Cleavage Of dsRNA Research Articles

Three alternative splicing variants of Loquacious play different roles in miRNA- and siRNA-mediated RNAi pathways in Locusta migratoria

ABSTRACT RNA interference (RNAi) is a specific post-transcriptional gene-silencing phenomenon, which plays an important role in the regulation of gene expression and the protection from transposable elements in eukaryotic organisms. In Drosophila melanogaster, RNAi can be induced by microRNA (miRNA), endogenous small interfering RNA (siRNA), or exogenous siRNA. However, the biogenesis of miRNA and siRNA in these RNAi pathways is aided by the double-stranded RNA binding proteins (dsRBPs) Loquacious (Loqs)-PB, Loqs-PD or R2D2. In this study, we identified three alternative splicing variants of Loqs, namely Loqs-PA, -PB, and -PC in the orthopteran Locusta migratoria. We performed in vitro and in vivo experiments to study the roles of the three Loqs variants in the miRNA- and siRNA-mediated RNAi pathways. Our results show that Loqs-PB assists the binding of pre-miRNA to Dicer−1 to lead to the cleavage of pre-miRNA to yield matured miRNA in the miRNA-mediated RNAi pathway. In contrast, different Loqs proteins participate in different siRNA-mediated RNAi pathways. In exogenous siRNA-mediated RNAi pathway, binding of Loqs-PA or LmLoqs-PB to exogenous dsRNA facilitates the cleavage of dsRNA by Dicer−2, whereas in endogenous siRNA-mediated RNAi pathway, binding of Loqs-PB or Loqs-PC to endogenous dsRNA facilitates the cleavage of dsRNA by Dicer−2. Our findings provide new insights into the functional importance of different Loqs proteins derived from alternative splicing variants of Loqs in achieving high RNAi efficiency in different RNAi pathways in insects.

RNA Interference: Promising Approach to Combat Plant Viruses.

Plant viruses are devastating plant pathogens that severely affect crop yield and quality. Plants have developed multiple lines of defense systems to combat viral infection. Gene silencing/RNA interference is the key defense system in plants that inhibits the virulence and multiplication of pathogens. The general mechanism of RNAi involves (i) the transcription and cleavage of dsRNA into small RNA molecules, such as microRNA (miRNA), or small interfering RNA (siRNA), (ii) the loading of siRNA/miRNA into an RNA Induced Silencing Complex (RISC), (iii) complementary base pairing between siRNA/miRNA with a targeted gene, and (iv) the cleavage or repression of a target gene with an Argonaute (AGO) protein. This natural RNAi pathway could introduce transgenes targeting various viral genes to induce gene silencing. Different RNAi pathways are reported for the artificial silencing of viral genes. These include Host-Induced Gene Silencing (HIGS), Virus-Induced Gene Silencing (VIGS), and Spray-Induced Gene Silencing (SIGS). There are significant limitations in HIGS and VIGS technology, such as lengthy and time-consuming processes, off-target effects, and public concerns regarding genetically modified (GM) transgenic plants. Here, we provide in-depth knowledge regarding SIGS, which efficiently provides RNAi resistance development against targeted genes without the need for GM transgenic plants. We give an overview of the defense system of plants against viral infection, including a detailed mechanism of RNAi, small RNA molecules and their types, and various kinds of RNAi pathways. This review will describe how RNA interference provides the antiviral defense, recent improvements, and their limitations.

Two Piwis with Ago-like functions silence somatic genes at the chromatin level

ABSTRACT Most sRNA biogenesis mechanisms involve either RNAse III cleavage or ping-pong amplification by different Piwi proteins harbouring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from targeted endogenous loci. Our data does not indicate any signatures from ping-pong amplification but Dicer cleavage of long dsRNA. Ptiwi13 and 14 prefer different sub-cellular localizations and different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and show a general preference for uridine-rich sRNAs along the entire sRNA length. In addition, Ptiwi14-loaded siRNAs show a 5´-U signature. Our data indicates both Ptiwis and 2´-O-methylation contributing to strand selection of Dicer cleaved siRNAs. This unexpected function of the two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. We describe an unusual mode of action of Piwi proteins extending not only the great variety of Piwi-associated RNAi pathways but moreover raising the question whether this could have been the primordial one.

