Analysis of the 3-hydroxyl group in Drosophila siRNA function

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3^′-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed “degradative PCR” and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3^′-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.     

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。     

On the role of RNA amplification in dsRNA-triggered gene silencing.

We have investigated the role of trigger RNA amplification during RNA interference (RNAi) in Caenorhabditis elegans. Analysis of small interfering RNAs (siRNAs) produced during RNAi in C. elegans revealed a substantial fraction that cannot derive directly from input dsRNA. Instead, a population of siRNAs (termed secondary siRNAs) appeared to derive from the action of a cellular RNA-directed RNA polymerase (RdRP) on mRNAs that are being targeted by the RNAi mechanism. The distribution of secondary siRNAs exhibited a distinct polarity (5'-->3' on the antisense strand), suggesting a cyclic amplification process in which RdRP is primed by existing siRNAs. This amplification mechanism substantially augments the potency of RNAi-based surveillance, while ensuring that the RNAi machinery will focus on expressed mRNAs.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells

The first evidence for gene disruption by double-stranded RNA (dsRNA) came from careful analysis in Caenorhabditis elegans. This phenomenon, called RNA interference (RNAi), was observed subsequently in various organisms, including plants, nematodes, Drosophila, and protozoans. Very recently, it has been reported that in mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (small inhibitory RNAs, siRNAs) exhibit an RNAi effect. This is because siRNAs are not recognized by the well-characterized host defense system against viral infections, involving dsRNA-dependent inhibition of protein synthesis. However, the current method for introducing synthetic siRNA into cells by lipofection restricts the range of applications of RNAi as a result of the low transfection efficiencies in some cell types and/or short-term persistence of silencing effects. Here, we report a vector-based siRNA expression system that can induce RNAi in mammalian cells. This technical advance for silencing gene expression not only facilitates a wide range of functional analysis of mammalian genes but might also allow therapeutic applications by means of vector-mediated RNAi.     

Structural diversity repertoire of gene silencing small interfering RNAs

Since the discovery of double-stranded (ds) RNA-mediated RNA interference (RNAi) phenomenon in Caenorhabditis elegans, specific gene silencing based upon RNAi mechanism has become a novel biomedical tool that has extended our understanding of cell biology and opened the door to an innovative class of therapeutic agents. To silence genes in mammalian cells, short dsRNA referred to as small interfering RNA (siRNA) is used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs. An early structure-activity relationship study performed in Drosophila melanogaster embryonic extract suggested the existence of strict siRNA structural design rules to achieve optimal gene silencing. These rules include the presence of a 3' overhang, a fixed duplex length, and structural symmetry, which defined the structure of a classical siRNA. However, several recent studies performed in mammalian cells have hinted that the gene silencing siRNA structure could be much more flexible than that originally proposed. Moreover, many of the nonclassical siRNA structural variants reported improved features over the classical siRNAs, including increased potency, reduced nonspecific responses, and enhanced cellular delivery. In this review, we summarize the recent progress in the development of gene silencing siRNA structural variants and discuss these in light of the flexibility of the RNAi machinery in mammalian cells.     

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.     

Inputs and outputs for chromatin-targeted RNAi

Plant gene silencing is targeted to transposons and repeated sequences by small RNAs from the RNA interference (RNAi) pathway. Like classical RNAi, RNA-directed chromatin silencing involves the cleavage of double-stranded RNA by Dicer endonucleases to create small interfering RNAs (siRNAs), which bind to the Argonaute protein. The production of double-stranded RNA (dsRNA) must be carefully controlled to prevent inappropriate silencing. A plant-specific RNA polymerase IV (Pol IV) initiates siRNA production at silent heterochromatin, but Pol IV-independent mechanisms for making dsRNA also exist. Downstream of siRNA biogenesis, multiple chromatin marks might be targeted by Argonaute-siRNA complexes, yet mechanisms of chromatin modification remain poorly understood. Genomic studies of siRNA target loci promise to reveal novel biological functions for chromatin-targeted RNAi.     

RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.     

Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21-23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in DROSOPHILA: DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform.     

Dual-target gene silencing by using long,synthetic siRNA duplexes without triggering antiviral responses

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of nonspecific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without nonspecific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.