To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two ~21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. In Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway (Tomari, 2004b).
In Drosophila lysates, siRNAs are loaded into the RISC by an ordered pathway in which one of the two siRNA strands, the guide strand, is assembled into the RISC, whereas the other strand, the passenger strand, is excluded and destroyed. A central step in RISC assembly is formation of the RISC-loading complex (RLC, previously designated complex A), which contains (1) double-stranded siRNA, (2) the double-stranded RNA binding protein R2D2, and (3)Dicer-2 (Dcr-2), as well as additional unidentified proteins. The function of Dicer in loading siRNA into the RISC is distinct from its role in generating siRNA from long double-stranded RNA (dsRNA). Both R2D2 and Dcr-2 are required to form RLC (Tomari, 2004a) and to unwind siRNA, but recombinant Dcr-2/R2D2 heterodimer or Dcr-2 alone cannot catalyze siRNA unwinding. Thus, the Dcr-2/R2D2 heterodimer is necessary but not sufficient to unwind siRNA (Tomari, 2004b).
If siRNA unwinding is initiated in the RLC, then the RLC should contain some single-stranded siRNA. To test this idea, siRNA duplex was briefly incubated in lysate, the complexes formed by native gel electrophoresis were resolved, the gel was divided into 11 parts, and the structure of the siRNA in each gel slice was analyzed. A peak of double-stranded siRNA comigrated with both the RLC and complex B, which is thought to be a precursor to RLC (Tomari, 2004a). A small peak of single-stranded siRNA also comigrated with the RLC, but not with complex B, which suggests that the RLC initiates siRNA unwinding. Similar peaks of single-stranded siRNA comigrated with the RLC for the passenger strand of this siRNA and for the guide and passenger strands of a second siRNA. It is concluded that the RLC initiates siRNA unwinding (Tomari, 2004b).
The RLC also senses siRNA thermodynamic asymmetry, thereby determining which strand enters the RISC. siRNA containing 5-iodouracil at the 20th nucleotide (p20) can be photocrosslinked to R2D2 and Dcr-2. Photocrosslinking is position-specific: An siRNA containing 5-iodouracil at position 12 was not cross-linked to R2D2 or Dcr-2. Photocrosslinking attaches the radiolabel of the siRNA to the protein, identifying proteins that lie near p20 of the substituted siRNA strand. The relative efficiency of photocrosslinking to R2D2 and Dcr-2 was evaluated for three types of siRNA: a luciferase-specific siRNA whose sequence makes the 5' end of the antisense strand less thermodynamically stable than the 5' end of the sense strand; a nearly symmetric siRNA targeting human Zn, Cu superoxide dismutase 1 (sod1), in which the stabilities of the 5' ends are essentially the same; and a series of highly asymmetric sod1-directed siRNAs in which the first nucleotide of the guide strand is mismatched to the passenger strand, causing the guide strand to be loaded into the RISC almost exclusively. When the partially asymmetric luciferase-specific siRNA was used, R2D2 was more efficiently photocrosslinked when the 5-iodouracil was on the strand more frequently incorporated into the RISC, whereas when the 5-iodouracil was on the strand less often incorporated into the RISC, Dcr-2 was more efficiently photocrosslinked. Because a 5-iodouracil at p20 of one siRNA strand is near the 5' end of the other strand, Dcr-2 must lie near the 5' end of the strand entering the RISC (the guide strand), whereas R2D2 binds near the 5' end of the strand destined for destruction (Tomari, 2004b).
When the symmetric sod1 siRNA was used, siRNA a, Dcr-2 and R2D2 were photocrosslinked with nearly equal efficiency to the 5-iodouracil strand: this finding suggests that each protein binds about half the time to one or the other end of the siRNA. In contrast, when derivatives of this siRNA were used that contained single-nucleotide mismatches that made them highly asymmetric, the 5-iodouracil strand was photocrosslinked to either Dcr-2 or R2D2, but not to both. With the asymmetric siRNA sequence, the photocrosslinking data suggest that Dcr-2 is almost always near the 5' end of the guide strand and R2D2 near the 5' end of the passenger strand. As expected when both siRNA strands contained p20 5-iodouracil and 5'-[32P]phosphate groups, both proteins were photocrosslinked. When a reciprocal series of siRNAs was used in which the strands assembled into and excluded from the RISC were reversed, Dcr-2 was again found near the 5' end of the guide strand and R2D2 near the 5' end of the passenger strand (Tomari, 2004b).
