InteractiveFly: GeneBrief

r2d2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - r2d2

Synonyms -

Cytological map position - 28C4

Function - RNA-binding protein

Keywords - RNAi, siRNA loading onto RISC, RISC-loading complex, Dicer-2-interacting protein

Symbol - r2d2

FlyBase ID: FBgn0031951

Genetic map position - 2L

Classification - double-stranded RNA binding

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Liang, C., Wang, Y., Murota, Y., Liu, X., Smith, D., Siomi, M. C. and Liu, Q. (2015). TAF11 assembles the RISC loading complex to enhance RNAi efficiency. Mol Cell 59: 807-818. PubMed ID: 26257286
Summary:
Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila. However, the precise composition and action mechanism of Drosophila RLC remain unclear. This study identified the missing factor of RLC as TATA-binding protein-associated factor 11 (TAF11) by genetic screen. Although it is an annotated nuclear transcription factor, TAF11 also associated with Dcr-2/R2D2 and localizes to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11-/- ovary extract, the RLC was reconstituted in vitro using the recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, this study showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, these genetic and biochemical studies define the molecular nature of the Drosophila RLC and elucidate a cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency.
Dowling, D., Pauli, T., Donath, A., Meusemann, K., Podsiadlowski, L., Petersen, M., Peters, R. S., Mayer, C., Liu, S., Zhou, X., Misof, B. and Niehuis, O. (2017). Phylogenetic origin and diversification of RNAi pathway genes in insects. Genome Biol Evol. PubMed ID: 28062756
Summary:
RNA interference (RNAi) refers to the set of molecular processes found in eukaryotic organisms in which small RNA molecules mediate the silencing or down-regulation of target genes. In insects, RNAi serves a number of functions, including regulation of endogenous genes, anti-viral defense, and defense against transposable elements. Despite being well studied in model organisms, such as Drosophila, the distribution of core RNAi pathway genes and their evolution in insects is not well understood. This study presents the most comprehensive overview of the distribution and diversity of core RNAi pathway genes across 100 insect species, encompassing all currently recognized insect orders. The phylogenetic origin of insect-specific RNAi pathway genes was inferred, and also several hitherto unrecorded gene expansions were identified using whole-body transcriptome data from the international 1KITE (1000 Insect Transcriptome Evolution) project as well as other resources such as i5K (5000 Insect Genome Project). Specifically, the origin of the double stranded RNA binding protein R2D2 was traced to the last common ancestor of winged insects (Pterygota), the loss of Sid-1/Tag-130 orthologs in Antliophora (fleas, flies and relatives, and scorpionflies in a broad sense), and confirm previous evidence for the splitting of the Argonaute proteins Aubergine and Piwi in Brachyceran flies (Diptera, Brachycera). This study offers new reference points for future experimental research on RNAi-related pathway genes in insects.
BIOLOGICAL OVERVIEW

The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). This process involves three proteins -- Dicer-2 (DCR-2), R2D2, whose name derives from the fact that it contains two dsRNA-binding domains (R2) and is associated with DCR-2 (D2), and Argonaute-2, the core component of the RISC. The siRNA-generating enzyme was purified from Drosophila S2 cells and consists of two stoichiometric subunits: DCR-2 and the newly discovered R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer (carrying out the initiation step) to RISC, which carrys out the effector step (Liu, 2003). A model for RISC assembly is presented. Initially, R2D2 orients the Dcr-2/R2D2 heterodimer on the siRNA within the RISC-loading complex (RLC). As siRNA unwinding proceeds, the heterodimer is exchanged for Argonaute-2, the core component of the RISC. Indeed, single-stranded siRNA was not detected in the RLC assembled in mutant 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).

RNAi is a form of posttranscriptional gene silencing whereby double-stranded RNA (dsRNA) molecules trigger the sequence-specific degradation of cognate mRNA. The biological importance of RNAi is underscored by its wide conservation throughout metazoans and the existence of closely related systems in plants (called cosuppression) and fungi (called quelling). Emerging evidence indicates that the RNAi and related pathways function in many fundamental biological processes, including antiviral defense, development, and maintenance of genomic stability (Liu, 2003 and references therein).

