Drosophila Polycomb group (PcG) proteins silence homeotic genes through binding to Polycomb group response elements (PREs). Fab-7 is a PRE-containing regulatory element from the homeotic gene Abdominal-B. When present in multiple copies in the genome, Fab-7 can induce long-distance gene contacts that enhance PcG-dependent silencing. Components of the RNA interference (RNAi) machinery are involved in PcG-mediated silencing at Fab-7 and in the production of small RNAs at transgenic Fab-7 copies. In general, these mutations do not affect the recruitment of PcG components, but they are specifically required for the maintenance of long-range contacts between Fab-7 copies. Dicer-2, PIWI, and Argonaute1, three RNAi components, frequently colocalize with PcG bodies, and their mutation significantly reduces the frequency of PcG-dependent chromosomal associations of endogenous homeotic genes. This suggests a novel role for the RNAi machinery in regulating the nuclear organization of PcG chromatin targets (Grimaud, 2006).
The RNAi machinery has been implicated in a wide variety of biological processes. One of these processes is the formation of heterochromatin. In S. pombe, this involves bidirectional transcription of RNA molecules from repetitive sequences and their cleavage into short interfering RNAs (siRNAs) of 21–23 nt by an RNase III enzyme called Dicer-1. siRNAs guide the RNA-induced initiation of transcriptional gene silencing (RITS) complex to homologous sequences in the nucleus (Noma, 2004; Verdel, 2004). Clr4, the homolog of the histone methyltransferase Su(Var) 3-9, is recruited along with the RITS complex to chromatin, where it methylates lysine 9 of histone H3 (H3K9). This epigenetic mark promotes the formation of heterochromatin by recruiting the heterochromatin protein Swi6, the homolog of HP1, via its chromodomain (Grewal, 2004). Consistent with these data, a redistribution of H3K9 methylation has been observed in Drosophila chromosomes in flies mutant for components of the RNAi machinery (Pal-Bhadra, 2004; Grimaud, 2006).
The RNAi machinery is also required for cosuppression, a phenomenon whereby the introduction of multiple transgenic copies of a gene phenocopies its loss of function instead of increasing its expression. In Drosophila, cosuppression can act at either the transcriptional or posttranscriptional level and involves PcG proteins as well as the RNAi machinery (Pal-Bhadra, 1997, Pal-Bhadra, 1999 and Pal-Bhadra, 2002; Grimaud, 2006 and references therein).
The Drosophila RNAi machinery includes two Dicer proteins encoded by the dicer-1 (dcr-1) and dicer-2 (dcr-2) genes. Dcr-2 is specifically required to process double-stranded RNAs into siRNAs and mediates the assembly of siRNAs into the RNA-induced silencing complex (RISC). Dcr-1 is involved in the metabolism of siRNAs as well as the processing of pre-microRNAs into microRNAs. RNA silencing also involves several highly conserved genes coding for PAZ-domain proteins. Argonaute1 (AGO1) and Argonaute2 (AGO2) are involved in microRNA biogenesis and RNA interference (RNAi). piwi is involved in cosuppression, silencing of retrotransposons, and heterochromatin formation. aubergine (aub) was first isolated based on its role in germline development but is also responsible for maintaining the silenced state of an X-linked male fertility gene locus (Stellate) via RNAi. The Aub protein is required for RNAi and RISC assembly in ovaries. In addition, homeless/spindle-E (hls) is involved in silencing of Stellate and in heterochromatin formation. This study tested whether RNAi components are involved in the PcG pathway. The results show that the RNAi machinery affects the PcG response via a novel regulatory function in nuclear organization (Grimaud, 2006).
The role of a variety of RNAi components in a specific transgenic line called Fab-X was tested. This line contains a construct carrying a 3.6 kb fragment from the Fab-7 region, cloned upstream of a mini-white reporter and inserted into the X chromosome. In the Fab-X line, the presence of the Fab-7 sequence is sufficient to induce PcG-dependent silencing, both of the mini-white eye-color reporter gene and of the endogenous scalloped (sd) gene, which is required for wing-blade morphogenesis and is located 18.4 kb downstream of Fab-7. These two repressed phenotypes are abolished in the presence of mutations in PcG genes and are not present in heterozygous females and hemizygous males, indicating that both mini-white and sd expression are subject to PSS (Grimaud, 2006).