Purified, recombinant Dcr-2/R2D2 heterodimer alone can also sense the thermodynamic stabilities of the ends of an siRNA duplex. At physiologically relevant concentrations of the proteins, photocrosslinking reflected siRNA asymmetry. Like heterodimer binding to an siRNA, differential photocrosslinking of recombinant Dcr-2/R2D2 heterodimer to an siRNA did not require adenosine triphosphate (ATP). In contrast, formation of the RLC requires ATP (Tomari, 2004b). The orientation of Dcr-2 and R2D2 on the siRNA duplex was less asymmetric for the recombinant heterodimer than for embryo lysate. It is proposed that siRNA asymmetry is initially sensed by the Dcr-2/R2D2 heterodimer in an ATP-independent manner but is later amplified by the ATP-dependent action of other proteins (Tomari, 2004b).
Photocrosslinking of R2D2, but not Dcr-2, to the two ends of an siRNA duplex was influenced by the presence of a 5'-phosphate group on the siRNA. A series of highly asymmetric siRNAs were prepared in which the strand containing the p20 5-iodouracil was radiolabeled with 32P at the 5' end and the other strand contained either a 5'-hydroxyl or 5'-phosphate group. In four trials, R2D2 photocrosslinking to the nearby p20 5-iodouracil of the guide strand was greater by a factor of 4.6 ± 0.4 (average ± SD) when the passenger strand contained a 5'-phosphate rather than a hydroxyl group. R2D2 photocrosslinking in ATP-depleted embryo lysate likewise required a 5'-phosphate at the more thermodynamically stable siRNA end. Thus, R2D2 can sense two aspects of siRNA structure: the stability of an siRNA 5' end, and the presence of a 5'-phosphate group. In contrast, Dcr-2 photocrosslinking is unperturbed by a 5'-hydroxyl group on the guide strand, both for the purified protein and in ATP-depleted lysate (Tomari, 2004b).
Active siRNAs contain 5'-phosphate groups on both strands. A 5'-phosphate on the guide strand is essential for siRNA function, but blocking 5'-phosphorylation of the passenger strand impairs rather than eliminates siRNA activity. The results suggest a molecular explanation for this observation: a 5'-phosphate on the passenger strand enhances R2D2 binding, thereby facilitating efficient incorporation of an siRNA into the RLC and consequently into the RISC. Thus, R2D2 is a licensing factor that ensures that only authentic siRNAs enter the RNAi pathway in Drosophila (Tomari, 2004b).
Dcr-2 alone does not efficiently bind siRNA, nor can Dcr-2 alone be photocrosslinked to any of the siRNAs in this study. Taken together, these results suggest that orientation of the Dcr-2/R2D2 heterodimer is determined largely by R2D2 binding to the siRNA end with the most double-stranded character. This binding is presumably mediated by one or both of the R2D2 double-stranded RNA binding domains. A 5' mismatch on an siRNA strand may therefore be an antideterminant for R2D2 binding, acting to direct the R2D2 protein to the 5' end of the passenger strand and positioning Dcr-2 near the 5' end of the strand to be loaded into the RISC. In this model, R2D2, as a component of the Dcr-2/R2D2 heterodimer, is the primary protein sensor of siRNA thermodynamic asymmetry (Tomari, 2004b).
How does the RLC, with the Dcr-2/R2D2 heterodimer positioned asymmetrically on the siRNA, progress to the RISC? Argonaute 2 (Ago2) is a ~130-kD protein that is a core component of the RISC and is required for siRNA unwinding. An ~130-kD protein was crosslinked to siRNA when the guide strand contained 5-iodouracil at p20. The ~130-kD protein was photocrosslinked only to the guide strand of the siRNA, which suggests that this protein is a component of the RISC. The ~130-kD protein was immunoprecipitated with antibodies to Ago2 but not to Ago1 and was not observed in embryos lacking both maternal and zygotic Ago2 (ago2414). Thus, the ~130-kD protein is Ago2. When R2D2 and Ago2 were photocrosslinked to siRNAs that contain 5-iodouracil at p20 of the passenger or the guide strand, R2D2 is bound to the 3' end of the guide strand and Dcr-2 to the 3' end of the passenger strand at early times in the reaction. Later, binding of R2D2 and Dcr-2 decrease concurrently, accompanied by a corresponding increase in binding of Ago2 to the 3' end of the guide strand. In ago2414 lysates, R2D2 binding to the 3' end of the guide strand and Dcr-2 binding to the 3' end of the passenger strand did not decrease with time; this finding suggests that binding of Ago2 facilitates the release of the heterodimer from siRNA (Tomari, 2004b).
