Novel modes of protein-RNA recognition in the RNAi pathway

Gene silencing mediated by RNA interference (RNAi) depends on short interfering RNAs (siRNAs) and micro RNAs (miRNAs). These RNAs have unique features, namely a defined size of 19-21 base pairs, and characteristic two-nucleotide single-stranded 3' overhangs and 5' monophosphate groups. These molecular features of siRNAs and miRNAs are produced by RNase III enzymes, which are a hallmark of gene silencing induced by double-stranded RNA. Recent structural studies of components of the RNAi pathway, including PAZ, Piwi and RNase III domains, as well as full-length Argonaute and viral p19 proteins, have revealed distinct and novel modes of sequence-independent recognition of the characteristic features of siRNAs and miRNAs in the RNAi pathway.     

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.     

Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing

RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.     

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.     

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.     

Cell-free translation system from Drosophila S2 cells that recapitulates RNAi

RNA interference (RNAi) is a fundamental mechanism of gene regulation in a variety of organisms. In Drosophila cells, long double-stranded RNAs (dsRNAs) are processed into 21- to 23-nucleotide double-stranded fragments, termed short interfering RNAs (siRNAs). The siRNAs trigger sequence-specific mRNA degradation, which results in the inhibition of gene expression. These phenomena can be recapitulated in vitro in lysates of Drosophila syncytial blastoderm embryos. In the present work, we used the common Drosophila cell line, Schneider Line 2 (S2), as a source to establish a cell-free translation system. We demonstrate here that the S2 cell-free translation system can recapitulate RNAi. Both long dsRNAs and siRNAs can trigger RNAi in this system, and the silencing effects are significant. This system should provide an important tool for biochemical analyses of the RNAi mechanism.     

RNA干扰在疾病治疗方面的应用研究

RNA干扰是由双链RNA引起的序列特异的基因沉默现象。由于RNA干扰能在细胞组织及动物模型中沉默疾病相关基因,因此,RNA干扰也是各种疾病治疗的有效手段。在哺乳动物细胞内诱导RNA干扰可以通过导入小干扰RNA（siRNA）,或是以质粒、病毒为载体表达短的发夹RNA（shRNA）而实现。本文介绍了RNA干扰在疾病治疗方面的应用,并就其面临的挑战进行讨论。     

Analysis of gene function in somatic mammalian cells using small interfering RNAs

RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response. Here we provide a collection of protocols for siRNA-mediated knockdown of mammalian gene expression. Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible.     

Illuminating the silence: understanding the structure and function of small RNAs

RNA interference (RNAi) is triggered by double-stranded RNA helices that have been introduced exogenously into cells as small interfering (si)RNAs or that have been produced endogenously from small non-coding RNAs known as microRNAs (miRNAs). RNAi has become a standard experimental tool and its therapeutic potential is being aggressively harnessed. Understanding the structure and function of small RNAs, such as siRNAs and miRNAs, that trigger RNAi has shed light on the RNAi machinery. In particular, it has highlighted the assembly and function of the RNA-induced silencing complex (RISC), and has provided guidelines to efficiently silence genes for biological research and therapeutic applications of RNAi.     

siRNA-like double-stranded RNAs are specifically protected against degradation in human cell extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time.     

Inhibition of herpesvirus-6B RNA replication by short interference RNAs

RNA interference (RNAi) is a process of sequence-specific gene silencing, which is initiated by double-stranded RNA (dsRNA). RNAi may also serve as an antiviral system in vertebrates. This study describes the inhibition of herpesvirus-6B (HHV-6B) replication by short interference RNAs (siRNAs) that are targeted to the U38 sequence that encodes DNA polymerase. When virus-infected SupT1 cells were treated by siRNA, these cells blocked the cytopathic effect (CPE) and detected the HHV-6B antibody-negative in indirect immunofluorescence assays (IFA). Our result suggests that RNAi can efficiently block Herpesvirus-6B replication.     

A technique to enzymatically construct libraries which express short hairpin RNA of arbitrary stem length

Short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) usually used for RNA interference (RNAi) are double-stranded RNAs (dsRNAs) of 21 base pairs. However, siRNAs and shRNAs of longer stem length have been reported to show more potent gene silencing. Here, we report a new technique to enzymatically construct shRNA libraries containing clones from firefly luciferase cDNA and Jurkat cDNA. The technique allowed the efficacious generation of shRNAs of arbitrary stem length as desired, providing the clones which potently silenced the specified gene expression and presenting a high efficiency rate of gene silencing. Our results indicate that the technique permits the rapid, efficient, and low-cost preparation of genomewide shRNA expression libraries not only for humans and mice but also for sorts of biological species and that the relevant libraries are applicable for the search of genes related to phenotype changes and of new targets for drug discovery.