The eye-color and wing phenotypes were used as a basis to analyze the effect of the RNAi machinery on PcG-dependent repression. Mutations in RNAi components were introduced into the Fab-X line and placed over a balancer chromosome containing a GFP marker. As the AGO1 mutant alleles involve P element insertions containing the mini-white reporter gene, they could not be tested using the eye phenotype. A null mutation in dcr-2 (dcr-2L811fsX) decreased silencing of the mini-white reporter gene relative to the Fab-X line when in the homozygous state. Likewise, two different mutant alleles of piwi (piwi1 and piwi2) decreased mini-white silencing, with the effect being more pronounced in piwi2 mutant flies. This effect was not restricted to the Fab-X line since it was also observed when piwi2 was recombined into another Fab-7-containing line. In contrast to the effects seen for dcr-2 and piwi alleles, Fab-X females homozygous mutant for hlsE1 or that carried the heteroallelic hlsE1/hlsE616 combination silenced mini-white like wt Fab-X females (Grimaud, 2006).
The sd phenotype was then analyzed in all mutant backgrounds at 28.5°C, a temperature inducing a strong wing phenotype in Fab-X. A preselection of non-GFP female larvae was carried out in order to selectively analyze homozygous or trans-heterozygous mutant adults. This analysis revealed that mutating any of the components of the RNAi machinery, except for hls and the heterozygous dcr-1 mutation, leads to a strong decrease in the sd phenotype. These data show that the RNAi machinery can affect PcG-mediated silencing. The fact that hls mutants had no effect suggests that this process might be mechanistically distinct from the role of RNAi components in heterochromatin formation (Grimaud, 2006).
While most RNAi components are not required for binding of PcG proteins to PREs, they are required to mediate long-range contacts between multiple copies of the Fab-7 element. Moreover, Dcr-2, PIWI, and AGO1 colocalize with PH in the cell nucleus, and their effect correlates with the presence of small RNAs homologous to Fab-7 sequences. Finally, in addition to their effects on transgenic Fab-7 copies, mutations in these genes also reduce the frequency of long-distance contacts between endogenous PcG target genes. Taken together, these results reveal a novel and unexpected role for the RNAi machinery in the regulation of euchromatic genes in the nuclear space (Grimaud, 2006).
The effects caused by mutations in different RNAi components suggest the existence of distinct molecular roles for these proteins in the regulation of PcG function. First, the hls gene does not seem to play a major role in silencing at the Fab-7 PRE or maintaining long-distance Fab-7 contacts. Since Hls has been shown to play a central role in heterochromatin formation (Pal-Bhadra, 2004), there may be different subtypes of nuclear RNAi machineries for heterochromatin formation and for regulating PcG function. No effect was found when a mutation in the dcr-1 gene was analyzed at the heterozygous state, but the elucidation of the function of dcr-1 in PcG-mediated silencing awaits further analysis in a homozygous mutant background. A second class of RNAi components that participate in PcG-mediated repression contains dcr-2, AGO1, and the aub gene. Loss of any of these RNAi gene products affects PcG-dependent silencing at Fab-7, although it does not impact the binding of PcG proteins to Fab-7. Only mutations in piwi affected the binding of PcG proteins to Fab-7, at least in polytene chromosomes, but even PIWI did not affect recruitment of PcG factors at endogenous genes. In S. pombe, both RNAi components as well as DNA binding proteins are involved in recruiting heterochromatin proteins to the mating-type region (Jia, 2004). At PREs, multiple DNA binding factors and chromatin-associated proteins are known to contribute to PcG protein recruitment, such as PHO, the GAGA factor, DSP1, and the CtBP proteins. Their combinatorial action might play a key role in the robust and specific chromatin tethering of PcG proteins, while, in contrast to the situation in S. pombe, the RNAi machinery might play a relatively minor role (Grimaud, 2006).
It is interesting to note that piwi mutations affected recruitment of E(Z) and PC to Fab-7 in polytene chromosomes but had no effects on PH, another PRC1 component. In the current model for recruitment of PcG proteins to PREs, histone H3 methylation by the E(Z) protein recruits PRC1 via the chromodomain of PC. The current results indicate that multiple mechanisms might be used to anchor different PRC1 components to PREs and that the loss of PC does not necessarily lead to the disintegration of the entire PRC1 complex at PREs (Grimaud, 2006).