The Drosophila RNAi pathway consists of initiation and effector steps. Initially, long dsRNA molecules are cleaved into 21- to 23-nucleotide (nt) small interfering RNA (siRNA) duplexes. Next, the siRNA is incorporated into a nuclease complex named RNA-initiated silencing complex (RISC) and functions as a guide RNA to direct RISC-mediated sequence-specific mRNA degradation. The endonucleases that process dsRNA have been identified as Dicers, a family of large noncanonical ribonuclease (RNase) III enzymes. Although only one Dicer enzyme is found in Caenorhabditis elegans and humans, two (DCR-1 and DCR-2) have been identified in Drosophila. It remains unclear how siRNA is transferred from Dicer to RISC (Liu, 2003).

This problem was studied by purifying the siRNA-generating activity from the cytoplasmic (S100) extract of S2 cells through a six-step chromatographic procedure. A single major peak of activity was observed at all steps and was followed throughout purification. Two proteins, ~190 kD and ~36 kD, showed perfect correlation with the enzymatic activity after the final gel filtration step. They were identified by mass spectrometry to be DCR-2 and a previously unknown protein (Flybase CG7138 or R2D2), respectively. R2D2 bears 20.9% identity and 33.4% similarity to the C. elegans RNAi protein RDE-4, which also contains tandem dsRNA-binding domains and interacts with Dicer (Liu, 2003).

To confirm these purification results, immunodepletion experiments were performed with antiserum directed against the carboxyl-terminal 150 amino acids of R2D2. This R2D2 antibody depleted both R2D2 and DCR-2 proteins and removed the majority of siRNA-generating activity from S100, whereas the level of DCR-1 remained unchanged in the supernatant. Furthermore, when S100 was fractionated on a QSepharose column, DCR-2 and R2D2, but not DCR-1, correlated perfectly with the siRNA-generating activity (Liu, 2003).

To determine the contribution of DCR-1 and DCR-2 to siRNA production in Drosophila cells, either protein was depleted from S2 cells by dsRNA soaking. Interestingly, DCR-2 dsRNA also caused a substantial reduction in the level of R2D2 protein. This was not simply due to cross-targeting because the level of R2D2 mRNA was unaffected. Likewise, R2D2 dsRNA also reduced, although not as dramatically, the level of DCR-2 protein. These results indicate that DCR-2 and R2D2 form a stable complex and that either protein alone is unstable. Although depletion of DCR-1 made no difference, knocking down DCR-2 reduced the siRNA-generating activity by fivefold in lysates and interfered with RNAi in vivo. Together, these results demonstrate that the DCR-2/R2D2 complex is the principal siRNA-generating enzyme responsible for initiation ofRNAi in Drosophila S2 cells (Liu, 2003).

To determine whether R2D2 is required for RNAi in vivo, 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).

The siRNA-generating activity was reconstituted in vitro with the use of purified His6-tagged DCR-2 and R2D2 recombinant proteins expressed in insect cells. A mutant form of R2D2 was also created by point mutations in the dsRNA-binding domains that abolish its ability to bind dsRNA. DCR-2 protein alone efficiently cleaved dsRNA into siRNA in an adenosine triphosphate (ATP)- and dose-dependent manner. The DCR-2/R2D2 complex is also ATP-dependent and shows activity equivalent to DCR-2 alone. Kinetic studies were performed with DCR-2 and DCR-2/R2D2 recombinant proteins and no statistically significant difference was found in their Km or Kcat (a measure of catalytic activity). Furthermore, the mutant DCR-2/R2D2M complex was as active in siRNA production as the wild-type complex. These results indicate that association with R2D2 does not affect the ability of DCR-2 to recruit or cleave dsRNA. This finding is inconsistent with the proposed function of RDE-4 to recruit dsRNA to DCR-1 (the single Dicer in worms) for processing (Tabara, 2002). However, R2D2 may stabilize DCR-2 and thereby positively regulate siRNA production in Drosophila cells. Consistently, DCR-2 and R2D2 were expressed at much higher levels in insect cells when expressed jointly than separately (Liu, 2003).

To follow the fate of siRNA, a gel shift assay was developed to identify proteins that interact with siRNA. When radiolabeled synthetic siRNA was incubated with S2 lysate, a distinct mobility shift was observed on a native polyacrylamide gel. The formation of this siRNA-protein complex did not require ATP hydrolysis because it could be carried out efficiently at 4°C. Furthermore, when S100 was fractionated by a QSepharose column, the peak of siRNA-binding activity correlated well with that of the siRNA-generating activity. The gel shift assays were then performed in the presence of antibodies against DCR-2 or R2D2. Both antibodies resulted in a supershift that was absent when their preimmune sera were used instead. This indicated that DCR-2 and R2D2 were present in this siRNA-protein complex (Liu, 2003).

