Double-stranded RNA-mediated signaling pathway and gene silencing

描述（由申请人提供）：已知细胞中双链RNA （dsRNA）的产生在几乎所有真核生物中激活RNA干扰（RNAi）途径，并在哺乳动物中触发基于转录的干扰素反应。丝状真菌粗神经孢子菌（Neurospora crassa）是一种广泛利用基因沉默来调控基因表达的生物，它为理解RNAi途径及其在真核生物中的功能提供了一个独特而强大的系统。我们发现神经孢子虫的RNAi通路，像那些在高级真核生物，使用小rna介导转录后基因沉默。我们还发现dsRNA导致神经孢子虫中许多基因的转录激活，包括大多数RNAi成分、假定的抗病毒基因和干扰素刺激基因的同源基因。此外，我们发现了一种新型的小RNA，它在DNA损伤后被显著诱导。此外，我们发现DNA损伤导致产生小RNA的位点产生异常RNA。这些研究提示了RNAi在基因沉默和防御机制中的进化保守作用，以及RNAi途径与DNA修复/复制过程之间的新联系。在Specific Aim 1中，我们将确定dsrna诱导基因转录的信号通路。我们将通过正向和反向遗传方法确定这一途径的组成部分。这项研究将有助于了解一种可能与哺乳动物相关的新型dsRNA激活信号通路。在特异性目标2中，我们将确定DNA损伤诱导的小RNA的生物发生和功能。本研究将揭示RNAi在DNA修复和维持基因组稳定性中的作用和机制。此外，我们将探讨DNA损伤后异常RNA产生的机制。这些拟议的研究结合了遗传、生化和生理学的方法，将对理解真核生物基因沉默和防御反应具有重要意义。RNA干扰（RNAi）及其相关途径调控着人类基因的表达、发育、基因组的稳定性和防御反应。dsrna诱导的干扰素应答是人类对病毒感染的先天免疫的重要组成部分。这些通路的损伤可导致发育异常、癌症或免疫缺陷。此外，RNAi技术的发展和采用已广泛应用于药物靶点验证和治疗开发。我们的目标是通过一个简单的真核模型系统来了解这些途径在人类中的功能和调控。更好地了解这些途径将有可能导致治疗人类疾病的新治疗方法。

DESCRIPTION (provided by applicant): Double-stranded RNA (dsRNA) production in cells is known to activate the RNA interference (RNAi) pathway in almost all eukaryotic organisms and to trigger the transcription-based interferon response in mammals. The filamentous fungus Neurospora crassa, an organism that broadly employs gene silencing in regulation of gene expression, offers a unique and powerful system for understanding the RNAi pathway and its function in eukaryotes. We showed that the Neurospora RNAi pathway, like those in higher eukaryotes, uses small RNAs to mediate posttranscriptional gene silencing. We also showed that dsRNA leads to the transcriptional activation of many genes in Neurospora, including most of the RNAi components, putative antiviral genes, and homologs of the interferon stimulated genes. Furthermore, we discovered a novel type of small RNA that is dramatically induced after DNA damage. In addition, we found that DNA damage results in the production aberrant RNA from the loci where small RNAs are produced. These studies suggested evolutionarily conserved roles of RNAi in gene silencing and defense mechanisms and a novel link between RNAi pathway and DNA repair/replication processes. In Specific Aim 1, we will determine the signaling pathway responsible for dsRNA-induced gene transcription. We will identify the components of this pathway by both forward and reverse genetic approaches. This study will lead to the understanding of a novel dsRNA activated signaling pathway that may be relevant in mammals. In Specific Aim 2, we will determine the biogenesis and function of the DNA damage- induced small RNA. This study will reveal the role and the mechanism of RNAi in DNA repair and in maintaining genome stability. In addition, we will investigate the mechanism of aberrant RNA production after DNA damage. These proposed studies, using a combination of genetic, biochemical and physiological approaches, will have important implications for the understanding of eukaryotic gene silencing and defense responses in general. PUBLIC HEALTH RELEVANCE RNA interference (RNAi) and related pathways regulate gene expression, development, genome stability and defense responses in human. The dsRNA-induced interferon response is an important part of the human innate immunity toward viral infection. The impairment of these pathways can result in developmental abnormality, cancer or immune deficiency. Furthermore, development and adoption of RNAi technologies have been extensively used in pharmaceutical target validation and in therapeutic development. Our goal is to understand the function and regulation of these pathways in human using a simple eukaryotic model system. A better understanding of these pathways will potentially lead to new therapeutic approaches for treating human diseases.