To date, all transcriptional gene-silencing phenomena that depend on the RNAi machinery involve the production of small-RNA molecules. RNAi components were also shown to affect telomere clustering in S. pombe, although binding of Swi6 and H3K9me to individual telomeres is not affected. The production of siRNAs is believed to be essential for the nuclear clustering of telomeres since cells carrying a catalytically dead RNA-dependent RNA polymerase (which abolishes siRNA production) are defective in telomere clustering. Consistent with a role for small RNAs in mediating gene contacts, sense and antisense transcription of Fab-7 as well as small-RNA species were found in Fab-7 transgenic lines. Moreover, a mutant allele of dcr-2 producing a truncated polypeptide lacking the RNase III domain, which is required for dsRNA processing, is defective in long-range interactions of PcG target sequences as well as in accumulation of Fab-7 small RNAs. These data suggest that small-RNA species could be involved in these gene contacts. However, no small Fab-7 RNA species was detected in the wt situation, although RNAi mutants affect the contact of the endogenous Fab-7 locus with the Antp gene. This might indicate that other RNA species produced in the endogenous Hox genes could contribute to gene clustering. However, the possibility remains that RNA-independent functions of RNAi proteins contribute to the maintenance of gene contacts, in particular in the case of endogenous PcG target genes (Grimaud, 2006).
Interestingly, none of the RNAi mutants tested are defective in the establishment of long-distance chromosomal interactions. Fab-7 contacts are correctly established during embryogenesis but decay during later stages of development. This suggests that the RNAi machinery is not required to initiate contacts but rather to maintain them via the stabilization of gene clustering at specific nuclear bodies. This clustering could be important in cosuppression, where transgene silencing can occur at the transcriptional and posttranscriptional levels, both requiring the RNAi machinery (Pal-Bhadra, 2002). It is difficult, however, to understand how a relatively modest increase in transcript levels caused by an increase in the copy number of a gene could trigger a robust silencing of all copies. This is particularly puzzling considering that the transcript levels of endogenous single-copy genes can vary, e.g., during normal physiological gene regulatory processes, without triggering gene silencing. One explanation might be that cosuppressed genes are clustered in the cell nucleus. Indeed, clustering of multiple gene copies has been reported in plant cells (Grimaud, 2006).
It is proposed that the RNAi machinery, perhaps in conjunction with PcG proteins, might stabilize this gene-clustering phenomenon. Specifically, the colocalization of multiple gene copies with components of the RNAi machinery might increase the local concentration of RNA species. Once this concentration overcomes a critical threshold, double-stranded RNAs might assemble and be cleaved in situ by the enzymatic activity of the RNAi machinery. RNA molecules might contribute to hold together loci containing PcG proteins that produce noncoding transcripts encompassing PREs. This gene clustering might involve contacts with components of the RNAi machinery as well as PcG proteins assembled in the same nuclear compartments (Grimaud, 2006).
One important question is, what is the role of the RNAi machinery in the regulation of endogenous PcG target genes? The data indicate that RNAi components affect only a subset of these genes since the colocalization of PcG bodies with RNAi bodies is limited. Hox loci are characterized by extensive noncoding RNA transcription, and, recently, other PcG target genes have been shown to be associated to intergenic transcription. RNAi components might be targeted to this subset of PcG target genes, while other PcG target genes that are characterized by the absence of noncoding transcripts might be independent on RNAi factors (Grimaud, 2006).
The fact that no homeotic phenotypes are visible in RNAi mutant backgrounds suggests that the function of RNAi components can be rescued by other chromatin factors. Indeed, the decrease in the level of nuclear interaction between the homeotic complexes was incomplete in RNAi mutant backgrounds. The data suggest that, while the RNAi machinery does not act in the establishment of PcG-dependent gene silencing, RNAi factors might help stabilize silencing during development by clustering PcG target genes at RNAi nuclear bodies. Thus, in addition to its role in defending the genome against viruses, transposons, and gene duplications, the RNAi machinery might participate in fine tuning the expression of PcG target genes through the regulation of nuclear organization. Finally, it must be noted that the developmental expression profile of the components of the RNAi machinery is highly specific. The function of specific RNAi components is therefore likely to be highly variable in different cell types and as a function of time. It will be of great interest to explore this issue in the developmental context of the whole organism, in Drosophila as well as in other species (Grimaud, 2006).
