其他疾病的发生和发展过程中哺乳动物细胞中的非编码RNA 流程.