Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila

Identification of R3D1

Based on our previous studies of Dicer-2/R2D2 in the siRNA pathway (Liu et al. 2003), we hypothesized that Dicer-1 also functions in concert with an unknown dsRNA-binding protein in the miRNA pathway. Through a bioinformatics approach, we identified an open reading frame (ORF, CG6866) in the fly genome that showed considerable homology with R2D2 and RDE-4, an R2D2 homolog in C. elegans (Fig. 1A; Tabara et al. 2002; Liu et al. 2003). Furthermore, PSI-Blast ranked this ORF as the best hit among R2D2-like proteins in FlyBase, and vice versa. We named this protein R3D1 because it contained three putative dsRNA-binding domains (R3) and was later found to associate with Dicer-1 (D1) (Fig. 1; Kharrat et al. 1995). The R3D1 gene encodes two alternatively spliced proteins, R3D1-L (long; 465 amino acids) and R3D1-S (short; 419 amino acids) (Fig. 1A,B).

R3D1-L interacts with Dicer-1 and AGO1

To test physical association of endogenous Dicer-1 and R3D1, we performed coimmunoprecipitation (co-IP) experiments by using anti-Dicer-1 or anti-R3D1 antibodies in the cytosolic (S100) extracts of S2 cells. R3D1-L (∼55 kDa) was present in the IPs of anti-Dicer-1 but not anti-Dicer-2 antibodies (Fig. 1C). We could not detect the presence of R3D1-S (∼50 kDa) probably because it was absent or masked by the Immunoglobin (IgG) heavy chain (Fig. 1C). Reciprocal IP using anti-R3D1 antibody brought down Dicer-1 but not Dicer-2, whereas anti-R2D2 antibody only brought down Dicer-2 (Fig. 1D). In addition, both Dicer-1 and R3D1 interacted with AGO1 (Fig. 1C,D), a critical component of miRISC (Okamura et al. 2004). These studies indicate that endogenous R3D1-L specifically associates with Dicer-1 and AGO1, which are key components of the initiation and effector complexes of the miRNA pathway.

Purification of the miRNA-generating enzyme

To study miRNA biogenesis, we purified the miRNA-generating enzyme from S2 cell extracts by biochemical fractionation. A single peak of activity was observed on all columns tested and was followed throughout the purification. We performed Western blots to detect the presence of Dicer-1, R3D1, Dicer-2, and R2D2 among individual fractions following each step of purification. Both Dicer-1 and R3D1-L showed perfect correlation with the miRNA-generating activity after every chromatography step (Fig. 2; data not shown). This was not the case for R3D1-S, nor for Dicer-2/R2D2, which has been shown to generate siRNA in S2 cells (Fig. 2; Liu et al. 2003). Therefore, our previous and current results indicate that Dicer-1/R3D1-L and Dicer-2/R2D2 represent distinct initiation complexes of the miRNA and siRNA pathways in Drosophila cells.

R3D1-L is required for miRNA biogenesis in vivo

To determine if R3D1-L is required for miRNA biogenesis in vivo, we depleted Dicer-1, R3D1, or both in S2 cells by RNAi followed by Northern blotting to measure the levels of pre-bantam and bantam miRNA. Surprisingly, only R3D1-L (∼55 kDa), but not R3D1-S (∼50 kDa), protein was efficiently knocked down by treatment of R3D1 dsRNA (Fig. 3A, lanes 6,7). The R3D1 (∼1.1 kb) dsRNA should efficiently target both R3D1-L and R3D1-S mRNA, which differ by 138 nt. It was likely that R3D1-S comigrated with a cross-reacting protein on the Western blot. As shown previously (Okamura et al. 2004), depletion of AGO1, a key component of miRISC, resulted in a specific reduction of bantam miRNA in S2 cells (Fig. 3B, cf. lanes 3 and 1,2). On the other hand, knocking down Dicer-1 caused accumulation of pre-bantam but no reduction in bantam (Fig. 3B, cf. lanes 4 and 1,2). Targeting R3D1 produced a similar phenotype as Dicer-1 depletion (Fig. 3B, cf. lanes 4 and 6). When Dicer-1 and R3D1 were both targeted, there was a greater accumulation of pre-bantam and a modest reduction in bantam (Fig. 3B, lane 7). Since RNAi is transient and rarely a complete knockout, the lack of significant bantam reduction is probably because the remaining Dicer-1 is sufficient to maintain the level of miRNA production. Consistent with these results, the miRNA-generating activity was reduced by approximately twofold in Dicer-1- or R3D1-L-depleted cells and by ∼3.5-fold in cells of double RNAi treatment (Supplementary Fig. 1). Thus, like Dicer-1, R3D1 is required for miRNA maturation in S2 cells.