The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2 cells and consists of two stoichiometric subunits: Dicer-2 (Dcr-2) and a previously unknown protein that has been termed 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 to RISC (Liu, 2003).
RNA interference (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. (1) Long dsRNA molecules are cleaved into 21- to 23-nucleotide (nt) small interfering RNA (siRNA) duplexes. (2) 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 and references therein).
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 as Dcr-2 and a previously unknown protein (Flybase CG7138), respectively. This protein was named R2D2 because it contains two dsRNA-binding domains (R2) and is associated with Dcr-2. R2D2 bears 20.9% identity and 33.4% similarity to the C. elegans RNAi protein RDE-4 (Tabara, 2002; Grishok 2000), which also contains tandem dsRNA-binding domains and interacts with Dicer (Liu, 2003).
To confirm the 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 from S2 cells was depleted 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 of RNAi 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 they were 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 silences 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 cleaves 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 (a measure of substrate affinity) 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. 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 are 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, binds to siRNA and produces a mobility shift indistinguishable from that of S100. The ability of the Dcr-2/R2D2 complex to bind siRNA is 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: this probably represents 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; this binding is dependent upon the dsRNA-binding domains of R2D2. On the basis of this finding, it was 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 the partially purified RISC was combined 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; this remaining material could be activated by addition of siRNA for sequence-specific mRNA degradation. The wild-type Dcr-2/R2D2 complex was much more effective than 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 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 can enhance the siRNA- as well as the dsRNA-initiated RISC activities. This enhancement is not simply because of siRNA stabilization by Dcr-2/R2D2, as shown by comparing the stability of radiolabeled siRNA in RISC reactions (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, AGO2 protein was precipitated by streptavidin beads only when biotinylated siRNA was used, suggesting that interaction between AGO2 and siRNA was a specific interaction. 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. In these experiments, if dsRNA was processed from the 5' to 3' direction of the sense strand, it would generate RISC that can 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. 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).
As a RNA helicase involved in posttranscriptional gene silencing, armitage mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. These results suggest that armi is required for RISC maturation (Tomari, 2004).
Drosophila syncitial blastoderm embryo lysate has been used widely to study the RNAi pathway. However, armi flies lay few eggs, making it difficult to collect enough embryos to make lysate. To surmount this problem, lysates were prepared from ovaries manually dissected from wild-type or mutant females. Approximately 10 μl of lysate can be prepared from ∼50 ovaries (Tomari, 2004).
The well-characterized siRNA-directed mRNA cleavage assay was used to evaluate the capacity of ovary lysate to support RNAi in vitro. Incubation in ovary lysate of a 5' 32P-cap-radiolabeled firefly luciferase mRNA target with a complementary siRNA duplex yielded the 5' cleavage product diagnostic of RNAi. siRNAs containing 5' hydroxyl groups are rapidly phosphorylated in vitro and in vivo, but modifications that block phosphorylation eliminate siRNA activity. Replacing the 5' hydroxyl of the antisense siRNA strand with a 5' methoxy group completely blocks RNAi in the ovary lysate. In Drosophila, siRNAs bearing a single 2'-deoxy nucleotide at the 5' end are poor substrates for the kinase that phosphorylates 5' hydroxy siRNAs (Nykanen, 2001). A comparison of initial cleavage rates shows that in ovary lysate, target cleavage was slower for siRNAs with a 2'-deoxy nucleotide at the 5' end of the antisense strand than for standard siRNAs. Furthermore, the rate of target cleavage was fastest when the siRNA was phosphorylated before its addition to the reaction. A similar enhancement from pre-phosphorylation was reported for siRNA injected into Drosophila embryos. It is concluded that lysates from Drosophila ovaries faithfully recapitulate RNAi directed by siRNA duplexes (Tomari, 2004).
