Enzymatically Produced Pools of Canonical and Dicer-Substrate siRNA Molecules Display Comparable Gene Silencing and Antiviral Activities against Herpes Simplex Virus

RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers.     

Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants

RNA interference (RNAi) using short interfering RNAs (siRNAs) has been widely explored for the suppression of intracellular viral target mRNAs. On the basis of our previous work with stable silencing of Tomato leaf curl virus, in vivo by the antisense replicase gene (AC1) of the virus and characterizing AC4, as a small RNA regulator, besides its role in pathogenicity, we used four different plasmid vector-based siRNA generation strategies to silence viral genes (AC1 and AC4) of tomato leaf curl viruses. The RNAi target sequence were chosen from DNA A of the Tomato leaf curl virus (ToLCV) on the basis of conserved regions in AC1 with an overlapping sequences of the AC4 gene. Different hairpin RNA-mediated strategies like antisense, self-complementary inverted repeats, intron-spliced hairpin RNAs, and small hairpin RNAs were deployed for efficient and predictable resistance to the viruses. Here we present that appropriately designed siRNAs not only prevents RNAi suppression but also help in developing trait-stable transgenics. These strategies imply that ToLCV rep-driven RNAi, targeting AC4 and conserved viral sequences, provides a promising approach to suppress a wide spectrum ToLCV infection in the tomato.     

RNA interference: The molecular immune system

Introduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo- and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21- to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.     

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.     

siSPOTR: a tool for designing highly specific and potent siRNAs for human and mouse

RNA interference (RNAi) serves as a powerful and widely used gene silencing tool for basic biological research and is being developed as a therapeutic avenue to suppress disease-causing genes. However, the specificity and safety of RNAi strategies remains under scrutiny because small inhibitory RNAs (siRNAs) induce off-target silencing. Currently, the tools available for designing siRNAs are biased toward efficacy as opposed to specificity. Prior work from our laboratory and others’ supports the potential to design highly specific siRNAs by limiting the promiscuity of their seed sequences (positions 2–8 of the small RNA), the primary determinant of off-targeting. Here, a bioinformatic approach to predict off-targeting potentials was established using publically available siRNA data from more than 50 microarray experiments. With this, we developed a specificity-focused siRNA design algorithm and accompanying online tool which, upon validation, identifies candidate sequences with minimal off-targeting potentials and potent silencing capacities. This tool offers researchers unique functionality and output compared with currently available siRNA design programs. Furthermore, this approach can greatly improve genome-wide RNAi libraries and, most notably, provides the only broadly applicable means to limit off-targeting from RNAi expression vectors.     

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence 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.     

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.     

Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos

BACKGROUND: RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants. RESULTS: We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction. CONCLUSIONS: Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.     

RNA interference in infectious tropical diseases

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.