Dicer-1 and Dicer-2 have different substrate specificities and ATP requirements

Previous genetic and biochemical studies have suggested that Dicer-1 and Dicer-2 may possess different biochemical activities (Liu et al. 2003; Lee et al. 2004). It is also possible that associated proteins, such as R3D1-L and R2D2, can help define the functional specificity for Dicer-1 and Dicer-2. To distinguish the two possibilities, we expressed polyhistidine-tagged Dicer-1 or Dicer-1/R3D1-L and Dicer-2 or Dicer-2/R2D2 recombinant proteins by using an insect cell expression system (Fig. 4A; Liu et al. 2003). These recombinant proteins were highly purified by Ni-NTA columns followed by SP-Sepharose and Q-Sepharose chromatography as previously described (Liu et al. 2003).

Despite sharing extensive sequence homology, Dicer-1 and Dicer-2 displayed different substrate specificities. As shown in Figure 4B, Dicer-1 demonstrated striking pre-miRNA processing activity, whereas miRNA generation was not detected for Dicer-2 at these concentrations. In contrast, Dicer-2 was much more active than Dicer-1 in processing long dsRNA into siRNA. In addition, Dicer-1 and Dicer-2 had different ATP requirements. Like human Dicer, Dicer-1 or Dicer-1/R3D1-L generates miRNA or siRNA in an ATP-independent manner (Fig. 4C; data not shown), whereas Dicer-2 or Dicer-2/R2D2 required ATP hydrolysis for efficient siRNA production (Liu et al. 2003). Taken together, these reconstitution studies establish that Drosophila Dicer-1 and Dicer-2 are functionally distinct enzymes with different substrate specificities and ATP requirements.

R3D1-L enhances miRNA production by Dicer-1

Recombinant Dicer-1 and R3D1-L formed a stable complex and cofractionated on multiple columns (data not shown). Purified recombinant Dicer-1/R3D1-L complex was at least fivefold more active than Dicer-1 alone when measured in the pre-miRNA-processing assay (Fig. 4C). Consistent with this, addition of purified R3D1-L to Dicer-1 greatly enhanced its miRNA-generating activity in a dose-dependent manner (Fig. 4D). Purified R3D1-S has a similar role but to a lesser degree (data not shown). To compare the substrate affinity of Dicer-1 and Dicer-1/R3D1-L, we performed gel-shift experiments in the absence of Mg²⁺, which blocked cleavage of pre-miRNA by Dicer-1 (Fig. 4E). Addition of R3D1-L to Dicer-1 greatly enhanced its affinity for pre-miRNA in a dose-dependent manner (Fig. 4F). These studies suggest that R3D1-L can enhance Dicer-1's miRNA-generating activity by increasing its substrate affinity.

R3D1 mutant flies are defective for miRNA biogenesis

To study the physiological function of R3D1 in flies, we obtained a piggyBac (PB) fly strain in which the piggyBac transposon was inserted in the vicinity of the R3D1 gene (Bloomington, #18371). By cloning and sequencing the flanking sequences, we found that the PB-element was inserted within the first exon and 221 nt upstream of the translational start codon of the R3D1 gene (Fig. 5A; Supplementary Fig. 2). The levels of R3D1-L and R3D1-S mRNA were much reduced in the homozygous flies when compared with wild type or heterozygotes by semiquantitative RT–PCR (Fig. 5A). However, we could not verify corresponding reductions in R3D1 proteins by Western blots due to masking by cross-reacting proteins. Nevertheless, this suggests that the PB-insertion creates a hypomorphic mutant allele of the R3D1 gene by attenuating its transcription.

To examine if miRNA biogenesis was defective in the r3d1^PB mutant flies, we compared the levels of pre-miR277 in wild-type, heterozygous, and homozygous adult flies. As shown by Northern blots, there was significant accumulation of pre-miR277 in both male and female homozygotes (Fig. 5B). Consistent with this, there was an approximately sixfold reduction in the miRNA-generating activity in the whole fly extracts of r3d1^PB/r3d1^PB mutant females (Fig. 5C). The lack of reduction in mature miR277 can be explained by the fact that r3d1^PB is a partial loss-of-function allele. In addition, these data suggest that miRNA production may not be the rate-limiting step in the Drosophila miRNA pathway. Importantly, the miRNA-generating defect in the mutant lysates could be rescued by addition of recombinant R3D1-L, but not R3D1-S (Fig. 5D). Together, these results indicate that r3d1^PB mutant flies are defective for miRNA biogenesis.