Liu and colleagues have proposed that a heterodimeric complex, comprising Dicer-2 and the dsRNA binding protein R2D2, loads siRNA into RISC (Liu, 2003). Complex A contains the Dcr-2/R2D2 heterodimer. R2D2 and Dcr-2 are readily crosslinked to 32P-radiolabeled siRNA with UV light (Liu, 2003). An siRNA was synthesized containing a single photocrosslinkable nucleoside base (5-iodouracil) at position 20. The 32P-5-iodouracil siRNA was incubated with embryo lysate to assemble complexes, then irradiated with 302 nm light, which initiates protein-RNA crosslinking only at the 5-iodo-substituted nucleoside. Proteins covalently linked to the 32P-radiolabeled siRNA were resolved by SDS-PAGE. Two proteins -- ∼200 kDa and ∼40 kDa -- efficiently crosslinked to the siRNA. Both crosslinked proteins were coimmunoprecipitated with either α-Dcr-2 or α-R2D2 serum, but not with normal rabbit serum. Neither crosslink was observed in ovary lysates prepared from r2d2 homozygous mutant females, a result expected because Dcr-2 is unstable in the absence of R2D2 (Liu, 2003; Tomari, 2004).
The crosslinking was repeated, and the reaction analyzed by native gel electrophoresis to resolve complexes B, A, and RISC. Each complex was eluted from the gel and analyzed by SDS-PAGE. The R2D2 and Dcr-2 crosslinks were present in complexes A and RISC, but not B. In a parallel experiment, complexes B, A, and RISC were isolated (without crosslinking) and analyzed by Western blotting with either α-Dcr-2 or α-R2D2 antibodies. Again, complexes A and RISC, but not B, contained both Dcr-2 and R2D2. Finally, complex assembly was tested in ovary lysates prepared from r2d2 homozygous mutant females. Only complex B formed in these lysates. It is concluded that complex A contains the previously identified Dcr-2/R2D2 heterodimer (Liu, 2003, and that both Dcr-2 and R2D2 remain associated with at least a subpopulation of RISC, consistent with earlier reports that Dcr-2 in flies and both DCR-1 and the nematode homolog of R2D2, RDE-4, coimmunoprecipitate with Argonaute proteins (Tomari, 2004).
Native gel electrophoresis has been used to characterize complexes that mediate RNA interference (RNAi) in Drosophila. The data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. These results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 'holo-RISC' complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi (Pham, 2004).
Of these complexes, R1 forms the earliest. It does not require ATP to form and contains Dcr-2, which directly binds siRNAs. R1 corresponds to a previously identified 360 kDa siRNP complex that contains duplex siRNAs and is inactive for targeted mRNA cleavage (Nykanen, 2001). It is also probably equivalent to a dsRNA-processing complex isolated from Drosophila S2 cells that contains Dcr-2 and R2D2 (Liu, 2003). R1 shares many features with this complex and can also form in extracts prepared from S2 cells). Based on these results, as well as those of Liu (2003), it is concluded that the factors that form R1 process dsRNA and initiate RISC assembly. Furthermore, these results indicate that siRNAs must interact with Dcr-2 in order to reach RNAi effector complexes and that Dcr-2/siRNA association within the R1 complex is the first detectable step in the RISC-assembly pathway. Intriguingly, UV crosslinking results indicate that Dcr-2 can weakly or transiently bind 5'-hydroxyl-bearing siRNAs in the R1 complex. Nykanen (2001) proposed that the siRNA 5'-phosphate serves a 'licensing' function in the RNAi pathway, allowing the silencing machinery to recognize bona fide siRNAs; if this is true, these results indicate that siRNA 5'-phosphorylation status is monitored within the R1 complex after the initial Dcr-2/siRNA interaction has occurred. This could be achieved by fast dissociation of 5'-hydroxyl siRNAs from the Dcr-2 complex (Pham, 2004).
The R2 complex has not yet been characterized in detail because it neither survives gel filtration chromatography nor sediments cleanly in a sucrose gradient. For now, it is only possible to speculate about its composition and role in RNAi. Based on its kinetics and behavior in a chase experiment, R2 may be an intermediate that links R1 and R3, although it is not certain whether it is 'on pathway.' Since R2 forms so rapidly in this experiment, it might arise from R1 binding to a set of pre-associated factors (or to a single factor). This binding may then trigger the recruitment of still more factors, possibly accompanied by conformational rearrangements, because relative to the other complexes, R2's mobility in a native gel decreases slightly with time. siRNA unwinding may occur during this process, leading to formation of R3 (Pham, 2004).
The R3 complex is significantly enhanced by incubation with ATP, a known requirement for activated RISC formation. R3 is very large (80S) and cofractionates with the known RISC-associated components VIG, TSN, Ago2, and dFXR. The siRNAs associated with R3 are single stranded, and the complex co-sediments precisely with mRNA target cleavage activity. Finally, R3 can bind a cognate mRNA target. Based on these results, it is concluded that R3 is an RNAi effector complex that is significantly larger than those previously described (Pham, 2004).