Purified DCR-2, DCR-2/R2D2, and DCR-2/R2D2M recombinant proteins were examined for siRNA binding by the gel shift assay. Wild-type DCR-2/R2D2 complex, but not DCR-2 alone, bound to siRNA and produced a mobility shift indistinguishable from that of S100. The ability of the DCR-2/R2D2 complex to bind siRNA was greatly diminished by point mutations within the two dsRNA-binding domains of R2D2. Thus, R2D2 is important for binding to the product (siRNA) rather than the substrate (dsRNA) of DCR-2 (Liu, 2003).

Furthermore, when DCR-2/R2D2 proteins and radiolabeled siRNA were exposed to ultraviolet (UV) light, both DCR-2 and R2D2 were crosslinked to siRNA. This crosslinking was greatly diminished when the mutant DCR-2/R2D2M complex was used instead, although similar amounts of proteins were used. Interestingly, two R2D2-siRNA crosslinked bands were observed, one at ~45 kD and another at ~52 kD, which probably represented R2D2 proteins covalently linked to one or two siRNA strands (the mass of each 21-nt siRNA strand was ~7 kD). This was confirmed by the downshift of both R2D2-siRNA crosslinked bands to the original position of ~38 kD His6-R2D2 protein after RNase treatment. These results indicate that DCR-2 and R2D2 bind siRNA coordinately, which is dependent upon the dsRNA-binding domains of R2D2 (Liu, 2003).

On the basis of this finding, it is hypothesized that R2D2 might be involved in facilitating siRNA loading onto RISC. To test this hypothesis, the RISC activity was separated from the Dicer activity in S100 by polyethylene glycol (PEG) precipitation and then combined the partially purified RISC with recombinant Dicer proteins to reconstitute the RNAi reaction. Although the majority of siRNA-generating activity was precipitated by 10% PEG, a substantial amount of RISC remained in the supernatant, which could be activated by addition of siRNA for sequence-specific mRNA degradation. The wild-type DCR-2/R2D2 complex was much more effective than either DCR-2 alone or the mutant complex in promoting the dsRNA-initiated RISC activity in the 10% PEG supernatant. At 3 nM concentrations, wild-type DCR-2/R2D2 stimulated the RISC activity by more than sevenfold, whereas DCR-2 or DCR-2/R2D2M stimulated RISC activity only by twofold. Interestingly, the mutant complex blocked the RISC activity at 10 nM concentration, possibly as a result of dominant-negative effects. Furthermore, a similar phenomenon was observed in the siRNA-initiated RISC assay. Thus, the DCR-2/R2D2 complex could enhance the siRNA- as well as the dsRNA-initiated RISC activities. It was further shown that this enhancement was not simply because of siRNA stabilization by DCR-2/R2D2, by comparing the stability of radiolabeled siRNA in the RISC reactions described above (Liu, 2003).

To confirm that DCR-2/R2D2 facilitates siRNA loading onto RISC, the association between AGO2, an essential component of RISC, and a 3'-biotinylated siRNA was followed by precipitation using streptavidin beads. The biotinylated siRNA was as active as unmodified siRNA in inducing RISC activities in S100. However, streptavidin beads precipitated AGO2 protein only when biotinylated siRNA was used, suggesting that the siRNA RISC activity was specific. RISC assays were then performed with the use of biotinylated siRNA in 10% PEG supernatant alone or in combination with recombinant DCR-2, DCR-2/R2D2, and DCR-2/R2D2M proteins. Consistently, more AGO2 proteins were detected in the biotinylated siRNA precipitates when wild-type DCR-2/R2D2 complex was used instead of DCR-2 alone or the mutant complex. Together, these results indicate that DCR-2/R2D2 not only generates siRNA from dsRNA but also binds to nascent siRNA and facilitates its loading onto RISC. The latter activity is dependent on the dsRNA-binding domains of R2D2 (Liu, 2003).

This model is supported by previous studies in which the direction of dsRNA processing was limited to one end of the dsRNA molecule (Elbashir, 2001). In these experiments, if dsRNA was processed from the 5' to 3' direction of the sense strand, it would generate RISC that could mediate degradation of the sense but not antisense target mRNA, and vice versa. However, if synthetic siRNA was used instead, it produced RISC that could degrade either sense or antisense target mRNA. Therefore, the newly synthesized symmetric siRNA product must not become a free molecule once it is generated from dsRNA. Rather, the nascent siRNA is likely to be retained by DCR-2/R2D2 in a fixed orientation determined by the direction of dsRNA processing such that only the antisense strand can become the guiding RNA for RISC (Liu, 2003).

