RNA interference: The molecular immune system

Introduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo- and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21- to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.     

RNA干扰(RNAi)文库研究进展

RNAi是由双链RNA(dsRNA)引发的转录后基因沉默现象,由dsRNA产生的小分子siRNA会导致生物体内同源转录产物特异性降解,是基因表达调控的重要方式之一。目前RNAi技术已发展成为遗传分析强有力的工具,在基因功能分析鉴定方面发挥越来越大的作用。构建大规模的RNAi文库进而转变成RNAi突变体库是功能基因组学研究的重要手段,因此如何利用简单经济的方法构建特定物种的高效RNAi文库就成为关键问题。综述了目前构建RNAi文库的不同方法以及每种构建方法的优点和存在的不足,为不同研究目的的RNAi文库的构建提供参考。     

RNA interference against viruses: strike and counterstrike

RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA. RNAi holds great promise as a novel nucleic acid-based therapeutic against a wide variety of diseases, including cancer, infectious diseases and genetic disorders. Antiviral RNAi strategies have received much attention and several compounds are currently being tested in clinical trials. Although induced RNAi is able to trigger profound and specific inhibition of virus replication, it is becoming clear that RNAi therapeutics are not as straightforward as we had initially hoped. Difficulties concerning toxicity and delivery to the right cells that earlier hampered the development of antisense-based therapeutics may also apply to RNAi. In addition, there are indications that viruses have evolved ways to escape from RNAi. Proper consideration of all of these issues will be necessary in the design of RNAi-based therapeutics for successful clinical intervention of human pathogenic viruses.     

Dicing and slicing: the core machinery of the RNA interference pathway

RNA interference (RNAi) is broadly defined as a gene silencing pathway that is triggered by double-stranded RNA (dsRNA). Many variations have been described on this theme. The dsRNA trigger can be supplied exogenously, as an experimental tool, or can derive from the genome in the form of microRNAs. Gene silencing can be the result of nucleolytic degradation of the mRNA, or by translational suppression. At the heart of the pathway are two ribonuclease machines. The ribonuclease III enzyme Dicer initiates the RNAi pathway by generating the active short interfering RNA trigger. Silencing is effected by the RNA-induced silencing complex and its RNaseH core enzyme Argonaute. This review describes the discovery of these machines and discusses future lines of work on this amazing biochemical pathway.     

RNA interference for antiviral therapy

Silencing gene expression through a process known as RNA interference (RNAi) has been known in the plant world for many years. In recent years, knowledge of the prevalence of RNAi and the mechanism of gene silencing through RNAi has started to unfold. It is now believed that RNAi serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing RNAi have been found in various viral pathogens. During the past few years, it has been demonstrated that RNAi, induced by specifically designed double-stranded RNA (dsRNA) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. Furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. However, many key questions and obstacles in the translation of RNAi into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsRNA that induces the system. It is expected that for the specific examples in which the delivery issue could be circumvented or resolved, RNAi may hold promise for the development of gene-specific therapeutics.     

RNA干扰及其在动物繁殖研究中的应用

RNA干扰(RNAi)是指小分子双链RNA通过特异性降解与其同源的mRNA,而在mRNA水平上高效阻断体内特异性基因表达的现象,属于转录后水平基因沉默。利用该技术进行基因功能研究,经济有效,通用性好,已成为反向遗传学研究中最重要的工具之一。在动物繁殖领域,RNAi技术主要应用于体外研究哺乳类和禽类卵母细胞发育、胚胎发育和精子形成中重要基因的功能及其作用机制。随着该技术不断发展完善,RNAi必将在动物繁殖生产实践中发挥巨大的作用。     

Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.     

RNAi的作用机制及其临床应用研究

RNAi是指在特定因子的作用下,由导入细胞内的双链RNA(dsRNA)降解成的约22nt左右的siRNA。是近年来发现的可被人工诱导的一种生物体自身所具有的基因沉默现象。因为其相对于其他类似手段有更为突出的高效性、高特异性等优点,而越来越受到人们的重视。已经迅速发展成为研究基因功能的重要工具,并将在对病毒病、肿瘤病、遗传病等的治疗方面发挥着重要作用,因此被《Science》评选为2002年度最重要的科技突破。然而作为一种新的技术它仍有一些不足亟待完善,而在临床应用方面需要进一步深入的研究和验证。     

Targeting genes in living mammals by RNA interference

More than a decade has passed since the discovery of RNA interference (RNAi), an eukaryotic sequence-specific degradation of mRNA induced by complementary double-stranded RNA (dsRNA). RNAi became a common tool for controlled down-regulation of gene expression in cultured cells, as well as in various model organisms. This review summarizes RNAi-based tools for silencing genes in living mammals, which include: (i) transgenic RNAi strategies, where RNAi is triggered by a transgene transmitted through the germline and (ii) approaches, where an RNAi trigger is delivered into an adult animal.     

A phagocytic route for uptake of double-stranded RNA in RNAi

RNA interference (RNAi) has a range of physiological functions including as a defence mechanism against viruses. To protect uninfected cells in a multicellular organism, not only a cell-autonomous RNAi response is required but also a systemic one. However, the route of RNA spread in systemic RNAi remains unclear. Here we show that phagocytosis can be a route for double-stranded RNA uptake. Double-stranded RNA expressed in Escherichia coli induces robust RNAi in Drosophila S2 cells, with effectiveness comparable to that of naked dsRNA. We could separate this phagocytic uptake route from that for RNAi induced by naked dsRNA. Therefore, phagocytic uptake of dsRNA offers a potential route for systemic spread of RNAi.     

Feasibility,limitation and possible solutions of RNAi‐based technology for insect pest control

Abstract Numerous studies indicate that target gene silencing by RNA interference (RNAi) could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi‐mediated crop protection. However, there are many limitations using RNAi‐based technology for pest control, with the effectiveness target gene selection and reliable double‐strand RNA (dsRNA) delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome‐scale high‐throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro‐injection or by feeding as a dietary component. However, micro‐injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi‐mediated crop protection has been considered as a potential new‐generation technology for pest control, or as a complementary method of existing pest control strategies; however, further development to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi‐based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.     

RNA interference: potential therapeutic targets

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi methodologies hold the promise to selectively inhibit gene expression in mammals. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. The use of RNAi for genetic-based therapies has been widely studied, especially in viral infections, cancers, and inherited genetic disorders. As such, RNAi technology is a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics.