Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21–23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.     

RNA干涉及其应用前景

RNA干涉是指由特定双链RNA(dsRNA)引起的转录后基因沉默现象。研究表明,Dicer断裂dsRNA产生的小干涉RNA可以抑制哺乳动物体细胞和胚胎中的基因的表达。RdRP在扩增RNAi中起着关键性的作用,RdRP活性复制较长的触发性dsRNA或以一种非引物的方式复制短的siRNA,即以siRNA为引物的RdRP反应使靶mRNA转变为dsRNA,同时复制触发性dsRNA。所有的产物又可作为Dicer的底物,起始RdRP级联反应。本文综述了RNAi可能的作用机制,并对RNAi在分析功能基因组、药物治疗等方面的应用前景进行了展望。     

水稻中大麦Mlo和玉米Hm1抗病基因同源序列的分析和定位

大麦抗病基因Mlo和玉米抗病基因Hm1编码的产物不具有绝大多数植物抗病基因产物所含有的保守结构域。这两个抗病基因的作用机理也不符合基因对基因学说。从水稻中分离克隆了Mlo基因的同源序列OsMlo-1和玉米Hm1基因的同源序列DFR－1。利用水稻分子标记遗传连锁图,将OsMlo-1定位于水稻第六染色体的两俱RZ667和RG424之间;Osmlo-1距离这两个分子标记分别为20．6和6．0cM(centi-Morgan)。将DFR－1定位于水稻第一染色体两个分子标记R2635和RG462之间;DFR－1距离这两个分子标记分别为11．3和23．9cM。参照已发表的水稻分子标记连锁图,发现OsMlo-1和DFR－1的染色体位点分别与两个报道的水稻抗稻瘟病数量性状位点（QTL）有较好的对应关系。结果提示,水稻中与大麦Mlo 和玉米Hml同源的基因可能也参于抗病反应的调控。     

Transgenic RNAi in mouse oocytes: The first decade

RNA interference (RNAi), a sequence-specific mRNA degradation induced by double-stranded RNA (dsRNA), is a common approach employed to specifically silence genes. Experimental RNAi in plant and invertebrate models is frequently induced by long dsRNA. However, in mammals, short RNA molecules are used preferentially since long dsRNA can provoke sequence-independent type I interferon response. A notable exception are mammalian oocytes where the interferon response is suppressed and long dsRNA is a potent and specific trigger of RNAi. Transgenic RNAi is an adaptation of RNAi allowing for inducing sequence-specific silencing upon expression of dsRNA. A decade ago, we have developed a vector for oocyte-specific expression of dsRNA, which has been used to study gene function in mouse oocytes on numerous occasions. This review provides an overview and discusses benefits and drawbacks encountered by us and our colleagues while working with the oocytes-specific transgenic RNAi system.     

A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley

Small GTP-binding proteins such as those from the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture, secondary wall formation, meristem signaling, and defense against pathogens. We isolated a RacB homolog from barley (Hordeum vulgare) to study its role in resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei). RacB was constitutively expressed in the barley epidermis and its expression level was not strongly influenced by inoculation with B. graminis. However, after biolistic bombardment of barley leaf segments with RacB-double-stranded RNA, sequence-specific RNA interference with RacB function inhibited fungal haustorium establishment in a cell-autonomous and genotype-specific manner. Mutants compromised in function of the Mlo wild-type gene and the Ror1 gene (genotype mlo5 ror1) that are moderately susceptible to B. graminis showed no alteration in powdery mildew resistance upon RacB-specific RNA interference. Thus, the phenotype, induced by RacB-specific RNA interference, was apparently dependent on the same processes as mlo5-mediated broad resistance, which is suppressed by ror1. We conclude that an RAC small GTP-binding protein is required for successful fungal haustorium establishment and that this function may be linked to MLO-associated functions.     