The siRNA bound by Ago2 is single-stranded, because Ago2, when photocrosslinked to siRNA, was captured by a tethered 2'-O-methyl oligonucleotide complementary to the siRNA guide strand. R2D2 is not captured by the 2'-O-methyl oligonucleotide, but is instead recovered in the supernatant, consistent with R2D2 binding of double-stranded siRNA (Tomari, 2004b).
The data suggest a model for RISC assembly. Initially, R2D2 orients the Dcr-2/R2D2 heterodimer on the siRNA within the RLC. As siRNA unwinding proceeds, the heterodimer is exchanged for Ago2, the core component of the RISC. Indeed, single-stranded siRNA was not detected in the RLC assembled in ago2414 lysate. It is hypothesized that unwinding occurs only when Ago2 is available, so that siRNA in the RLC is unwound only when the RISC can be assembled (Tomari, 2004b).
Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer, alone, can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex (RLC), which also requires Ago2 for assembly into RISC. R2 was compared to the RLC and the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. The requirements for RISC formation were examined; the siRNA 5' termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2' position of the 5'-terminal nucleotide also affects RISC assembly because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference (RNAi) pathway (Pham, 2005).
To act as silencing triggers, double-stranded siRNAs must be channeled through an ordered RISC assembly pathway that results in the selection of one strand and the destruction of the other. The results indicate that siRNA ends are recognized at multiple steps in the pathway and that these recognition events determine whether the siRNAs will become incorporated into the RISC or blocked from further assembly. The first recognition event occurs at initiation, when Dcr-2 and R2D2 bind the siRNA to form the RDI complex. RDI formation requires 5'-phosphorylated siRNA; however, the siRNA need not be phosphorylated on both strands. siRNAs bearing a single 5'-phosphate group are ineffective at guiding mRNA cleavage. This is true for siRNAs phosphorylated only on the guiding strand as well as those phosphorylated only on the nonguiding strand. To explain these observations, a model has been involked in which R2D2 acts as a sensor for siRNA asymmetry. It has been suggest that a 5' phosphate on the thermodynamically disfavored 'passenger' strand is required for stable R2D2 binding, facilitating siRNA incorporation into the RLC and the RISC (Pham, 2005 and references therein).
When only one of two siRNA strands is phosphorylated, R2D2 and Dcr-2 can still avidly engage the siRNA, probably with R2D2 located at the phosphorylated end as predicted. Despite this, singly phosphorylated siRNA duplexes are still ineffective silencing triggers. The data indicate that a second 5'-recognition event occurs after R2D2 binding. RNA unwinding and activation can proceed, but only if the siRNA bears the second 5' phosphate at the end not occupied by R2D2. Since Ago2 is required both for siRNA unwinding and target mRNA cleavage, it may be the factor that recognizes the second 5' phosphate. An archaebacterial PIWI protein has been shown to have conserved residues that interact with the 5'-phosphate group of siRNA-like duplexes. Furthermore, when analogous residues were mutated in the human Ago2 PIWI domain, target mRNA cleavage is compromised. These results seem to implicate Ago2 in the second 5' recognition event. Alternatively, Ago2 may not engage the 5' phosphate until later in the assembly pathway. In this case, some other factor may interact with the second 5' phosphate upstream of Ago2, causing the RISC assembly defect that is observed with singly phosphorylated siRNA duplexes (Pham, 2005).
