RNA interference has a role in regulating Drosophila telomeres

Unlike many other organisms, Drosophila maintains its telomeres by the transposition of retrotransposons to chromosome ends. Recent work shows that proteins in the RNA interference pathway specifically regulate the expression of these retrotransposons and frequency of transposition in germline cells, but do not affect retrotransposon expression or telomere function in the soma.     

RNA干扰

RNA干扰（RNA interference,RNAi）现象是指,当与内源性mRNA编码区某段序列同源的双链RNA(dsRNA)导入细胞后,该mRNA发生特异性的降解,而导致该基因表达的沉寂。这可能反映了生物防范病毒或转座子诱导DNA突变的一种防御机制。RNA干扰已经成为一种重要的研究基因功能的有力工具,并且有希望在对疾病的防御及治疗中发挥重要的作用。     

RNA干涉的研究进展

生物体内导入双链RNA后会引起体内同源基因特异性的沉默,这种现象称为RNA干涉,本主要介绍RNA干涉的研究历史,作用机制和应用等方面的情况。     

RNA干涉技术

RNA干涉(RNAi)技术是利用一些小的双链RNA来高效、特异地阻断体内特定基因的表达,并促使mRNA降解,从而诱使细胞表现出特定基因缺失的表型。本从RNAi技术的历史、作用机制、研究策略、研究现状及应用前景等几个方面进行了综述,预测RNAi将会给基因治疗的发展带来新的希望。     

RNA干扰与技术

RNA干扰(RNA interference,RNAi)是由双链RNA诱导的、序列特异的基因沉默机制。它是自然存在于植物、线虫和果蝇中抵抗外来基因(包括病毒、转座子)入侵的方式。在哺乳动物细胞中,能够人工诱导RNA干扰,沉默有同源序列基因表达。这一新技术具有特异性、高效性。因此,正被用来研究人类基因组的功能、肿瘤和抗病毒感染等。     

Environmental RNA interference

The discovery of RNA interference (RNAi), the process of sequence-specific gene silencing initiated by double-stranded RNA (dsRNA), has broadened our understanding of gene regulation and has revolutionized methods for genetic analysis. A remarkable property of RNAi in the nematode Caenorhabditis elegans and in some other multicellular organisms is its systemic nature: silencing signals can cross cellular boundaries and spread between cells and tissues. Furthermore, C. elegans and some other organisms can also perform environmental RNAi: sequence-specific gene silencing in response to environmentally encountered dsRNA. This phenomenon has facilitated significant technological advances in diverse fields including functional genomics and agricultural pest control. Here, we describe the characterization and current understanding of environmental RNAi and discuss its potential applications.     

Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo

We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.     

Induction of RNA interference genes by double-stranded RNA; implications for susceptibility to RNA interference

Gene silencing by RNA interference (RNAi) can be a useful reverse genetics tool in eukaryotes. However, some species appear refractory to RNAi. To study the role of the differential expression of RNAi proteins in RNAi, we isolated partial dicer-2, argonaute-2 translin, vasa intronic gene (VIG) and tudor staphylococcus/micrococcal nuclease (TSN) genes from the tobacco hornworm, Manduca sexta, a well-studied insect model which we have found to be variably sensitive to RNAi. We found that the RNAi gene, translin, was expressed at minimal levels in M. sexta tissue and that there is a specific, dose-dependent upregulation of dicer-2 and argonaute-2 expression in response to injection with dsRNA, but no upregulation of the other genes tested. Upregulation of gene expression was rapid and transient. In order to prolong the upregulation we introduced multiple doses of dsRNA, resulting in multiple peaks of dicer-2 gene expression. Our results have implications for the design of RNAi experiments and may help to explain differences in the sensitivity of eukaryotic organisms to RNAi.     