The experiments suggest that R3 is a ribosome bound silencing complex. Indeed, several lines of evidence link RNAi to translation and therefore support this possibility. In Drosophila oocytes, untranslated mRNAs are refractory to RNAi, whereas those actively undergoing translation are not (Kennerdell, 2002), indicating that mRNAs most susceptible to RNAi are those that can also interact with the translational machinery. MicroRNAs have been identified within RISC (Hutvagner, 2002) and can also affect protein synthesis from target messages (Olsen, 1999), suggesting further functional links between RISC and ribosomes. In S2 cells, Drosophila RISC components pellet with ribosomes and other large complexes after high-speed centrifugation (Hammond, 2001; Caudy, 2002, 2003). Two of these components, Ago2 and dFXR, are found in complexes that include 5S rRNA as well as two ribosomal proteins, L5 and L11 (Ishizuka, 2002). siRNAs have also been found in association with the translational machinery (Djikeng, 2003), interacting with polyribosomes in the protozoan Trypanosoma brucei (Pham, 2004 and references therein).
If R3 is a ribosome bound complex, then it is likely that RISC binds during or after siRNA unwinding and activation. This assertion is based both on the apparent ATP dependence of R3 formation and on the preponderance of single-stranded siRNAs within the complex. It has been shown that siRNA unwinding requires ATP (Nykanen, 2001). Although the possibility cannot be rule out that a potential RISC/ribosome interaction requires ATP for unrelated reasons, the data indicate that siRNA unwinding is one source of that requirement. Once formed, the R3 complex may be biochemically separable since smaller, active RISCs have been identified (Hammond, 2001; Nykanen, 2001; Martinez, 2002). If so, then R3 may represent a holo-silencing complex that includes factors not absolutely required for mRNA cleavage activity in vitro. These factors may include regulatory elements or those involved in the miRNA silencing pathway (Pham, 2004).
Because dsRNA processing and mRNA target cleavage activities are biochemically separable, Dicer is generally thought to release siRNAs to RISC, playing no apparent role in the effector phase of RNAi. However, the presence of Dcr-2 and R2D2 in R3-containing sucrose gradient fractions raises interesting questions about their role in RNA silencing, especially since they are not detectable in the corresponding fractions collected from naive extracts. Liu (2003) has argued that the Dcr-2-associated protein R2D2 conveys siRNAs from the RNAi initiator complex to the effector complex. The current data instead indicate that the Dcr-2/R2D2 initiator complex, itself, is incorporated into the effector in a RISC assembly pathway (Pham, 2004).
Sucrose gradient experiments indicate that RISC components may associate with each other even in the absence of exogenous siRNAs. This association is not likely to be an apo-complex because fractionated naive extracts cannot form R3 when they are incubated with siRNA duplexes after fractionation. Rather, it may represent endogenous microRNA-programmed RISC complexes or perhaps a partially preformed 'holo-RISC' that lacks essential assembly factors (such as Dcr-2 and R2D2). Since the silencing activities of RISC and the miRNP are apparently governed by the degree of complementarity between trigger and target, and not by the identity of the silencing complex (Hutvagner, 2002; Doench, 2003; Zeng, 2003), R3 may be able to form on both siRNAs and miRNAs. If so, then there may be distinct mechanisms for incorporating siRNA and miRNA triggers into the silencing complex. siRNA incorporation into RISC is likely to proceed on a Dcr-2/R2D2-dependent pathway since Dcr-2 and R2D2 can process dsRNAs (Liu, 2003) but are not detectable in sucrose gradient fractions collected from naive extracts. However, miRNA incorporation may proceed on a different pathway to the silencing complex, possibly relying on Dcr-1. There is support for this model in the apparent division of labor between Dcr-1 and Dcr-2 in processing miRNA and dsRNA triggers (Lee, 2004). If this model is correct, then the observed size of R3 may reflect the diverse roles it may play, from mRNA cleavage to translational control (Pham, 2004).