Both R2D2 and RDE-4 contain tandem dsRNA-binding domains, interact with Dicer, and are required for the RNAi pathway. Thus, it is concluded that R2D2 is homologous to RDE-4. RDE-4 also interacts with RDE-1, an AGO2 homolog and a RISC component. Therefore, it is proposed that R2D2 and RDE-4 play a similar role in bridging the initiation and effector steps of the Drosophila and C. elegans RNAi pathways, respectively (Liu, 2003).


REGULATION

R2D2 acts as a sensor for siRNA asymmetry

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).

Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway

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 RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila

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).

Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes

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).


DEVELOPMENTAL BIOLOGY

Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1

The miRNA pathway has been shown to regulate developmentally important genes. Dicer-1 is required to cleave endogenously encoded microRNA (miRNA) precursors into mature miRNAs that regulate endogenous gene expression. RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) that protects organisms from parasitic nucleic acids. In Drosophila, Dicer-2 cleaves dsRNA into 21 base-pair small interfering RNA (siRNA) that are loaded into RISC (RNA induced silencing complex) that in turn cleaves mRNAs homologous to the siRNAs. Dicer-2 co-purifies with R2D2, a low-molecular weight protein that loads siRNA onto Ago-2 in RISC. Loss of R2D2 results in defective RNAi. However, unlike mutants in other RNAi components like Dicer-2 or Ago-2, r2d21 mutants have striking developmental defects. r2d21 mutants have reduced female fertility, producing less than 1/10 the normal number of progeny. These escapers have normal morphology. R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects are dramatically enhanced when one copy of the dcr-1 gene is missing and Dicer-1 protein co-immunoprecipitates with R2D2 antisera. These data show that r2d21 mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function, and Dicer-1 impacts this process. It is concluded that R2D2 functions beyond its role in RNA interference to include ovarian development in Drosophila (Kalidas, 2008).

R2D2 has a well characterized role with Dicer-2 and Ago-2 in RNA interference. R2D2 forms a stable heterodimeric complex with Dicer-2 in vivo and in vitro. R2D2 does not affect the enzymatic activity of Dicer-2, but instead orchestrates the transfer of siRNAs produced by Dicer-2 to Ago-2. Dicer-2 and Ago-2 physically interact in the same complex with R2D2 during RNA interference. dcr-2 mutants are defective for RNAi but have normal fertility. ago-2 mutants are also viable and fertile, but have been recently reported to have defects in nuclear divisions and migration in early embryonic development, with a reduction in the number of pole cells that give ultimately give rise to the germline. However, these subtle developmental defects in ago-2 mutants are compensated for during development so they have little effect on overall fertility, and were not reported in the initial characterization of the null mutants. By contrast, r2d21 mutants have striking fertility defects. These abnormalities are not observed in dcr-2 and have much higher penetrance and are qualitatively different from ago-2 mutants, suggesting there is a Dicer-2 and Ago-2-independent role for R2D2 in vivo. The genetic interaction observed with Dicer-1 provides the first insights into this role (Kalidas, 2008).

The hatching frequency of r2d21 null mutant embryos was approximately 14% of that of wild type embryos. This drop in hatching is due in part to defective fertilization. However, fertilized r2d21 mutant embryos generally failed to develop, showing no signs of nuclear divisions and were usually arrested before the blastoderm stage. Viability was improved in embryos fertilized by wild type sperm, indicating R2D2 is important both maternally and zygotically. Therefore, these data indicates that R2D2 contributes to viability early in development and embryogenesis, in addition to its established role in RNA interference (Kalidas, 2008).

r2d21 mutants have defective ovaries and are partially sterile. The ovaries from these mutants show a range of phenotypic defects, all of which are completely rescued by introduction of an r2d2 transgene into the mutant background, clearly demonstrating loss of R2D2 produces these defects in oogenesis. Clonal analysis reveals a requirement for R2D2 in the somatic follicle cells, but the data indicates there is not a germline requirement for R2D2. Removal of R2D2 specifically from somatic cells results in the typical ovarian architecture defects including loss of stalks observed in r2d21, while loss of R2D2 in germ cells did not affect follicle morphology. The observed follicle cell phenotypes, while broader in scope, are similar to those previously described for defective polar cell specification and formation. Once specified, polar cells are known to secrete a variety of signals that pattern the follicular epithelium. The robust expression of R2D2 protein in the stalk cells will focus future efforts on candidate signaling pathways expressed in these cells (Kalidas, 2008).

