Mechanisms of large non-coding RNA regulation of gene expression

项目总结 大的非编码RNA(LncRNAs)包括一类新发现的数千种功能RNA 在多种细胞通路中发挥重要作用的分子。这些LncRNA可能具有显著的 对基因表达的影响，并参与控制对细胞生长和 差异化。许多lncRNA在人类疾病，特别是人类癌症中扮演着重要角色。一个 新出现的假说是lncRNAs通过结合来调节基因表达和细胞功能。 功能蛋白质复合体，以组织和协调其活动。然而，一个主要的挑战是 了解了lncRNA介导的基因表达和细胞功能调节的机制 对他们的蛋白质相互作用伙伴缺乏了解。为了解决这一研究瓶颈，我有 开发了一种全面定义直接与lncRNA相互作用的蛋白质的方法，称为rna。 反义质谱仪(RAP-MS)。到目前为止，这种方法在 识别几个细胞RNA的直接和特定相互作用伙伴，包括Xist lncRNA，18S 核糖体RNA、U1小核RNA和45S前核糖体RNA。在提议的工作中，我将使用RAP-MS 为了确定三个lncRNAs(TSix、Neat1和MALAT1)的功能蛋白相互作用，然后确定 LncRNA和蛋白质的相互作用区域，并创建详细的高分辨率lncRNA结构模型 和蛋白质复合体。在基因水平上剖析LncRNA-蛋白质相互作用的序列决定因素 核苷酸和氨基酸的相互作用将使人们更好地理解 LncRNAs在分子水平上调控基因表达和调节细胞功能。这样做的目的是 研究是使用RAP-MS(目标1)鉴定直接和特定的lncRNA相互作用蛋白，确定 LncRNA相互作用蛋白的功能(目标2)，并剖析了lncRNA-2的分子结构 蛋白质复合体(目标3)。综上所述，这些实验的结果将揭示lncRNA是如何结合的 并将蛋白质协调成更高阶的复合体，以实现它们在调节基因表达和 细胞功能。在氨基酸和蛋白质水平上对功能性lncRNA-蛋白质相互作用的理解 核苷酸相互作用将深入了解lncRNAs的作用机制，并揭示分子 指导lncRNA-蛋白质组装的原则。此外，确定lncRNA的基本原理- 在详细的分子水平上的蛋白质功能将导致新的机会来控制和操纵这种行为 在癌症和其他疾病的发生和发展过程中哺乳动物细胞中非编码RNA的表达 流程。

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.