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Evolutionarily conserved developmental pathways

RNA interference influences biological processes by silencing expression of genes

Small RNAs influence a wide variety of biological processes by silencing the expression of genes within organisms. These RNAs, with a size of about 22 nucleotides, influence development, genome organization, viral and transposon defense, and disease (Hannon, 2002). There are two classes of small RNAs, and they exert their powers of silencing differently. One class is processed from longer double-stranded (dsRNA) precursor molecules with perfect complementarity. The dsRNAs are cleaved into small interfering RNAs (siRNAs) that are 21-23 nucleotide duplexes. They act as guides for a siRNA-induced silencing complex (siRISC) to target complementary mRNAs. If such an mRNA molecule is found, the base pairing interactions between siRNA and mRNA lead to cleavage of the mRNA molecule and its degradation. A second class of small RNAs, the microRNAs (miRNAs), is processed from stem-loop RNA precursors (pre-miRNAs) that are encoded within plant and animal genomes. The known functions of a few of these miRNAs indicate that they play widespread roles in growth and development (Abrahante, 2003; Brennecke, 2003; Lee, 1993; Lin, 2003; Llave, 2002; Palatnik, 2003; Reinhart, 2000). Animal miRNAs silence gene expression primarily by blocking the translation of mRNA transcripts into protein. They act as guides for a multiprotein complex, miRISC, which identifies mRNAs with imperfect complementarity in the 3' untranslated region of the message (Lee, 2004 and reference therein).

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).

date revised: 5 May 2005

Developmental Pathways conserved in Evolution

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