A Rapid Method for Sequencing the 5'- and 3'-Termini of Double-Stranded RNA Viral Templates using RLM-RACE

A protocol is described that permits rapid, inexpensive and reliable synthesis and amplification of 5′‐ and 3′‐terminal cDNAs using viral double‐stranded (ds) RNA templates. The method of RNA ligase‐mediated amplification of cDNA ends for amplification of single‐stranded RNAs ( Liu and Gorovsky, 1993 ) was adapted for dsRNAs to generate the amplicons. These amplicons can be easily cloned and sequenced to complete the sequencing of 5′‐ and 3′‐termini of any particular virus genome. The method is of great utility and can be used on viral genomic dsRNA or viral dsRNA replicative forms when the virion RNA is unavailable.     

Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo

RNA interference (RNAi) has become an essential technique in entomology research. However, RNAi efficiency appears to vary significantly among insect species. Here, the sensitivity of four insect species from different orders to RNAi was compared to understand the reason for this variation. A previously reported method was modified to monitor trace amounts of double-stranded RNA (dsRNA). After the administration of dsRNA, the dynamics of its content was determined in the hemolymph, in addition to the capability of its degradation in both the hemolymph and the midgut juice. The results showed that injection of dsRNA targeting the homologous chitinase gene in Periplaneta americana, Zophobas atratus, Locusta migratoria, and Spodoptera litura, with doses (1.0, 2.3, 11.5, and 33.0 μg, respectively) resulting in the same initial hemolymph concentration, caused 82%, 78%, 76%, and 20% depletion, respectively, whereas feeding doses based on body weight (24, 24, 36, and 30 μg) accounted for 47%, 28%, 5%, and 1% depletion. The sensitivity of insects to RNAi was observed to be as follows: P. americana > Z. atratus >> L. migratoria >> S. litura. In vivo monitoring revealed that RNAi effects among these insect species were highly correlated with the hemolymph dsRNA contents. Furthermore, in vitro experiments demonstrated that the hemolymph contents after dsRNA injection were dependent on hemolymph degradation capacities, and on the degradation capabilities in the midgut juice, when dsRNA was fed. In conclusion, the RNAi efficacy in different insect species was observed to depend on the enzymatic degradation of dsRNA, which functions as the key factor determining the inner target exposure dosages. Thus, enzymatic degradation in vivo should be taken into consideration for efficient use of RNAi in insects.     

Viral RNA recognition by the Drosophila small interfering RNA pathway

Viral RNA is a common activator of antiviral responses. In this review, we dissect the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster. This antiviral response in fruit flies can help understand general principles of nucleic acid recognition.     

RNA干涉在纤毛虫中的研究进展

RNA干涉是dsRNA介导的基因沉默现象,本文简要介绍了其作用的机制和生物学意义,重点阐述了RNA干涉在原生动物纤毛虫中的发现与应用,比较了RNA干涉与纤毛虫大核基因组重排机理的异同,并对RNA干涉在纤毛虫中传输的技术途径-RNAi喂饲法的原理也做了详细的介绍。     

RNAi在哺乳动物中的应用及研究进展

该文综述RNAi技术在哺乳动物中应用及研究方面的最新进展。     

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.     

RNAi技术在昆虫功能基因研究中的应用进展

RNA干扰（RNA interference,RNAi）是指外源或内源的双链RNA（dsRNA）特异性地引起基因表达沉默的现象,它作为一种有效的工具用来产生转录后沉默,从而抑制特定基因的表达,成为基因功能研究的一种新方法,除了在模式昆虫如果蝇Drosophila中广泛应用之外,也在非模式昆虫中得到成功应用。近年来,RNAi技术在导入方法和基因功能分析方面都取得了飞速发展,且与转基因技术相结合成功应用于害虫防治领域。本文综述了RNAi技术在导入方法、昆虫功能基因组功能分析及害虫防治等领域新近的研究成果,并展望了该技术的应用前景。     

Predicting siRNA activity based on back-propagation neural network

RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene.RNAi can be used to identify the function of genes or to knock down the targeted genes.In RNAi technology,19 bp double-stranded short interfering RNAs (siRNA) with characteristic 3' overhangs are usually used.The effects of siRNAs are quite varied due to the different choices in the sites of target mRNA.Moreover,there are many factors influencing siRNA activity and these factors are usually nonlinear.To find the motif features and the effect on siRNA activity,we carried out a feature extraction on some published experimental data and used these features to train a backpropagation neural network (BP NN).Then,we used the trained BP NN to predict siRNA activity.     

Nanoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management

RNA interference (RNAi) targeting lethal genes in insects has great potential for sustainable crop protection. Compared with traditional double-stranded (ds)RNA delivery systems, nanoparticles such as chitosan, liposomes, and cationic dendrimers offer advantages in delivering dsRNA/small interfering (si)RNA to improve RNAi efficiency, thus promoting the development and practice of RNAi-based pest management strategies. Here, we illustrate the limitations of traditional dsRNA delivery systems, reveal the mechanism of nanoparticle-mediated RNAi, summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management, and finally address the prospects of nanoparticle-based RNA pesticides.     

Functional analysis of the RNAi response in ovary-derived silkmoth Bm5 cells

Experiments of dsRNA-mediated gene silencing in lepidopteran insects in vivo are characterized by high variability although lepidopteran cell cultures have shown an efficient response to RNAi in transfection experiments. In order to identify the core RNAi factors that regulate the RNAi response of Lepidoptera, we employed the silkmoth ovary-derived Bm5 cells as a test system since this cell line is known to respond potently in silencing after dsRNA transfection. Two parallel approaches were used; involving knock-down of the core RNAi genes or over-expression of the main siRNA pathway factors, in order to study possible inhibition or stimulation of the RNAi silencing response, respectively. Components from all three main small RNA pathways (BmAgo-1 for miRNA, BmAgo-2/BmDcr-2 for siRNA, and BmAgo-3 for piRNA) were found to be involved in the RNAi response that is triggered by dsRNA. Since BmAgo-3, a factor in the piRNA pathway that functions independent of Dicer in Drosophila, was identified as a limiting factor in the RNAi response, sense and antisense ssRNA was also tested to induce gene silencing but proved to be ineffective, suggesting a dsRNA-dependent role for BmAgo-3 in Bombyx mori. After efficient over-expression of the main siRNA factors, immunofluorescence staining revealed a predominant cytoplasmic localization in Bm5 cells. This is the first study in Lepidoptera to provide evidence for possible overlapping of all three known small RNA pathways in the regulation of the dsRNA-mediated silencing response using transfected B. mori-derived Bm5 cells as experimental system.     

RNA干扰技术及其在植物研究中的应用

RNA干扰(RNA interference,RNAi)是最近几年发现和发展起来的一门新兴的在转录水平上的基因阻断技术,它是生物体内由双链RNA(double-stranded RNA,dsRNA)介导同源mRNA降解的现象。RNAi广泛存在于从真菌到高等植物、从无脊椎动物到哺乳动物各种生物中。研究表明通过转入目的基因序列的双链RNA可以诱导产生基因沉默现象。同时,RNAi能监控异常的或外源的遗传物质在机体内的水平,并调控基因的表达,是生物体抵御外在感染的一种重要的保护机制,这使得RNA干扰技术具有十分诱人的应用前景。介绍了RNAi的研究历史、作用机制、特点及其在植物研究中的应用。     

Predicting siRNA activity based on back-propagation neural network

RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene. RNAi can be used to identify the function of genes or to knock down the targeted genes. In RNAi technology, 19 bp double-stranded short interfering RNAs (siRNA) with characteristic 39 overhangs are usually used. The effects of siRNAs are quite varied due to the different choices in the sites of target mRNA. Moreover, there are many factors influencing siRNA activity and these factors are usually nonlinear. To find the motif features and the effect on siRNA activity, we carried out a feature extraction on some published experimental data and used these features to train a back-propagation neural network (BP NN). Then, we used the trained BP NN to predict siRNA activity. __________ Translated from Acta Biophysica Sinica, 2006, 22(6): 429–434 [译自: 生物物理学报]     

Mammalian RNA virus-derived small RNA: biogenesis and functional activity

The role of virus-derived small RNAs (vsRNAs) has been identified as an antiviral mechanism in plants, arthropods, and nematodes. Although mammalian DNA viruses have been observed to encode functional miRNAs, whether RNA virus infection generates functional vsRNAs remains under discussion. This article reviews the most recent reports regarding pathways for generating vsRNAs and the identified vsRNA activity in mammalian cells infected with RNA viruses. We also discuss several hypotheses regarding the roles of mammalian vsRNAs and comment on the potential directions for this research field.     

MicroRNA研究进展

在多细胞生物的基因组中都存在一类非编码RNA基因,能够产生长度约为22个核苷酸的小分子RNA,称为microRNA(miRNA),具有调节其他基因表达活性的功能。miRNA的发现,为我们理解复杂的基因调节网络开辟了新的空间。本文概述了miRNA的产生过程、转录抑制机理、研究并预测miRNA的方法等。