Like the 5'-phosphate group, the 2' hydroxyl of the 5'-terminal nucleotide can influence RISC assembly. A 5'-terminal 2'- deoxyribose modification on one siRNA strand inhibits the cognate strand from entering the RISC. Since the unmodified, cognate strand does not accumulate in an unwinding assay, it is probably degraded. In contrast, the modified strand has enhanced activity and is apparently favored for entry into the RISC. There are at least two (non-mutually exclusive) explanations for these observations. The 2'-deoxyribose may be a negative determinant for R2D2 binding. In support of this possibility, the dT-PO4-modified Pp-luc-switch siRNA forms very little of the RDI and R2 complexes, even though it has a relatively stable and phosphorylated terminus that R2D2 would be expected to bind. Decreased R2D2 binding would bias the 'sensor' of siRNA asymmetry, leading to increased selection of the modified strand and increased degradation of the unmodified strand, as is observed. Alternatively, the 2'-deoxyribose-modified strand may have an increased affinity for some downstream RNAi factor, thereby enhancing its entry into the RISC. This may also explain why a single-stranded dTPO4-modified siRNA is better at guiding target mRNA cleavage than the analogous rU-PO4 control. In either case, the observations are surprising because the RNAi apparatus generally tolerates limited modifications at the 2' position of siRNA duplexes, both on the sense and antisense strands. However, completely substituting siRNAs with 2'-deoxy modifications on either the sense or antisense strand abolishes their ability to guide target mRNA cleavage, perhaps by altering the helical geometry of the duplex. Since there does not seem to be a general requirement for 2' hydroxyls along the length of the siRNA nonguiding strand, the defect observed for 5'- terminal 2'-deoxyribose modifications could reflect a specific recognition event that cannot yet be fully defined (Pham, 2005).
Since a 5'-terminal 2'-deoxyribose modification on one siRNA strand can inhibit the activity of the cognate strand, it should reduce the off-target effects that can complicate RNAi-mediated gene silencing. Thus, this simple and inexpensive modification should be considered in the design of effective siRNA reagents for targeted gene knock-down (Pham, 2005).
Finally, progress has been made in the goal of developing a more unified understanding of RISC assembly in Drosophila. The R2D2/Dcr-2 heterodimer, alone, can initiate RISC assembly by binding siRNAs to form the RDI complex. Like the RDI, Complex B has been proposed to act at initiation. Since this complex can form in the absence of R2D2, it is upstream of the RDI and may play an auxiliary role in RISC assembly. Complex B may transfer bound siRNA directly to the RDI in addition to (or instead of) the RLC. Continuing investigation will be required to resolve this issue (Pham, 2005).
The results suggest that the RLC and R2 complexes have more in common than previously thought. Based on side-by-side comparisons, both complexes can form in extracts that lack Ago2 and in extracts depleted of ATP. Furthermore, both complexes can assemble on siRNAs that lack a phosphate on one of the two siRNA strands. Based on these observations, R2 and the RLC are probably identical complexes, and R2 is now referred to as the RLC (Pham, 2005).
Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. A Drosophila in vitro system was used in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro (Preall, 2006).
In Drosophila, the Dicer-2/R2D2 complex initiates RNA interference (RNAi) by processing long double-stranded RNA (dsRNA) into small interfering RNA (siRNA). Recent biochemical studies suggest that the Dcr-2/R2D2 complex also facilitates incorporation of siRNA into the RNA-induced silencing complex (siRISC). This study presents genetic evidence that R2D2 and Dcr-2 are both required for loading siRNA onto the siRISC complex. Consistent with this, only the Dcr-2/R2D2 complex, but neither Dcr-2 nor R2D2 alone, can efficiently interact with duplex siRNA. Furthermore, both dsRNA-binding domains of R2D2 are critical for binding to siRNA and promoting assembly of the siRISC complexes (Liu, 2006).
The current study strengthens the model that R2D2 bridges the initiation step (siRNA production) and the effector step (siRNA loading onto siRISC) of the Drosophila RNAi pathway. The assembly of the siRISC complex is abolished or diminished when Dcr-2 or R2D2 are genetically removed. The role of the Dcr-2/R2D2 complex to load siRNA onto the siRISC complex is dependent on its ability to bind siRNA. Only the Dcr-2/R2D2 complex, but neither Dcr-2 nor R2D2 alone, could efficiently interact with the siRNA duplex. In addition, both dsRNA-binding domains of R2D2 are critical for binding to siRNA and promoting siRISC assembly. Therefore, Dcr-2 and R2D2 coordinately bind siRNA and constitute the gateway for siRNA loading and RISC activation (Liu, 2006).
