Mechanism of RNA-Mediated Control of Transcription or Splicing

描述(申请人提供)：蛋白质控制哺乳动物细胞核中的关键调控通路，这些通路是正常生理和疾病的基础。一些蛋白质识别染色体DNA来调节转录，而另一些蛋白质则与前-mRNA结合来改变剪接。然而，蛋白质缺乏一种普遍的能力，无法高度选择性地识别一个基因，或者快速进化出对新基因的选择性。我们假设RNA可以在染色质上形成网络，起到补充蛋白质转录和剪接因子的作用。长的非编码RNA(LncRNAs)重叠在大多数mRNAs的3‘和5’末端。当lncRNAs被合成时，它们与其相关的蛋白质编码基因非常接近。与染色体DNA中的序列不同，lncRNAs以一种易于通过小RNA进行序列特异性碱基配对和随后影响基因表达的操作来显示序列的形式。目的：我们的长期目标是了解RNA与染色质相互作用以调节转录或剪接的潜力。我们将特别关注ArgAerte(AGO)蛋白，因为它在促进RNA-RNA结合方面发挥着关键作用。目的1.核AGO的作用机制。虽然RNAi在细胞质中得到了很好的研究，但对细胞核中的识别机制几乎一无所知。我们将检查亚细胞定位，链负载，以及在核提取液和染色质上进行标准切割的可能性。这些数据将揭示AGO是如何在细胞核中控制基因表达的。目的2.鉴定和验证与核抗原相关的蛋白质的作用。了解AGO和其他蛋白质之间的相互作用对于了解细胞核的功能是必不可少的。我们将使用质谱学来确定AGO的蛋白质伙伴，并建立一个详细的核控制网络的机制模型。目标3.识别与核抗原相关的RNA。核仁组成蛋白有可能成为细胞核内RNA控制网络的重要组织者。我们将使用抗AGO抗体的RNA免疫沉淀和高通量深度测序作为一种无偏见的方法来识别与AGO相互作用的细胞核中的RNA序列。我们将检查AGO与miRNAs、mRNA和长非编码转录本之间的关联，并预测染色质上的内源RNA相互作用。积极的影响包括：1)提高了对AGO和小RNA如何促进细胞核和染色质中非编码RNA的序列特异性识别的理解；2)确定了AGO的RNA伙伴，这是理解RNA识别如何导致转录剪接改变的关键信息；以及3)对内源RNA介导的细胞核调控途径以及小RNA在治疗或生物技术应用中控制基因表达的潜力的认识。

DESCRIPTION (provided by applicant): Proteins control critical regulatory pathways in mammalian cell nuclei that underlie normal physiology and disease. Some proteins recognize chromosomal DNA to regulate transcription while others bind to pre-mRNA to alter splicing. Proteins, however, lack a general capacity to be highly selective for recognition of just one gene and or rapidly evolve selectivity for new genes. We hypothesize that RNA can form networks on chromatin that act to complement protein transcription and splicing factors. Long noncoding RNAs (lncRNAs) overlap the 3' and 5' termini of most mRNAs. As lncRNAs are synthesized they are in close proximity to their related protein-encoding genes. Unlike sequences within chromosomal DNA, lncRNAs display sequences in a form that is readily accessible to sequence-specific base-pairing by small RNAs and subsequent action to affect gene expression. Objective: Our long-term goal is to understand the potential for RNA to interact with chromatin to regulate transcription or splicing. We will pay specific attention to argonaute (AGO) protein because of its key role in promoting RNA-RNA association. Aim 1. Mechanism of Nuclear AGO. While RNAi is well studied in the cytoplasm almost nothing is known about mechanisms for recognition in the nucleus. We will examine subcellular localization, strand loading, and the potential for stand cleavage in nuclear extracts and on chromatin. These data will reveal how AGO functions in the nucleus to control gene expression. Aim 2. Identify and Validate Action of Proteins Associated with Nuclear AGO. Understanding interactions between AGO and other proteins is essential for comprehending function in the nucleus. We will use mass spectrometry to identify protein partners for AGO and build a detailed mechanistic model of nuclear control networks. Aim 3. Identify RNAs Associated with Nuclear AGO. Nuclear AGO has the potential to be a critical organizer of RNA control networks in cell nuclei. We will use RNA immunoprecipitation with anti-AGO antibodies followed by high throughput deep sequencing as an unbiased approach to identify RNA sequences in nuclei that interact with AGO. We will examine associations between AGO and miRNAs, mRNA, and long noncoding transcripts and predict endogenous RNA interactions on chromatin. Positive impacts include: 1) Improved understanding for how AGO and small RNAs promotes sequence- specific recognition of noncoding RNAs in cell nuclei and on chromatin; 2) Identification of RNA partners for AGO, information critical for understanding how RNA recognition leads to altered splicing of transcription; and 3) An appreciation of endogenous RNA-mediated regulatory pathways in cell nuclei and the potential of small RNAs to control gene expression for therapeutic or biotechnological applications.