Several genes related to RNAi components have defects in ovarian development. These include piwi, aubergine and Spindle-E. However, the phenotypes associated with these other mutants are quite different from found here for r2d21. piwi is an Argonaut homologue involved in stem cell maintenance, co-suppression, heterochromatin formation, and transposon silencing. However piwi, while expressed in both somatic and germline cells, is required in the terminal filament for stem cell maintenance but is also needed for cell division in the germline. Aubergine and spindle-E are also involved in heterochromatin formation and aubergine plays a role in suppression of stellate required for male fertility. However, aubergine and spindle-E are required in the germline. Most recently Loquacious was reported to have a role in germline stem cell maintenance and stellate suppression. R2D2 appears unique among the RNAi-related components identified to date because it is required in the follicle tissue and not in the germline, and has no effect on male fertility. Therefore, the function of R2D2 in female fertility appears to be distinct from these other RNAi related proteins (Kalidas, 2008).

The genetic and physical interaction between Dicer-1 and R2D2 implies these gene products are operating in the same pathway to orchestrate female follicle cell patterning. Dicer-1 mutants are lethal, likely due to loss of mature miRNAs and subsequent regulation of endogenous gene function. r2d21 flies can survive to adulthood and are morphologically normal, so R2D2 is unlikely to have a significant role in processing most miRNAs. One possibility is that Dicer-1 and R2D2 are partnered in the ovary to process one or a small number of miRNAs in the follicle cells. Dicer-1 has three splicing variants, so specific splicing variants of Dicer-1 could potentially partner with R2D2. Alternatively, R2D2 and Dicer-1 may perform an unknown function independent of miRNA processing. Screens to identify suppressor mutations of r2d21 with increased fertility will prove valuable in providing future insights into R2D2-dependent follicle formation (Kalidas, 2008).


EFFECTS OF MUTATION

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).

RNA interference directs innate immunity against viruses in adult Drosophila

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).

The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation. This has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, the viral suppressors of RNA silencing B2 and P19 were used. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. It was hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. These findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila (Fagegaltier, 2009).

This study implicates components of the RNAi pathway in heterochromatin silencing during late Drosophila development. The study also provides correlative evidence supporting a functional link between endo-siRNAs and the formation or maintenance of somatic heterochromatin in flies. The viral proteins NLS-P19 and B2 suppress the silencing of PEV markers and induce aberrant distribution of H3m2K9 and H3m3K9 heterochromatic marks as well as histone H3 methylase Su(var)3-9 in larval tissues. Dcr2 and Ago2 mutations have similar effects. In striking contrast, cytoplasmic P19 has no noticeable effect on chromatin. It is proposed that B2 inhibits Dcr2-mediated processing of double-stranded TE read-through transcripts in the cytoplasm; it is further proposed that NLS-P19 sequesters TE-derived siRNA duplexes. This model implies that part of the cytoplasmic pool of TE-derived endo-siRNA (which might be involved in PTGS events) is translocated back into the nucleus to exert chromatin-based functions. In C. elegans, silencing of nuclear-localized transcripts involves nuclear transport of siRNAs by an NRDE-3 Argonaute protein. A similar siRNA nuclear translocation system, possibly mediated by Ago2, may also exist in flies. Alternatively, an as yet unidentified siRNA duplex transporter may be involved. Deep sequencing analyses show that the fraction of siRNAs sequestered by NLS-P19 is smaller as compared with the one bound by P19 in the cytoplasm. Thus, the poor effects of P19 on nuclear gene silencing may be explained if the cytoplasmic pool of siRNA competes with the pool of siRNA to be translocated in the nucleus (Fagegaltier, 2009).

The Dcr-1 partner Loquacious (Loqs), but not the Dcr-2 partner R2D2, was unexpectedly found to be required for biogenesis of siRNA derived from fold-back genes that form dsRNA hairpins. By contrast, it is noteworthy that loqs mutations had little or no impact on the accumulation of siRNA derived from TE. The finding that r2d2 but not loqs mutation suppresses the silencing of PEV reporters and delocalizes H3m2K9 and H3m3K9 heterochromatic marks agrees with these results and further suggests that siRNA involved in heterochromatin formation and siRNA derived from endogenous hairpins arise from distinct r2d2- and loqs-dependent pathways, respectively. One possible mechanism by which TE- or repeat-derived endo-siRNAs could promote heterochromatin formation is by tethering complementary nascent TE transcripts and guiding Su(var)3-9 recruitment and H3K9 methylation. Identifying which enzymes tether siRNAs to chromatin in animals is a future challenge. In addition, some endo-siRNAs could also impact on heterochromatin formation by posttranscriptionaly regulating the expression of chromatin modifiers, such as Su(var)3-9. In any case, the current results demonstrate the value of viral silencing suppressor proteins in linking siRNAs to heterochromatin silencing in the fly soma, as established in S. pombe and higher plants. Because silencing suppressors are at the core of the viral counterdefensive arsenal against antiviral RNA silencing in fly, whether they also induce epigenetic changes in chromatin states during natural infections by viruses deserves further investigation (Fagegaltier, 2009).