Resolving the Dynamics of the Double Stranded RNA Binding Protein TRBP

The TAR RNA binding protein (TRBP) is a double-stranded RNA binding proteins (dsRBP) that plays an important role in HIV replication, immune response and RNAi processing. As an important cofactor of Dicer, it supports the specific cleavage of dsRNA and the loading of this RNA into the Argonaut complex. Like other dsRBPs, TRBP consists of several copies of dsRNA binding domains that are connected by long unstructured linker regions, and TRBP is highly dynamic in its RNA-bound state. The role of this flexibility and the influence of these linker regions are still poorly understood.

Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease

The glycoprotein Erns plays a central role in the biology of the pestivirus bovine viral diarrhea virus (BVDV). This soluble endonuclease mediates the escape from an interferon (IFN) response in the infected fetus, thereby permitting the establishment of persistent infection. Viral single-stranded (ss) and double-stranded (ds) RNA act as potent IFN inducing signals and we previously showed that Erns efficiently cleaves these substrates, thereby inhibiting an IFN response that is crucial for successful fetal infection. Considering that a large variety of RNases and DNases require dimerisation to cleave double-stranded substrates, the activity of Erns against dsRNA was postulated to depend on homodimer formation mediated by disulfide bonds involving residue Cys171. Here, we show that monomeric Erns is equally able to cleave dsRNA and to inhibit dsRNA-induced IFN synthesis as the wild-type form. Furthermore, both forms were able to degrade RNA within a DNA/RNA- as well as within a methylated RNA/RNA-hybrid, with the DNA and the methylated RNA strand being resistant to degradation. These results support our model that Erns acts as ‘nicking endoribonuclease’ degrading ssRNA within double-stranded substrates. This efficiently prevents the activation of IFN and helps to maintain a state of innate immunotolerance in persistently infected animals.

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.

Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry

RNA interference has provided valuable insight into a wide range of biological systems and is a powerful tool for the analysis of gene function. The exploitation of this pathway to block the expression of specific gene targets holds considerable promise for the development of novel RNAi-based insect management strategies. In addition, there are a wide number of future potential applications of RNAi to control agricultural insect pests as well as its use for prevention of diseases in beneficial insects. The potential to synthesise large quantities of dsRNA by in-vitro transcription or in bacterial systems for RNA interference applications has generated significant demand for the development and application of high throughput analytical tools for the rapid extraction, purification and analysis of dsRNA. Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry.

Sequence-specific cleavage of dsRNA by Mini-III RNase.

Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.

Different roles for RNA silencing and RNA processing components in virus recovery and virus-induced gene silencing in plants.

A major antiviral mechanism in plants is mediated by RNA silencing, which relies on the cleavage of viral dsRNA into virus-derived small interfering RNAs (vsiRNAs) by DICER-like enzymes. Members of the Argonaute (AGO) family of endonucleases then use these vsiRNA as guides to target viral RNA. This can result in a phenomenon known as recovery, whereby the plant silences viral gene expression and recovers from viral symptoms. Endogenous mRNAs can also be targeted by vsiRNAs in a phenomenon known as virus-induced gene silencing (VIGS). Although related to other RNA silencing mechanisms, it has not been established if recovery and VIGS are mediated by the same molecular mechanisms. We used tobacco rattle virus (TRV) carrying a fragment of the phytoene desaturase (PDS) gene (TRV-PDS) or expressing green fluorescent protein (TRV-GFP) as readouts for VIGS and recovery, respectively, in Arabidopsis ago mutants. Our results demonstrated roles for AGO2 and AGO4 in susceptibility to TRV, whereas VIGS of endogenous genes appeared to be largely mediated by AGO1. However, recovery appeared to be mediated by different components, as all the aforementioned mutants were able to recover from TRV-GFP inoculation. TRV RNAs from recovered plants associated less with ribosomes, suggesting that recovery involves translational repression of viral transcripts. Translationally repressed RNAs often accumulate in RNA processing bodies (PBs), where they are eventually processed by decapping enzymes. Consistent with this, we found that viral recovery induced increased PB formation and that a decapping mutant (DCP2) showed increased VIGS and virus RNA accumulation, indicating an important role for PBs in eliminating viral RNA.

Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi.

RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.

Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain

In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5' terminal phosphate and a two-nucleotide, 3' overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer-2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5' terminal phosphate of short substrates, blocking their use and restricting pre-miRNA processing in flies to Dicer-1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme.

Molecular dissection of Flock house virus protein B2 reveals that electrostatic interactions between N-terminal domains of B2 monomers are critical for dimerization

Flock house virus (FHV) encodes a suppressor protein B2 to overcome antiviral RNA silencing during infection. Biochemical analyses have shown that a homodimer of B2 binds to double-stranded RNA to inhibit dicer-mediated cleavage of dsRNA and incorporation of small interfering RNAs into the RNA-induced silencing complex. In this study, using FHV-Nicotiana benthamiana system, we identified that the charged amino acids at the N-terminus of B2 are critical for dimerization. Interestingly, B2 mutants defective in dimerization exhibited enhanced silencing suppressor activity, Furthermore, we found that the C-terminal charged amino acids are dispensable for B2 dimerization and viral RNA silencing suppression but are critical for transgene silencing suppression. Additional yeast two hybrid assays revealed that dimerization of B2 is not essential for interacting with the RNA silencing machinery. Taken together, our data provide evidence that both monomeric and dimeric B2 proteins function in different modes to suppress RNA silencing.

A forward genetic screen to study mammalian RNA interference - essential role of RNase IIIa domain of Dicer1 in 3′ strand cleavage of dsRNA in vivo

RNA interference is a major post-transcriptional regulatory pathway in many eukaryotes. The RNase III enzyme Dicer1 processes precursor RNAs into small RNA duplexes to be loaded onto Argonaute proteins, the effector components of RNA-induced silencing complex. Biochemical studies have shown that the RNase IIIa and RNase IIIb domains of Dicer1 cleave the 3' and 5' strands of dsRNAs, respectively, although the in vivo functional significance of this activity remains unclear. Genetic screening of mammalian cells is useful for studying molecular mechanisms at the cellular level. In the present study, we conducted a novel forward genetic screen for mammalian RNA interference components using Chinese hamster ovary cells and successfully obtained several Dicer1 mutant lines. One mutant bore an intriguing Dicer1 allele in which a conserved glutamic acid in the RNase IIIa domain was substituted with a lysine. Our detailed cell biological study demonstrated that the RNase IIIa domain of Dicer1 was essential for generating small RNAs embedded in the 3' stem of exogenous hairpin-like RNAs. In the mutant cells, the expression of endogenous mature microRNAs derived from the 3' stem of pre-microRNA was repressed more severely than that from the 5' stem. Moreover, appropriate processing and loading of small RNAs were required for the dissociation of Argonaute 2 from Dicer1. The data obtained in the present study demonstrate that this screening method represents a promising strategy for the identification of unknown components of mammalian RNA interference pathways and the study of the biological significance of these components at the cellular level.

Functional Role of Glutamine 28 and Arginine 39 in Double Stranded RNA Cleavage by Human Pancreatic Ribonuclease

Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.

Comparison of Dengue Virus Type 2-Specific Small RNAs from RNA Interference-Competent and –Incompetent Mosquito Cells

The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

In C. elegans, High Levels of dsRNA Allow RNAi in the Absence of RDE-4

C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.

Dicer Cuts the Kidney

We should occasionally stop, take a breath, and reflect about what is happening at the interface between medicine and science. Collectively, we are uncovering paradigms that are relevant to understanding disease at a frantic pace. One can only turn to wonder, in this moment of reflection, when

DsRNA Binding Properties of RDE-4 and TRBP Reflect Their Distinct Roles in RNAi

Double-stranded RNA (dsRNA)-binding proteins facilitate Dicer functions in RNA interference. Caenorhabditis elegans RDE-4 facilitates cleavage of long dsRNA to small interfering RNA (siRNA), while human trans-activation response RNA-binding protein (TRBP) functions downstream to pass siRNA to the RNA-induced silencing complex. We show that these distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA length. These properties are mechanistically based on the fact that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds noncooperatively. Our studies offer a paradigm for how dsRNA-binding proteins, which are not sequence specific, discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity using its dsRNA-binding motif 2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation.