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。     

Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila adult flies

Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence-specific manner. Here we show that the direct injection of dsRNA can be used in adult Drosophila flies to disrupt function of endogenous genes in vivo. As a proof of principle, we have used this method to silence components of a major signaling cascade, the Toll pathway, which controls fruit fly resistance to fungal and Gram-positive bacterial infections. We demonstrate that the knockout is efficient only if dsRNA is injected in 4- or more day-old flies and that it lasts for at least 1 week. Furthermore, we report dsRNA-based epistatic gene analysis via injection of a mixture of two dsRNAs and propose that injection of dsRNA represents a powerful method for rapid functional analysis of genes in Drosophila melanogaster adults, particularly of those whose mutations are lethal during development.     

Molecular analysis of a second functional A1 gene (dihydroflavonol 4‐reductase) in Zea mays

Some genes involved in anthocyanin biosynthesis in Zea mays are duplicated and differentially expressed. From the analysis of the A1 gene (dihydroflavonol 4-reductase), which is involved in this pathway, no molecular evidence for gene duplication was known to date. Isolation and analysis of A1 homologous genomic clones revealed the presence of a second A1 gene in maize and also two copies of the gene in Teosinte guerrero. The duplicated genes are structurally very similar and, at least in maize, the second gene is expressed.     

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。     

Analysis of the role of RecQ helicases in RNAi in mammals

The identity of mammalian genes involved in RNA interference (RNAi), the targeted sequence-specific mRNA degradation by double-stranded RNA (dsRNA), is poorly defined. Here we report the analysis of mice with null mutations of Wrn, Blm, and RecQ1 genes that are related to Mut-7 and Qde3, two genes essential for RNAi in Caenorhabditis elegans and quelling in Neurospora, respectively. Our results suggest that Wrn, Blm, and RecQ1 are not involved in sequence-specific mRNA degradation in mammals in response to dsRNA, suggesting potential differences in the mammalian RNAi pathway.     

Gerbera hybrida (Asteraceae) imposes regulation at several anatomical levels during inflorescence development on the gene for dihydroflavonol-4-reductase

In the ornamental cut flower plant Gerbera hybrida the spatial distribution of regulatory molecules characteristic of differentiation of the composite inflorescence is visualized as the various patterns of anthocyanin pigmentation of different varieties. In order to identify genes that the plant can regulate according to these anatomical patterns, we have analysed gene expression affecting two enzymatic steps, chalcone synthase (CHS) and dihydroflavonol-4-reductase (DFR), in five gerbera varieties with spatially restricted anthocyanin pigmentation patterns. The dfr expression profiles vary at the levels of floral organ, flower type and region within corolla during inflorescence development according to the anthocyanin pigmentation of the cultivars. In contrast, chs expression, although regulated in a tissue-specific manner during inflorescence development, varies only occasionally. The variation in the dfr expression profiles between the varieties reveals spatially specific gene regulation that senses the differentiation events characteristic of the composite inflorescence.     

Isolation of a Suppressor-mutator/Enhancer-like transposable element, Tpn1, from Japanese morning glory bearing variegated flowers.

The Japanese morning glory has an extensive history of genetic studies. Many mutants in the colors and shapes of its flowers and leaves have been isolated since the 17th century, and more than 200 genetic loci have been localized for the 10 linkage groups. They include over 20 mutable loci, several with variegated flower phenotypes. In a line of Japanese morning glory bearing variegated flowers called flecked, a transposable element of 6.4 kb, termed Tpn1, was found within one of the anthocyanin biosynthesis genes encoding dihydroflavonol-4-reductase (DFR). The 6.4-kb element carries 28-bp perfect terminal inverted repeats, the outer 13 bp being identical to those of the maize transposable element Suppressor-mutator/Enhancer. It is flanked by 3-bp direct repeats within the second intron of the DFR gene, 9 bp upstream of the third exon. When somatic and germinal excision occurs, it produces excision sequences characteristic of plant transposable elements. Cosegregation data of the variegated flower phenotype and the DFR gene carrying Tpn1 indicated that the mutable phenotype is due to excision of Tpn1 from the DFR gene. Sequences homologous to Tpn1 are present in multiple copies in the genome of Japanese morning glory.     

Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.