EVOLUTIONARY HOMOLOGS

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. The C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. These findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing (Tabara, 2002).

RNAi and related posttranscriptional gene silencing (PTGS) pathways have been implicated in silencing transposons and/or viruses, suggesting that these pathways may function as a form of sequence-directed immunity. The two strongest mutants discovered in screens carried out to discover C. elegans mutants deficient in RNAi, (rde-1, encoding an Argonaute family member and rde-4) were both completely deficient in RNAi but failed to exhibit transposon activation or any other discernible phenotypes. Subsequent genetic studies suggested that rde-1 and rde-4 may function upstream in the initiation of RNAi (Tabara, 2002 and references therein).

To shed more light on the role of the RDE-4 protein in RNAi, the corresponding gene was positionally cloned. The rde-4 gene is predicted to encode a 385 amino acid protein that contains two copies of a conserved motif found in dsRNA binding proteins. The genetic lesions in rde-4 appear likely to be null mutations, and consistent with this idea, the ne299 allele, when placed in trans to a chromosomal deficiency, exhibits an RNAi-deficient phenotype and no other additional phenotypes (Tabara, 2002).

The RDE-4 protein contains two copies of a dsRNA binding motif (dsRBM) that is found in numerous other proteins that interact with dsRNA in a sequence-nonspecific manner. Therefore, it was first asked whether recombinant RDE-4 could bind to dsRNA by using an in vitro gel shift assay. Labeled dsRNA corresponding to a portion of a GFP cDNA was incubated with GST-fused RDE-4, or with the GST protein alone, and was then run on a native polyacrylamide gel. The GST-fused RDE-4 efficiently bound to the dsRNA, causing a mobility shift, while the GST protein did not. In competition assays, dsRNA but not single-stranded RNA efficiently competed for RDE-4 binding. Similarly, it was asked whether the labeled dsRNA could bind to the recombinant RDE-4 immobilized on a membrane. This North-Western blot analysis also showed that the dsRNA binds efficiently to the GST-fused RDE-4 but not to the GST protein alone (Tabara, 2002).

It was then asked if RDE-4 interacts with dsRNA during RNAi in vivo. To address this question, polyclonal antibodies were raised against a recombinant RDE-4 protein. Animals were then exposed to dsRNA targeting the gene pos-1. Extracts from these animals and from control animals not exposed to dsRNA were prepared, and RDE-4-specific antibodies were used to precipitate the RDE-4 protein complex. The precipitates were extracted to purify any associated RNA and analyzed by agarose and acrylamide gel electrophoresis followed by Northern blotting using pos-1 sense and antisense radiolabeled RNA probes. Both the sense and antisense probes detected approximately equal amounts of pos-1 RNA that migrated below 350 bases on an agarose gel and migrated in a size range of 50 to 200 bases on an acrylamide gel. The coprecipitated pos-1 RNA was resistant to treatment with a mixture of the single-strand-specific RNaseA and RNaseT1 enzymes, suggesting that the pos-1 RNA is double stranded. Little or no pos-1 RNA coimmunoprecipitated with RDE-4, which was purified from populations either not exposed to dsRNA or exposed for only 15 min. As expected, rde-4(ne299) mutants failed to exhibit pos-1 dsRNA in the precipitate. The RDE-4 immune complex recovered from the rde-1(ne300) and rde-3(ne298) mutant strains exhibited greatly reduced quantities of pos-1 dsRNA (Tabara, 2002).

dsRNA is thought to exist and function at multiple steps in RNAi: (1) it functions in the initiation step when long dsRNA is recognized as foreign; (2) it functions in the execution step when target mRNA is bound by siRNA and cleaved, and (3) it functions in a potential amplification step in which target mRNA sequences are copied by an RNA-dependent RNA polymerase activity. In the above experiments, the probes and techniques used were designed to detect long dsRNA sequences contained within the trigger dsRNA, and thus could not address the question of whether siRNAs or target mRNA sequences might also coimmunoprecipitate with RDE-4. To address these questions, sequences in the 3′ UTR of pos-1 that were not included in the dsRNA trigger were screened for. These experiments failed to detect significant coprecipitating RNA, indicating that sequences within the target mRNA are not stable components of the RDE-4 immune complex (Tabara, 2002).