R3D1 deficiency causes severe sterility in male and female flies

We suspected that r3d1 mutant flies might display developmental phenotypes because miRNAs play essential roles in animal development (Bartel 2004). Since r3d1^PB mutants survived to adulthood, we decided to examine their fertility by setting up crosses between r3d1^PB homozygous males or females and their wild-type counterparts. Interestingly, while r3d1^PB mutant females were completely sterile, the males were ∼60%–70% sterile when compared with the control crosses between heterozygotes and wild-type flies (Fig. 5E). To further analyze this phenotype, we dissected and examined the testes and ovaries from r3d1^PB mutant flies. Although mutant testes appeared normal, mutant ovaries contained a few maturing egg chambers and a shriveled germarium with few healthy germline stem cells (Fig. 5F). This is a classic “germ cell loss” phenotype because a few egg chambers can develop from primordial germ cells when the adult ovary first forms (McKearin and Ohlstein 1995). However, the mutant ovary did not sustain continuous egg chamber production since germline stem cells could not be properly maintained. These results indicate that R3D1 is required for normal reproductive development in male and female flies and suggest that specific miRNAs may play essential roles in maintaining germline stem cells in the fly ovary. We suspect that R3D1 deficiency, and hence the miRNA biogenesis defect, is far more severe in mutant testes and ovaries than other parts of the fly body.

Dicers in flies, worms, and humans

miRNA and siRNA can be viewed as two parallel branches of the RNAi pathway. Our biochemical studies demonstrate that Dicer-1/R3D1-L and Dicer-2/R2D2 are used as distinct initiation complexes for the miRNA and siRNA pathways, respectively, in Drosophila cells. The same concept can also be applied to species containing a single Dicer, such as C. elegans and humans. In C. elegans, DCR-1/RDE-4 functions as the initiation complex for the siRNA pathway. However, RDE-4 is not required for the miRNA pathway. It is likely that DCR-1 functions in concert with another dsRNA-binding protein in the miRNA pathway.

By reconstitution, we establish that Drosophila Dicer-1 and Dicer-2 enzymes are functional distinct enzymes with different ATP requirements and substrate specificities. Like Dicer-1, human Dicer generates miRNA or siRNA in an ATP-independent manner (Provost et al. 2002; Zhang et al. 2002). Like Dicer-2, the C. elegans DCR-1 requires ATP hydrolysis for efficient siRNA production (Hutvagner et al. 2001; Tabara et al. 2002; Liu et al. 2003). While Dicer-1 is more suited for processing pre-miRNA, Dicer-2 favors long dsRNA as its ideal substrate. Thus, it will be important to identify the sequence and structural features that determine the evolutionary and functional differences between Dicer-1 and Dicer-2.

Does Dicer-1/R3D1-L facilitate miRISC assembly?

The Dicer-2/R2D2 complex not only generates siRNA, but also binds siRNA and facilitates siRNA loading onto the siRISC complex (Liu et al. 2003; Tomari et al. 2004). It is likely that the Dicer-1/R3D1-L complex plays a similar role in facilitating miRNA loading onto the miRISC complex. Consistent with this hypothesis, recombinant Dicer-1/R3D1-L complex efficiently binds to the synthetic miRNA/miR* duplex (data not shown). Since the majority of the miRNA/miR* duplexes have different stability at the two ends, this thermodynamic asymmetry is believed to cause preferential loading of miRNA onto miRISC and destruction of the miR* strand (Tomari et al. 2004). It is reasonable to speculate that the Dicer-1/R3D1-L complex also functions as a sensor for the asymmetry of nascent miRNA/miR* duplex and helps to select the miRNA strand as the guide RNA for miRISC. Since R3D1-L interacts with both Dicer-1 and AGO1, it may play a similar role as R2D2 by bridging the initiation and effector steps of the miRNA pathway.

Partnerships of RNase III enzymes and dsRNA-binding proteins

While Dicer-2/R2D2 cleaves long dsRNA into siRNA, Drosha/Pasha (DGCR8 in human) and Dicer-1/R3D1-L catalyze sequential steps of miRNA biogenesis, processing of pri-miRNA into pre-miRNA and of pre-miRNA into miRNA, respectively (Liu et al. 2003; Denli et al. 2004; Gregory et al. 2004; Han et al. 2004; Landthaler et al. 2004). Although R2D2 does not regulate siRNA production, it facilitates the role of Dicer-2 in loading siRNA onto siRISC (Liu et al. 2003). While Pasha^fly/DGCR8^human is essential for Drosha to process pri-miRNA (Gregory et al. 2004; Han et al. 2004), R3D1-L greatly enhances miRNA generation by Dicer-1. Taken together, these studies indicate that all known RNase III enzymes (Drosha, Dicer-1, and Dicer-2) are paired with specific dsRNA-binding proteins (Pasha, R3D1-L, and R2D2) in catalyzing small RNA biogenesis and/or function in Drosophila. It remains uncertain if the same pattern will repeat in other species.

