Small Regulatory RNA Functions in the Nucleus

描述(申请人提供)：1993年，Victor Ambros和他的同事发现了第一个小的调节性RNA，LIN-4。自那时以来，以及随后由Andy Fire、Craig Mello和David Baulcombe发现的RNA干扰(RNAi)和小干扰RNAs(SiRNAs)，小调节RNA领域以任何人无法预测的方向扩展。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在非常广泛的生物学过程中发挥作用，包括但不限于：异染色质形成、发育时序、对寄生核酸的防御和基因组重排。RNAi的机械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RNAs 1。这些小的调控RNAs与一个保守的蛋白质家族有关，被称为ArgAertes(AGOS)2。AGO蛋白和它们相关的小RNAs一起，通过识别和抑制互补核酸来负调控基因的表达。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正向遗传筛查，试图确定核内小RNA介导的沉默所需的因素(核RNAi)。到目前为止，我们的筛选已经确定了两个进化保守的因子，核RNAi缺陷-2(nrde-2)和nrde-3，这是核RNAi所必需的。NRDE-3编码一种AGO蛋白，将siRNAs从细胞质运输到细胞核3。NRDE-2编码一种在进化上与NRDE-3有关的蛋白质，由NRDE-3/siRNA核糖核蛋白复合体定向到RNAi靶向的新生转录产物。我们的数据表明，小RNA与NRDE-2和NRDE-3共同作用，指导了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，我们的基因筛查针对的是一个专门的核沉默途径，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。我们在这项资助中提出的实验旨在识别和表征核RNAi途径的其他成分，解开核RNAi的机制，并开始阐明小RNA介导的后生动物核基因沉默的理由。这些实验将为我们和其他人提供一个框架，以询问这些过程在哺乳动物中是否机械保守。利用siRNA靶向致癌和病毒mRNAs的初步成功已经引起了人们的兴奋，即siRNAs最终可能被用于治疗人类疾病4。理论上，siRNA治疗可以用于下调存在于人体内的任何RNA分子，无论是外来的还是内源性的。然而，在合理使用siRNAs治疗人类疾病之前，我们必须了解：小RNA是如何产生的，小RNA如何发挥作用以及在哪里发挥作用，小RNA驱动沉默的特异性，以及小RNA在内源生物过程中的作用。我们的研究正在解决这些问题。与公共卫生相关：siRNA疗法最终可能被用于调节人类的基因表达。然而，在合理使用siRNAs治疗人类疾病之前，我们必须了解：小RNA是如何产生的，小RNA如何发挥作用以及在哪里发挥作用，小RNA驱动沉默的特异性，以及小RNA在内源生物过程中的作用。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.