Several studies have suggested that RNAi may involve a mechanism for the amplification of the dsRNA signal and provide evidence that sequences in the target mRNA that lie 5′ of the trigger sequence can be amplified. In order to ask if the RDE-4 immune complex contains antisense RNA sequences derived from regions of the mRNA located 5′ of the trigger dsRNA, transgenic animals were utilized that expressed dsRNA corresponding to a portion of unc-22 gene. RNA probes were then used to assay for sequences in the RDE-4 immune complex that were derived either from the trigger region or from the region located just 5′ of the trigger. As expected, the RDE-4 immune complex contained unc-22 antisense RNA corresponding to the targeted region of unc-22. In contrast, no significant hybridization was observed when a sequence derived from the region located just 5′ of the trigger dsRNA was used as a probe. As expected, precipitates prepared from nontransgenic wild-type animals did not exhibit significant hybridization to the probes designed to detect unc-22 antisense RNA (Tabara, 2002).

Finally, in order to ask if siRNA sequences precipitate with RDE-4, the RNA species in the RDE-4 immune complex and in the RDE-4-depleted supernatant were fractionated by acrylamide gel electrophoresis. RNA species smaller than 150 bases were analyzed by Northern blotting using a pos-1 sense probe. It was found that the RNA species in the supernatant included abundant molecules of less than 150 bases, including a prominent signal corresponding in size to siRNAs, that migrated at approximately 24 to 25 bases. RNA species of 24 to 25 bases were absent in the RDE-4 immune complex, suggesting that RDE-4 preferentially interacts with longer dsRNAs. Taken together, these studies indicate that RDE-4 binds to long dsRNA sequences that are restricted to the region present within the trigger dsRNA. These findings support a model in which RDE-4 interacts with the trigger dsRNA and functions in the initiation step of RNAi (Tabara, 2002).

In order to determine what fraction of the total dsRNA was bound to RDE-4 and to examine the requirements for the stability of the dsRNA bound to RDE-4, RNA extracts from wild-type and rde mutant strains were analyzed. Wild-type animals showed an accumulation of antisense and sense RNA corresponding to the pos-1 trigger dsRNA sequence that appeared to be very similar to the dsRNA sequences found in the RDE-4 immune complex. This RNA species was similar in size but slightly smaller, on average, than the major pos-1 RNA species detected in the bacterial expression strain used to induce RNAi, suggesting that some digestion of the bacterially expressed dsRNA may have occurred during transit through the intestine of the animals. Single-stranded RNA-specific nucleases digested the endogenous pos-1 mRNA but failed to digest the accumulated RNA species, suggesting that the accumulated RNA species is indeed double-stranded (Tabara, 2002).

Interestingly, the analysis of total RNA extracts showed that animals lacking rde-1, rde-3, and rde-4 activities exhibit markedly reduced levels of dsRNA. In contrast, animals lacking rde-2 and mut-7 accumulate dsRNA to levels that are intermediate (rde-2) or similar to wild-type (mut-7). The finding that the accumulation of this dsRNA species requires some but not all rde(+) activities supports the idea that this RNA species is an intermediate in the RNAi process (Tabara, 2002).

To ask what fraction of the accumulating dsRNA in the total RNA extract from wild-type animals is associated with RDE-4, the immunoprecipitations were repeated with either RDE-4-specific antibodies or with control nonspecific IgG obtained from preimmune serum and the amount of the dsRNA present in the immune complex and supernatant of each lysate was compared. Approximately 60% of the pos-1 dsRNA species present in the total RNA coprecipitates with the RDE-4 immune complex, indicating that a majority of the accumulating dsRNA is associated with RDE-4 (Tabara, 2002).

Previous studies have suggested that RDE-1 and RDE-4 function at an upstream step in the initiation of RNAi. This observation, and the finding that RDE-1 and RDE-4 are both required for the accumulation of dsRNA, prompted a test to see if RDE-4 forms a complex in vivo with RDE-1. For these studies a transgenic strain was generated expressing RDE-1 tagged with the HA epitope and RDE-4 tagged with the FLAG epitope. RDE-4 was immunoprecipitated with RDE-4-specific polyclonal antibodies, and RDE-1 via the epitope tag, and the precipitates were analyzed by immunoblotting. In reciprocal assays, RDE-1 and RDE-4 were found to coprecipitate. Based on a comparison of the RDE-1 present in total extracts and in the RDE-4 immune complex, it is estimated that approximately 5% of the RDE-1 molecules stably associate with the RDE-4 complex (Tabara, 2002).