Cloning, expression, and purification of recombinant proteins

The cDNAs of Dicer-1, R3D1-L, and R3D1-S were cloned from total RNA of S2 cells by RT–PCR using the RLM-RACE kit from Ambion. Polyhistidine (His)-tagged recombinant proteins were expressed in insect cells by using the BAC-to-BAC baculovirus expression system (Invitrogen) as described (Liu et al. 2003). Both R3D1-L and R3D1-S were His-tagged at the N terminus, whereas Dicer-1 was double His-tagged at both ends.

RNAi and Northern blots

In a 24-well dish, 10⁶ S2/GFP cells were soaked in 0.5 mL serum-free media (Invitrogen) containing 10–15 μg dsRNA for 36 h followed by a second dose for another 36 h. Following RNAi treatment, whole cell lysates were prepared in 2% SDS buffer for Western blotting, and total RNA was isolated using TRIzol (Invitrogen) for Northern blotting as previously described (Aravin et al. 2003; Liu et al. 2003). For Northern blotting, a DNA oligo, 5′-GGCCGACAAAATCAGCTTTCAAAATGAT CTCACTTGT-3′, was used for bantam (S2 cells); a DNA oligo, 5′-TAC AACCCTCAACCATATGTAGTCCAAGCA-3′, for 2SrRNA; and an RNA oligo, 5′-UCGUACCAGAUAGUGCAUUUUCA-3′, for miR277 (flies). All probes were 5′-end-labeled with γ-³²P by T4 polynucleotide kinase.

Pre-miRNA-processing and gel-shift assays

A 61-nt synthetic pre-let7, 5′-UGAGGUAGUAGGUUGUAUAGU AGUAAUUACACAUCAUACUAUACAAUGUGCUAGCUUUCUU-3′ (Dharmacon), was 5′-end-labeled by γ-³²P by polynucleotide kinase (NEB). In the pre-miRNA-processing assay, 4 × 10⁴ cpm pre-let7 was incubated with extracts or recombinant proteins in 110 mM KOAc, 10 mM HEPES (pH 7.4), 10 mM Mg(OAc)₂, and 5 mM DTT. Typically, a 10-μL reaction was performed at 30°C for 30 min in the absence or presence of a 1× ATP-regenerating system (1 mM ATP, 30 mM creatine phosphate [Fluka], and 30 U creatine phosphokinase [Sigma]). The gel-shift assay was performed as the processing assay except for using 10⁵ cpm pre-let7 in the presence of 0.2 mM Mg²⁺ and 2.5 mM EDTA. The reaction mixture was resolved by a 5% native PAGE.

Antibodies and co-IP

The anti-R3D1 and anti-AGO2 antibodies were raised in rabbits against full-length recombinant R3D1-L and a truncated AGO2. The anti-Dicer-2 and anti-R2D2 antibodies have been described (Liu et al. 2003). The anti-Dicer-1 antibodies were a gift from Dr. Gregory Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (used for IPs) or purchased from Abcam (used for Western blots). The anti-GFP and anti-AGO1 antibodies were from Molecular Probes and Abcam, respectively. Co-IP experiments were carried out in 500 μL volume by incubating 2 mg of S100, 10 μL of affinity-purified antibodies, and 10 μL protein A-Sepharose beads (Santa Cruz) for 2 h in RISC buffer [110 mM KOAc, 10 mM HEPES at pH 7.4, 2 mM Mg(OAc)₂, 5 mM DTT, 0.05% NP-40]. The beads were washed six times with 1 mL RISC buffer containing 50 mM NaCl, and boiled in 2× SDS sample buffer prior to Western blot analysis.

RT–PCR

Total RNA was isolated from 30 female flies by TRIzol (Invitrogen). RT–PCR reactions were performed using the SuperScript One-Step RT–PCR kit from Invitrogen. In brief, 100 ng RNA was used as template with one round of reverse transcription and 30 cycles of PCR. The primers included 5′-GGGCAAAAGCAAGAAGATAG-3′ and 5′-GCGGCCGCT CACTTCTTGGTCATGATCTTC-3′ for amplifying R3D1-L (508 bp) and R3D1-S (370 bp), or 5′-CATATGGATAACAAGTCAGCCGTATC-3′ and 5′-AGAACGCGGCCGCTTAGTAGGTGGTAAATTTCTTTCTG-3′ for amplifying R2D2 (631 bp). All PCR products were resolved by 1.5% agarose gel electrophoresis.