Virus meets RNAi

The European Science Foundation (ESF)–European Molecular Biology Organization (EMBO) Symposium on Antiviral Applications of RNA Interference took place between 5 and 10 April 2008, in Sant Feliu de Guixols, Spain, and was organized by J. Kurreck and B. Berkhout. ![][1] Small RNAs are important regulators of gene expression that operate through a range of RNA‐silencing mechanisms. Of these, RNA interference (RNAi)—gene silencing through small‐interfering RNAs (siRNAs) derived from exogenous double‐stranded RNA (dsRNA)—and the microRNA (miRNA) pathway—gene silencing guided by small RNAs encoded in the genome—are the best known. The discovery that dsRNA induces RNAi (Fire et al , 1998) and the appreciation of its antiviral activity revolutionized our view of viruses in many ways. The European Science Foundation (ESF)–European Molecular Biology Organization (EMBO) Symposium on Antiviral Applications of RNA Interference brought together about 50 scientists working at the interface between virology and RNAi. Here, I review the main issues that were discussed at the meeting. RNAi provides an antiviral defence mechanism in plants, nematodes and insects. The cleavage of viral dsRNA by the ribonuclease Dicer generates viral siRNAs (v‐siRNAs), which are incorporated into the RNA‐induced silencing complex (RISC) and direct RISC activity onto messenger RNAs (mRNAs) in a sequence‐specific manner. Recognition of a complementary sequence then triggers the endonucleic cleavage (slicer activity) or translational inhibition of the viral target RNAs by an Argonaute family member (Fig 1). Figure 1. RNA silencing in virus infections. ( A ) RNA interference (RNAi) as an antiviral defence mechanism. Viruses might produce double‐stranded RNAs (dsRNAs) that are processed by the RNAi machinery and thereby programme the RNA‐induced silencing complex (RISC) to degrade viral RNAs. ( B ) Cellular or viral microRNAs (miRNAs), or viral RNAs with structural similarity to pre‐miRNAs, regulate viral and host gene expression, and contribute to viral pathogenesis. miRISC, microRNA‐induced silencing complex; pre‐miRNA, precursor miRNA; pri‐miRNA, primary miRNA … [1]: /embed/graphic-1.gif

RNAi‐mediated gene silencing in fishes?

The recent discovery that small regulatory RNAs have diverse roles in eukaryotic intracellular life, such as fine tuners of endogenous gene expression, as inhibitors of viral replication and as suppressors of transposon activity, is now widely recognized as a major breakthrough in the understanding of cellular biology. The pathways used by these small RNAs seem to build upon a few central mechanisms in which small double‐stranded RNAs (dsRNAs) are generated from various precursor RNAs of exogenous or endogenous origin and used for inhibiting expression of genes with sequence complementarity to the small RNA. In particular, one class of small gene‐regulatory RNAs, known as small interfering RNAs (siRNAs), has the potential for highly specific gene silencing. These are c. 21 nucleotide perfectly complementary dsRNAs with 3′ end overhangs. They are produced by cleavage of long dsRNA believed to be of viral origin. Subsequently, a large cytoplasmic protein complex known as the RNA‐induced silencing complex (RISC) uses the antisense strand of the siRNAs for targeting complementary mRNA strands followed by cleavage of these transcripts in the mechanism called RNA interference (RNAi). Accordingly, synthetic siRNAs may have a potential for treating diseases caused by intracellular pathogens like viruses by specifically targeting the expression of their molecular components. Furthermore, as target sequences need not be of viral origin, siRNAs hold a promise for the treatment of various cellular diseases and as a tool in reverse genetic studies. SiRNAs are currently exploited for such purposes in diverse organisms ranging from plants and invertebrates to human beings. But due to initial difficulties, when using RNAi for gene silencing in fishes, only a few such studies have been conducted in fishes. The purpose of this review is to inspire further experimental work.