Depletion of Dcr-2 by RNAi also diminishes the level of R2D2 protein, whereas RNAi of R2D2 cause a modest reduction in Dcr-2 protein in S2 cells. Moreover, recombinant Dcr-2 and R2D2 proteins are produced at much higher levels when expressed jointly than individually in insect cells. Both results suggest that Dcr-2 and R2D2 proteins stabilize each other in vivo. In the current study, it was found that the level of Dcr-2 protein remains the same in r2d21 null flies, whereas both Dcr-2 and R2D2 proteins are missing in dcr-2R416X null flies. One possible explanation for the difference seen in S2 cells and flies is that the level of Dcr-2 protein may be compensated when Drosophila cells permanently lack R2D2. Therefore, it is concluded that Dcr-2 stabilizes R2D2, but the stability of Dcr-2 is largely independent of R2D2 in the fruitfly. Consistent with this, His-tagged Dcr-2, but not R2D2, recombinant proteins can be successfully produced in insect cells. This is probably because R2D2 becomes unfolded or unstable in the absence of Dcr-2 (Liu, 2006).
The results indicate that Dcr-2 and R2D2 bind siRNA coordinately. Only the Dcr-2/R2D2 complex, but neither Dcr-2 nor R2D2 alone, can efficiently interact with siRNA duplex in the gel-shift assay. R2D2 contains tandem dsRNA-binding domains (dsRBDs). The first, but not the second, dsRBD of R2D2 is capable of binding long dsRNA. However, both dsRBDs of R2D2 are necessary for siRNA binding by the Dcr-2/R2D2 complex. Thus, the second dsRBD of R2D2 is critical for binding to siRNA rather than long dsRNA. It is possible that, without the second dsRBD, the first dsRBD of R2D2 only possesses low affinity for the 21–22-nt siRNA duplex (Liu, 2006).
It has been shown that R2D2 as well as Dcr-2 can be efficiently cross-linked to radiolabeled siRNA by ultraviolet (UV) light. Thus, both Dcr-2 and R2D2 are in close contact with siRNA strands. Dcr-2 contains an RNA helicase domain, a DUF283 domain, and a PAZ domain at the N terminus as well as tandem RNase III motifs and a dsRBD motif at the C terminus. It is unclear which of these domains physically contact siRNA. Since neither Dcr-2 nor R2D2 bind siRNA alone, it is possible that siRNA is bound at the interface between Dcr-2 and R2D2. It is also possible that association of Dcr-2 and R2D2 triggers conformational change in either or both proteins, allowing them to bind siRNA cooperatively (Liu, 2006).
It has been reported that Dcr-2 is required for siRNA-initiated RISC assembly in vivo. This study presents genetic evidence that R2D2 is also required for loading siRNA onto the siRISC complex in vivo. It is possible that the Dcr-2/R2D2 complex helps recruit the siRNA duplex to Ago2 for siRISC assembly. However, it remains unclear exactly how the Dcr-2/R2D2 complex facilitates incorporation of siRNA into the siRISC complex. While newly synthesized siRNA is double-stranded, siRNA exists as a single-stranded form in an active siRISC complex. Thus, the nascent siRNA duplex must be unwound during siRISC assembly. It is reasonable to speculate that the Dcr-2/R2D2 complex facilitates unwinding of the siRNA duplex, thereby promoting incorporation of single-stranded siRNA into the siRISC complex. Dcr-2 is a candidate for the siRNA-unwinding helicase because it carries a putative DExH helicase domain and physically contacts the siRNA end that is easier to unwind. However, two dcr-2 mutations in the helicase domain have been isolated that do not affect the siRNA-initiated RISC activity, suggesting that a functional helicase activity is not required for Dcr-2 to promote siRISC assembly. In addition, recombinant Dcr-2 or Dcr-2/R2D2 complex cannot unwind the siRNA duplex in vitro. Alternatively, the Dcr-2/R2D2 complex may recruit an unknown helicase to unwind the siRNA duplex. In Caenorhabditis elegans, the DCR-1/RDE-4 (an R2D2 homolog) complex is associated with two highly related RNA helicases, DRH-1 and DRH-2, that are necessary for RNAi. Several RNA helicases, such as Armitage and Dmp68, have also been implicated in the Drosophila RNAi pathways (Liu, 2006).
