Small Regulatory RNA Functions in the Nucleus

描述（由申请人提供）：1993年，维克托安布罗斯和同事发现了第一个小的调节RNA，lin-4。从那时起，以及随后安迪·法尔、克雷格·梅洛和大卫·鲍尔科姆发现了RNA干扰（RNAi）和小干扰RNA（siRNA），小调控RNA领域朝着没有人能预测的方向发展。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，其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、发育时机、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。dsRNA被Dicer样酶切割成短的20-25个核苷酸的RNA 1。这些小的调节RNA与称为Argonautes（Agos）2的保守蛋白质家族相关。Ago蛋白及其相关的小RNA通过识别和抑制互补核酸来负调控基因表达。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，我们已经在后生动物中进行了第一次正向遗传筛选，试图鉴定细胞核中小RNA介导的沉默（核RNAi）所需的因子。到目前为止，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，这是核RNAi所必需的。nrde-3编码Ago蛋白，其将siRNA从细胞质转运至细胞核3。nrde-2编码一种进化上保守的蛋白质，该蛋白质与核中的NRDE-3相关，并被NRDE-3/siRNA核糖核蛋白复合物引导至已被RNAi靶向的新生转录物。我们的数据表明，小RNA与NRDE-2和NRDE-3共同作用，指导一种新的基因调控模式：RNA聚合酶II转录的终止。这些数据表明，我们的遗传筛选是针对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鉴定和表征细胞核RNAi途径的其他组分，解开细胞核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，以询问这些过程是否在哺乳动物中机械地保守。利用siRNA靶向致癌和病毒mRNA的初步成功已经产生了siRNA最终可用于治疗人类疾病的兴奋4。siRNA处理理论上可用于下调存在于人体内的任何RNA分子，无论是外源的还是内源的。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如何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疗法最终可能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如何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.