The interaction between RDE-1 and RDE-4 occurred in animals that were not exposed to exogenous dsRNA, and the interaction was not abolished by treatment with either double-stranded RNA-directed nuclease, RNaseV1, or a mixture of RNaseA and T1 that are single-stranded RNA-specific nucleases. These findings suggest that RDE-1 and RDE-4 form a complex in the absence of dsRNA, but the possibility cannot be ruled out that dsRNA promotes the interaction or is required for the interaction but is protected from digestion in the immune complex (Tabara, 2002).

In order to identify additional proteins that interact with RDE-4 in vivo, large quantities of the RDE-4 immune complex were isolated and then its components were resolved using SDS polyacrylamide gel electrophoresis (SDS-PAGE). rde-4(ne299) mutant animals were used that were rescued via an RDE-4::GFP fusion protein. Protein extracts prepared from these transgenic animals and from control wild-type animals were then passed over a column conjugated with anti-GFP monoclonal antibody. After electrophoresis, the gel was silver stained to detect components of the RDE-4::GFP immune complex. These experiments identified prominent bands migrating at 210 kilodaltons (kDa) and 110 kDa as well as several additional polypeptides that were present in the RDE-4::GFP immune complex but absent in wild-type control animals. A minor band at 125 kDa was identified, corresponding in size to RDE-1, but it contained insufficient protein for further analysis. The 210 kDa and 110 kDa bands were subjected to tryptic digestion, and the resulting fragments were then analyzed by mass spectrometry. The peptide mass fingerprints of p210 and p110 were compared with in silico-digested sequences from protein databases. A total of 21 peptides from the tryptic digestion of p210 matched DCR-1, while 11 peptides from p110 matched a DExH-box RNA helicase-related protein F15B10.2 (Tabara, 2002).

The finding that RDE-4 forms a complex in vivo with DCR-1 suggests that RDE-4 may function to present the foreign trigger dsRNA to DCR-1 for processing. DCR-1 also acts to process the stem-loop precursors of the developmental regulators lin-4 and let-7. However, rde-4 mutants do not exhibit developmental defects, and thus are not required for the activities of lin-4 or let-7. Furthermore, no lin-4 or let-7 precursor RNAs associated with the RDE-4 immune complex were detected. Thus, although RDE-4 interacts strongly with DCR-1 in vivo, RDE-4 is not essential for the developmental functions of DCR-1 and may rather serve as an adaptor protein that recruits DCR-1 to the RNAi pathway (Tabara, 2002).

The RDE-4 protein interacts with a 110 kDa protein, which as described in the previous section corresponds to the predicted gene F15B10.2, one of two closely related C. elegans DExH-box RNA helicase genes. F15B10.2, along with its homolog C01B10.1, map directly adjacent to one another on chromosome IV and appear likely to be expressed together from a single promoter. Consistent with this idea, it was found that some F15B10.2 cDNAs were spliced to the trans-spliced leader sequence (sl2), which is often spliced onto the 5′ end of transcripts encoded by downstream genes in an operon. The C01B10.1 gene and a cDNA clone (yk226c6) both appear to encode a frame-shifted open reading frame predicted to terminate the protein prior to the helicase domain, raising the question of whether or not this represents a functional gene. An alternative conceptual splicing of C01B10.1, or utilization of an alternative initial methionine, could encode a protein with overall 74% identity to F15B10.2 (Tabara, 2002).

These C. elegans genes have close homologs in mammals, including a gene named RHIV-1 induced in response to viral infection in pigs. Notably, these helicase proteins are more similar to the DCR-1 helicase domain than to any other helicase-related proteins in C. elegans or other organisms. Consequently, these genes have been named drh-1 and drh-2 (for F15B10.2 and C01B10.1, respectively) which stands for dicer-related helicase (Tabara, 2002).


REFERENCES

Search PubMed for articles about Drosophila r2d2

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

Fagegaltier, D., et al. (2009). The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila. Proc. Natl. Acad. Sci. 106(50): 21258-63. PubMed Citation: 19948966

Kalidas, S., et al. (2008). Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1. Mech. Dev. 125(5-6): 475-85. PubMed Citation: 18299191

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


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date revised: 28 December 2011

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