Recent studies also suggest an alternative model for separation of siRNA strands and activation of the siRISC complex. After the Dcr-2/R2D2 complex recruits duplex siRNA to Ago2, the PIWI domain of Ago2 cleaves the passenger strand and facilitates the formation of an active siRISC complex containing only the guide strand. It is likely that the orientation of siRNA binding by the Dcr-2/R2D2 complex allows Ago2 to access and cleave only one of the two siRNA strands. Therefore, the Dcr-2/R2D2 complex determines which strand of siRNA duplex becomes the guide strand or passenger strand. The two mechanistic models of siRISC assembly are not mutually exclusive. In either model, the Dcr-2/R2D2 complex plays a critical role in facilitating the strand separation of the duplex siRNA. Since not all Ago proteins possess the slicer activity, there must be more than one mechanism for siRNA loading and RISC activation (Liu, 2006).
r2d2 deletion mutant flies were generated by P element mobilization and crossed with transgenic flies expressing green fluorescent protein (GFP) under the ubiquitin promoter to derive homozygous r2d2; Ub-GFP mutant flies. Then, 0- to 2-hour wild-type or r2d2 mutant embryos were collected for microinjection of GFP dsRNA. Whereas introduction of GFP dsRNA effectively silenced GFP expression in wild-type embryos, r2d2 mutant embryos were completely defective for the dsRNA-initiated RNAi response (Liu, 2003).
Innate immunity against bacterial and fungal pathogens is mediated by Toll and immune deficiency (Imd) pathways, but little is known about the antiviral response in Drosophila. This study demonstrates that an RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. The work also describes a molecular framework for the viral immunity, in which viral double-stranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, respectively. These findings establish a Drosophila model for studying the innate immunity against viruses in animals (Wang, 2006).
RNA interference (RNAi) silences gene expression through small interfering RNAs (siRNAs) and microRNAs (miRNAs). In Drosophila, Dicer-2 (Dcr-2) produces siRNAs, whereas Dicer-1 (Dcr-1) recognizes precursors of miRNAs. The small RNAs are assembled with an Argonaute (Ago) protein into related effector complexes, such as RNA-induced silencing complex (RISC), to guide specific RNA silencing (Wang, 2006).
RNA silencing provides an antiviral mechanism in plants and animals. Plant viruses have evolved diverse strategies for evading the RNA silencing immunity, and expression of viral suppressors of RNAi (VSRs) is essential for infection and virulence. However, it is unknown whether antiviral silencing in plants is controlled by a specific small RNA pathway targeted by plant VSRs. Bacterial and fungal infections of Drosophila induce Toll and immune deficiency (Imd) pathways, leading to transcriptional induction of antimicrobial peptide effectors via NF-KappaB)–like signaling processes. However, it has been unclear whether either pathway plays a role in Drosophila innate immunity against viruses. Previous work in cell culture has indicated that RNAi might mediate viral immunity in Drosophila. This study investigated whether RNAi indeed provides protection against virus infection in Drosophila embryos and adults (Wang, 2006).
Flock house virus (FHV) contains an RNA genome divided among two plus-strand molecules, RNAs 1 and 2. RNA2 (R2) encodes the single virion structural protein, whereas RNA1 (R1) encodes protein A, the viral RNA–dependent RNA polymerase (RdRP), and B2, a VSR expressed after RNA1 replication from its own mRNA, RNA3. In the absence of R2, R1 replicates autonomously, accumulates to high levels, and produces abundant RNA3 in wild-type (WT) Drosophila embryos 30 hours after injection with R1 transcripts synthesized in vitro. No FHV RNAs accumulate in WT embryos injected with R1fs transcripts that contain a frameshift mutation in the RdRP open reading frame (ORF). FHV RNAs are also not readily detected in WT embryos injected with a second mutant of R1, R1DeltaB2, which does not express the VSR. However, abundant accumulation of R1DeltaB2 but not FR1fs occurs in mutant Drosophila embryos that carry a homozygous null mutation in ago-2 (ago-2414), which is essential for RNAi in Drosophila. These data indicated that viral RNA replication in Drosophila embryos triggers an RNAi-mediated virus clearance in an Ago-2–dependent manner and that effective RNAi suppression by B2 is necessary to achieve normal accumulation of FHV RNAs (Wang, 2006).
