Small Regulatory RNA Functions in the Nucleus

描述（由申请人提供）：1993年Victor Ambros及其同事发现了第一个小调控RNA lin-4。从那时起，以及随后Andy Fire、Craig Mello和David Baulcombe发现RNA干扰（RNAi）和小干扰RNA （sirna）之后，小调控RNA领域向无人能预测的方向扩展。我们现在知道，大多数真核细胞表达各种各样的内源性小调控rna，这些rna在非常广泛的生物过程中起作用，包括但不限于：异染色质形成、发育定时、对寄生核酸的防御和基因组重排。RNAi的机制基础在真核生物中广泛保守。dsRNAs被dicer样酶切割成短的20-25个核苷酸的rna 1。这些小的调控rna与一个被称为Argonautes (Agos) 2的保守蛋白家族有关。总之，Ago蛋白及其相关的小rna通过识别和抑制互补核酸负性调节基因表达。我们的长期目标是了解小rna和RNAi的方式和原因。为了实现这一目标，我们在后生动物中进行了第一次前向遗传筛选，试图确定核中小RNA介导的沉默（核RNAi）所需的因素。迄今为止，我们的筛选已经确定了两个进化上保守的因子，核RNAi缺陷-2 （nrde-2）和nrde-3，它们是核RNAi所必需的。nrde-3编码一种Ago蛋白，将sirna从细胞质转运到细胞核。nrde-2编码一种进化上保守的蛋白，该蛋白在细胞核中与NRDE-3结合，并由NRDE-3/siRNA核糖核蛋白复合物引导到RNAi靶向的新生转录物。我们的数据表明，小RNA与NRDE-2和NRDE-3一起作用，指导一种新的基因调控模式：终止RNA聚合酶II的转录。这些数据表明，我们的基因筛选是针对一个专用的核沉默途径，并暗示我们正在定义的核沉默途径代表了一种新的和保守的基因调控模式。我们在本基金中提出的实验旨在鉴定和表征核RNAi途径的其他成分，揭示核RNAi的机制，并开始阐明后生动物细胞核中小rna介导的基因沉默的原因。这些实验将为我们和其他人提供一个框架，来询问这些过程在哺乳动物中是否在机械上保守。利用sirna靶向致癌和病毒mrna的初步成功让人兴奋，sirna最终可能用于治疗人类疾病4。siRNA处理理论上可以用于下调人体内存在的任何RNA分子，无论是外来的还是内源性的。然而，在合理使用sirna治疗人类疾病之前，我们必须了解：小rna是如何产生的，小rna是如何和在哪里起作用的，小rna驱动沉默的特异性，以及小rna在内源性生物过程中的作用。我们的研究正在解决这些问题。公共卫生相关性：siRNA疗法可能最终用于调节人类基因表达。然而，在合理使用sirna治疗人类疾病之前，我们必须了解：小rna是如何产生的，小rna是如何和在哪里起作用的，小rna驱动沉默的特异性，以及小rna在内源性生物过程中的作用。我们的研究正在解决这些问题。

DESCRIPTION (provided by applicant): In 1993 Victor Ambros and colleagues discovered the first small regulatory RNA, lin-4. Since that time and the subsequent discovery of RNA interference (RNAi) and small interfering RNAs (siRNAs) by Andy Fire, Craig Mello, and David Baulcombe, the small regulatory RNA field has expanded in directions no one could have predicted. We now know that most eukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in a remarkably wide range of biological processes, including but not limited to: heterochromatin formation, developmental timing, defense against parasitic nucleic acids, and genome rearrangement. The mechanistic underpinnings of RNAi are broadly conserved across eukaryotes. dsRNAs are cleaved by Dicer-like enzymes into short 20-25 nucleotide RNAs 1. These small regulatory RNAs associate with a conserved family of proteins termed the Argonautes (Agos) 2. Together, Ago proteins, and their associated small RNAs, negatively regulate gene expression by recognizing and inhibiting complementary nucleic acids. Our long-term goal is to understand the how and why of small RNAs and RNAi. Towards this goal we have conducted the first forward genetic screen in metazoans seeking to identify factors required for small RNA- mediated silencing in the nucleus (nuclear RNAi). To date our screen has identified two evolutionarily conserved factors, nuclear RNAi defective-2 (nrde-2) and nrde-3, which are required for nuclear RNAi. nrde-3 encodes an Ago protein that transports siRNAs from the cytoplasm to the nucleus 3. nrde-2 encodes an evolutionarily conserved protein that associates with NRDE-3 in the nucleus and is directed by NRDE-3/siRNA ribonucleoprotein complexes to nascent transcripts that have been targeted by RNAi. Our data indicate that small RNAs, acting in conjunction with NRDE-2 and NRDE-3, direct a novel mode of gene regulation: termination of RNA Polymerase II transcription. These data suggest that our genetic screen is targeting a dedicated nuclear silencing pathway and hint that the nuclear silencing pathway we are defining represents a novel and conserved mode of gene regulation. The experiments we propose in this grant are designed to identify and characterize additional components of the nuclear RNAi pathway, unravel the mechanism of nuclear RNAi, and begin to elucidate the raison d'jtre of small RNA-mediated gene silencing in metazoan nuclei. These experiments will provide a framework for us, and others, to ask if these processes are mechanistically conserved in mammals. Initial successes utilizing siRNAs to target oncogenic and viral mRNAs have generated excitement that siRNAs may be utilized eventually to treat human disease 4. siRNA treatment could theoretically be used to down- regulate any RNA molecule, either foreign or endogenous, present within the human body. Prior to the rational use of siRNAs in the treatment of human disease, however, it is essential that we understand: how small RNAs are generated, how and where small RNAs function, the specificity of small RNA-driven silencing, and the role of small RNAs in endogenous biological processes. Our research is addressing these questions. PUBLIC HEALTH RELEVANCE: siRNA therapeutics may eventually be used to regulate gene expression in humans. Prior to the rational use of siRNAs in the treatment of human disease, however, it is essential that we understand: how small RNAs are generated, how and where small RNAs function, the specificity of small RNA-driven silencing, and the role of small RNAs in endogenous biological processes. Our research is addressing these questions.