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. This study used a Drosophila in vitro system 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, Dcr enzymes are required for RISC assembly as well as dsRNA processing, suggesting that the two phases of RNAi might be functionally coupled in a manner that affects siRNA strand selection. However, these experiments indicate that Drosophila RISC assembly and siRNA strand selection are not significantly influenced by the dsRNA processing step and that the thermodynamic asymmetry rules apply equally well with processed and unprocessed siRNAs in this system. This suggests that Drosophila Dcr enzymes do not channel newly generated siRNAs directly into RISC, but rather release the siRNAs into solution (or onto another factor) before they enter the RISC assembly pathway (Preall, 2006).
Several observations have suggested that thermodynamic asymmetry governs strand selection for processed RNAi triggers. MicroRNAs (miRNAs) are diced from stem-loop precursors, and in most instances only one strand of the processed miRNA duplex is stably incorporated into RISC. The mature strand can be present at either the 5' or the 3' end of the stem-loop, but either way, the selected strand is generally compatible with the thermodynamic asymmetry guidelines. In addition, artificial dsRNAs introduced into plant cells give rise to a stable set of siRNAs that adhere to the asymmetry rules. Similar results have been reported with natural dsRNAs in plants. However, interpretation of these results is difficult because the Dcr processing polarities were not defined, and it is also not clear whether small RNA stability is always a suitable surrogate measure of RISC assembly. Furthermore, plant cells (unlike insect and mammalian cells) export siRNAs into the vasculature to enable systemic RNAi, and therefore the plant dsRNA processing machinery may have specifically evolved the propensity to release newly processed siRNAs. Thus the applicability of the plant analyses to insects and mammals has not been clear (Preall, 2006).
While this work was in progress, Rose (2005) characterized modified ~27 nt duplexes that force a defined Dcr processing polarity and give rise to specific, predictable 21 nt siRNAs. Experiments with these Dcr substrate RNAs revealed that hDcr processing polarity can in fact influence siRNA strand selection in transfected human cells, although it does not completely supercede thermodynamic asymmetry. The reasons for the discrepancy between the results and those of Rose are not clear, although one possibility is that different Dcr enzymes may vary in their tendencies to remain associated with newly generated siRNAs. It is noteworthy that Drosophila Dcr-2 (which is primarily devoted to the siRNA pathway) appears to lack the canonical PAZ domain that normally provides Dcr with a binding pocket for 2 nt 3' overhangs. A PAZ domain is present in hDcr, and mutational analyses indicate that the hDcr PAZ domain assists with dsRNA binding and processing when a 2 nt 3' overhang is present. The apparent lack of a PAZ domain in Dcr-2 may compromise its ability to remain bound to newly cleaved siRNA. It is curious that Drosophila Dcrs are required for RISC assembly but do not appear to couple dsRNA processing to siRNA strand selection, whereas mammalian Dcrs are not required for RISC assembly but apparently do couple dsRNA processing to siRNA strand selection (Preall, 2006).
Finally, it remains to be determined whether Dcr enzymes associate with long dsRNA processing substrates and siRNA RISC-assembly substrates in the same way. This issue is undoubtedly important for understanding the functional relationship between Dcr's roles in the initiator and effector phases of RNAi. Crystal structures of E. coli RNase III, an ancestor of eukaryotic Dcrs, are likely to be informative. The structural data, and models derived from them, depict a protein that can engage dsRNAs in a dynamic fashion. A single dsRBD on each subunit of the RNase III homodimer is tethered to the endonuclease domain by a flexible linker that can rotate roughly 90° around the catalytic core. Thus, there are likely to be at least two binding modes for dsRNA in complex with an RNase III enzyme: one in which the dsRBD braces the RNA helix from either side as it is channeled into the catalytic cleft, and another where the dsRBD holds the dsRNA above and orthogonal to the active site. It is possible that Dcr enzymes also exhibit alternate dsRNA binding modes depending on whether they are actively processing dsRNA or channeling siRNA into RISC. Interconversion between these two conformations may require at least transient release of the siRNA product. Additional dynamic dsRNA/protein interactions during dsRNA processing and RISC assembly presumably involve the dsRNA binding proteins Loquacious/R3D1, R2D2, and TRBP, which associate with Dcr-1, Dcr-2, and hDcr, respectively. Further functional analysis of Dcr's PAZ, RNase III, and dsRNA binding domains, aided by recent advances in the structural biology of Dcr, will be necessary to understand Dcr's roles in the transition between the initiation and effector phases of RNAi (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).
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