Modeling recursive RNA interference

An important application of the RNA interference (RNAi) pathway is its use as a small RNA-based regulatory system commonly exploited to suppress expression of target genes to test their function in vivo. In several published experiments, RNAi has been used to inactivate components of the RNAi pathway itself, a procedure termed recursive RNAi in this report. The theoretical basis of recursive RNAi is unclear since the procedure could potentially be self-defeating, and in practice the effectiveness of recursive RNAi in published experiments is highly variable. A mathematical model for recursive RNAi was developed and used to investigate the range of conditions under which the procedure should be effective. The model predicts that the effectiveness of recursive RNAi is strongly dependent on the efficacy of RNAi at knocking down target gene expression. This efficacy is known to vary highly between different cell types, and comparison of the model predictions to published experimental data suggests that variation in RNAi efficacy may be the main cause of discrepancies between published recursive RNAi experiments in different organisms. The model suggests potential ways to optimize the effectiveness of recursive RNAi both for screening of RNAi components as well as for improved temporal control of gene expression in switch off-switch on experiments.     

昆虫的RNA干扰

RNA干扰(RNAi)是一种强有力的分子生物学技术, 在昆虫研究中得到了较多的应用。目前, RNAi技术主要应用于昆虫功能基因和功能基因组研究, 已在多个目的19种昆虫上实现了RNAi。在昆虫上实现RNAi的方法主要有注射、浸泡、喂食、转基因和病毒介导等方法, 这些方法各有特点, 其中喂食法因其简单而最有应用前景。昆虫RNAi的系统性较为复杂, 只有部分昆虫具有RNAi的系统性。昆虫中RNAi信号传导的基因可能是sid-1, 但昆虫RNAi的系统性机理还不是很清楚。转基因植物产生的dsRNA实现了对作物的保护, 证实了RNAi技术可用于害虫控制, 为害虫控制开辟了新领域。昆虫的RNAi研究处在起步阶段, 研究昆虫RNAi的机理, 特别是RNAi在昆虫体内的系统性扩散机理, 改进实现RNAi的方法, 提高RNAi技术在昆虫研究中的应用, 有利于昆虫基因功能鉴定和害虫控制, 促进昆虫学科的发展。     

RNA interference in cancer

In the recent years, RNA interference (RNAi) has emerged as a major regulatory mechanism in eukaryotic gene expression. The realization that changes in the levels of microRNAs are directly associated with cancer led to the recognition of a new class of tumor suppressors and oncogenes. Moreover, RNAi has been turned into a potent tool for artificially modulating gene expression through the introduction of short interfering RNAs. A plethora of individual inhibitory RNAs as well as several large collections of these reagents have been generated. The systems for stable and regulated expression of these molecules emerged as well. These tools have helped to delineate the roles of various cellular factors in oncogenesis and tumor suppression and laid the foundation for new approaches in gene discovery. Furthermore, successful inhibition of tumor cell growth by RNAi aimed at oncogenes in vitro and in vivo supports the enthusiasm for potential therapeutic applications of this technique. In this article we review the evidence of microRNA involvement in cancer, the use of short interfering RNAs in forward and reverse genetics of this disease, and as well as both the benefits and limitations of experimental RNAi.     

RNA干扰的研究进展

RNA干扰是指外源双链RNA进入细胞后引起与其同源的mRNA特异性降解的现象,它是真核生物在长期进化中形成的一种保守的防御机制,对真核生物有着重要的意义,它参与真核生物抵御病毒侵染、阻断转座子的异常活动,调控基因表达。RNA干扰已成为一种进行基因功能分析的强有力的工具,并有望成为最有潜力的基因干预治疗方法。     

RNA interference: it's a small RNA world

Short RNAs regulate gene expression in many species. Some are generated from any double-stranded RNA and degrade complementary RNAs; others are encoded by genes and repress specific mRNAs. Both, it turns out, are processed and handled by similar proteins. These pathways offer a glimpse into a world of small RNAs.     

RNA binding proteins in the RNA interference phenomenon

The ability of short RNAs (21-27 nucleotides) to silence genes containing homologous nucleotide sequences is related to RNA silencing. The pathways of short RNAs (siRNA and microRNA) biogenesis from their precursors, double stranded and hairpin RNAs respectively, are briefly reviewed. The functioning of specific RNA binding domains found for the first time in the proteins operating in RNA interference (RNAi) is considered. The interactions of these domains with the earlier well known RNA binding modules in RNAi proteins are described.     

Suppression of RNA interference by adenovirus virus-associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.