In Drosophila, Ago-2 acts downstream of Dicer-2 (Dcr-2) to recruit siRNAs, the products of Dcr-2 activity, into the siRNA-dependent RISC (siRISC). Thus, a genetic requirement for ago-2 in FHV RNA clearance implicates Dcr-2 in the RNAi antiviral effector mechanism. To test this hypothesis, R1, R1fs, and R1DeltaB2 transcripts were injected into embryos carrying a homozygous dcr-2 null mutation, dcr-2L811fsX. Northern blot hybridizations showed that, although FHV RNAs remained undetectable in dcr-2L811fsX embryos injected with R1fs, viral RNA accumulation is rescued in the dcr-2L811fsX embryos injected with R1DeltaB2 transcripts. This result shows that Dcr-2 is required to initiate RNAi-mediated clearance of FHV RNAs in Drosophila embryos (Wang, 2006).
To investigate whether the RNAi pathway protects Drosophila from virus infection, adult flies of either WT or dcr-2L811fsX genotype were injected with purified FHV virions. The FHV isolate was of low virulence in WT flies, because about 50% of infected flies survived 15 days postinoculation (dpi) despite a detectable virus load. Inoculation with the same dose of FHV resulted in 60% mortality by 6 dpi and 95% by 15 dpi in dcr-2L811fsX flies. Mock inoculation with buffer had little effect on either WT or dcr-2L811fsX flies for as long as observations were made. Both Northern and Western blot analyses revealed that the virus accumulated more rapidly and to much greater levels in dcr-2L811fsX than WT flies. Thus, dcr-2 mutants exhibit enhanced disease susceptibility to FHV in comparison with WT flies, demonstrating that Dcr-2 is also required to mount an immune response that protects adult Drosophila against FHV infection (Wang, 2006).
R2D2 contains tandem double-stranded RNA (dsRNA)–binding domains and forms a heterodimer with Dcr-2 in vivo that is required for siRNA loading into RISC. Flies homozygous for a loss-of-function mutation in r2d2 exhibit a phenotype of enhanced disease susceptibility to FHV infection similar to that of dcr-2L811fsX. Thus, R2D2 also participates in the innate immunity pathway that protects adult flies from FHV infection. Notably, although FHV accumulates to extremely high levels in both dcr-2 and r2d2 mutant flies, abundant viral siRNAs were detected only in r2d2 mutant flies, and viral siRNAs were below the level of detection in dcr-2L811fsX flies. Thus, FHV infection is detected by Dcr-2, leading to production of FHV siRNAs. However, R2D2 is not required for the production but is essential for the function of viral siRNAs, which is consistent with the genetic requirements for processing the artificially introduced dsRNA (Wang, 2006).
To investigate whether the RNAi pathway in Drosophila is specific against nodaviruses and not other classes of RNA viruses, the susceptibility of WT, dcr-2L811fsX, and r2d2 mutant flies to cricket paralysis virus (CrPV) was assessed. CrPV contains a nonsegmented plus-strand RNA genome but belongs to a group of picorna-like viruses. CrPV is substantially more virulent than FHV in Drosophila; injection of CrPV at much lower titers resulted in mortality of 70% of WT flies by 15 dpi. CrPV was also found to induce enhanced disease susceptibility in both dcr-2 and r2d2 mutant flies. About 60% of the infected mutant flies were dead by 6 dpi, and more than 95% were dead by 15 dpi. In addition, Northern blots indicated that the virus accumulated more rapidly and to greater levels in the mutant flies. Thus, both dcr-2 and r2d2 are required for protection of Drosophila against CrPV (Wang, 2006).
CrPV infection of cultured S2 cells induced antiviral silencing, illustrated by detection of CrPV-specific siRNAs. Antiviral silencing against FHV in S2 cells induced by FR1gfp is suppressed by CrPV superinfection, leading to derepression of green fluorescent protein (GFP). Two ORFs are encoded by the CrPV RNA genome. No suppression of antiviral silencing was observed in S2 cells cotransfected with a plasmid expressing either the entire downstream ORF of CrPV or the individual mature virion proteins processed from the polyprotein. In contrast, RNAi suppression was detected after cotransfection with a plasmid expressing either the entire upstream ORF of CrPV or its N-terminal 140 codons. However, the suppressor activity was not detected after a frameshift mutation was introduced into pA, thus identifying the N-terminal fragment of 140 amino acids of the CrPV nonstructural polyprotein as a VSR (Wang, 2006).
