Mechanism of RNA-Mediated Control of Transcription or Splicing

描述（由申请人提供）：蛋白质控制哺乳动物细胞核中的关键调控途径，构成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，而另一些蛋白质结合前体mrna来改变剪接。然而，蛋白质缺乏对单一基因的高度选择性识别和对新基因的快速选择性进化的一般能力。我们假设RNA可以在染色质上形成网络，以补充蛋白质转录和剪接因子。长链非编码rna （lncRNAs）重叠在大多数mrna的3‘和5’端。当lncrna被合成时，它们与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以一种易于由小rna进行序列特异性碱基配对并随后影响基因表达的形式显示序列。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argonaute （AGO）蛋白，因为它在促进RNA-RNA结合中起着关键作用。目的1。核AGO机制。虽然在细胞质中对RNAi进行了很好的研究，但对其在细胞核中的识别机制几乎一无所知。我们将研究亚细胞定位，链负载，以及在核提取物和染色质上的分裂潜力。这些数据将揭示AGO如何在细胞核中起作用来控制基因表达。目标2。鉴定和验证核AGO相关蛋白的作用。了解AGO与其他蛋白质之间的相互作用对于理解细胞核中的功能至关重要。我们将使用质谱法鉴定AGO的蛋白质伙伴，并建立核控制网络的详细机制模型。目标3。鉴定与核AGO相关的rna。核AGO具有成为细胞核中RNA控制网络的关键组织者的潜力。我们将使用抗AGO抗体的RNA免疫沉淀，然后使用高通量深度测序作为一种无偏倚的方法来鉴定与AGO相互作用的细胞核中的RNA序列。我们将研究AGO与mirna、mRNA和长链非编码转录物之间的关系，并预测内源性RNA在染色质上的相互作用。积极影响包括：1)提高了对AGO和小rna如何促进细胞核和染色质上非编码rna序列特异性识别的理解；2)鉴定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；3)细胞核内源性rna介导的调控途径的认识，以及小rna在治疗或生物技术应用中控制基因表达的潜力。

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.