In Drosophila, Imd signaling is stimulated by Gram negative (Gram–) bacterial infection, whereas Toll signaling is triggered by Gram positive (Gram+) bacterial infection. To determine whether loss of the RNAi pathway initiated by Dcr-2 has an impact on the Toll and Imd signaling processes, WT, dcr-2L811fsX, and r2d2 mutant flies were subjected to immune challenge by inoculation with Escherichia coli (Gram–) or Micrococcus luteus (Gram+). Northern blot hybridizations detected substantial transcriptional induction of the antimicrobial peptide gene Diptericin A 6 hours postimmune challenge (hpi) with either E. coli or M. luteus, which declined at 24 hpi as described. Similar induction patterns for Diptericin A were observed in dcr-2L811fsX and r2d2 mutant flies inoculated with Gram+ and Gram– bacteria. Furthermore, it was found that induction of either Attacin A or Drosomycin by Gram+ and Gram– bacteria was also not altered in dcr-2L811fsX and r2d2 mutant flies as compared to WT flies. These results indicate that induction of antimicrobial peptide genes via Toll and Imd signaling pathways is not compromised in dcr-2L811fsX and r2d2 mutant flies (Wang, 2006).
Nodaviruses and the polio-like CrPV belong to two different superfamilies of animal RNA viruses. The same set of RNAi pathway genes is required for Drosophila defense against FHV and CrPV and both viruses encode a potent VSR. These results collectively show that RNAi pathway functions as a common viral immunity mechanism in Drosophila and that RNAi suppression represents a general counterdefensive strategy used by insect viruses. Furthermore, a genetic requirement for Dcr-2, R2D2, and Ago-2 in antiviral silencing establishes a molecular framework for the innate immunity against viruses in Drosophila. None of Dcr-2, R2D2, and Ago-2 plays a detectable role in either the production or function of miRNAs in Drosophila. Thus, this work identifies the dsRNA-siRNA pathway of RNAi as providing the innate immunity against virus infection in Drosophila and establishes that dsRNA produced during virus replication acts as the pathogen trigger whereas Dcr-2 and Ago-2 act as host sensor and effector of the immunity, respectively. These results support and extend the previous findings on antiviral silencing in C. elegans (Wang, 2006).
Although NF-KappaB-like signaling in the Toll and Imd pathways do not appear to play a role in the RNAi-directed viral immunity mechanism in Drosophila, the fly mutant defective in the Janus kinase (JAK) Hopscotch exhibit a modest increase in susceptibility to infection with Drosophila C virus, suggesting an antiviral role for JAK–signal transducer and activator of transcription (STAT) signaling. Nonetheless, it is believed that RNAi-based immunity is independent of JAK-STAT signaling, because virus infection is not known to induce the RNAi pathway in Drosophila and FHV induction of the JAK-STAT responsive gene vir-1 is unaltered in the dcr-2 and r2d2 mutants. Because the Toll and Imd pathways are highly conserved in vertebrates, the Drosophila model established for RNAi may also be useful for the analyses of the innate antiviral immunity in vertebrates (Wang, 2006).
Elbashir, S. M., Lendeckel, W. and Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15(2): 188-200. 11157775
Liu, Q., et al. (2003). R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301: 1921-1925. 14512631
Liu, X., Jiang, F., Kalidas, S., Smith, D. and Liu, Q. (2006). Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes. RNA 12(8): 1514-20. 16775303
Pham, J. W. and Sontheimer, E. J. (2005). Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway. J. Biol. Chem. 280(47): 39278-83. 16179342
Preall, J. B., He, Z., Gorra, J. M. and Sontheimer, E. J. (2006). Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr. Biol. 16(5): 530-5. 16527750
Tabara, H., Yigit, E., Siomi, H. and Mello. C. C. (2002). The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109: 861-871. 12110183
Tomari, Y., Du, T., Haley, B., Schwarz, D. S., Bennett, R., Cook, H. A., Koppetsch, B. S., Theurkauf, W. E. and Zamore, P. D. (2004a). RISC assembly defects in the Drosophila RNAi mutant armitage. Cell 116(6): 831-41. 15035985
Tomari, Y., Matranga, C., Haley, B., Martinez, N. and Zamore, P. D. (2004b). A protein sensor for siRNA asymmetry. Science 306(5700): 1377-80. 15550672
Wang, X.-H., et al. (2006). RNA interference directs innate immunity against viruses in adult Drosophila. Science 312: 452-454. 16556799
date revised: 20 November 2006
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