Processing of Pre-microRNAs by the Dicer-1–Loquacious Complex in Drosophila Cells

Depletion of Loqs and Dicer-1 by RNAi Resultsin Pre-miRNA Accumulation

Dicer-1 has been shown to be the pre-miRNA processing factor in Drosophila [58]. We have previously shown that depletion of Dicer-1 by RNAi resulted in a marked accumulation of pre-miR-bantam (pre-miR-ban) [60]. Depletion of Loqs by RNAi resulted in a similar effect to Dicer-1 depletion for miR-ban (Figure 2A). Loqs dsRNAs caused the suppression of Loqs mRNA (Figure 2B). RNAi against Loqs does not appear to affect Dicer-1 protein levels (lower panel in Figure 2B), suggesting that the observed pre-miRNA accumulation in Loqs-depleted cells is not simply due to destabilizing Dicer-1. Similar effects on miR-8 were seen in Dicer-1- and Loqs-depleted S2 cells (Figure 2C). Depletion of Dicer-2 and R2D2, which form the enzyme complex predominantly responsible for generating siRNAs from long dsRNA [59], had no significant effect on pre-miRNA processing (Figure 2A and 2C). These results show that along with Dicer-1, Loqs is essential for efficient pre-miRNA processing in vivo.

Loqs Associates with Dicer-1 In Vivo and In Vitro

This observation prompted us to ask if Loqs forms a complex in vivo with Dicer-1. For these studies, we simultaneously expressed Dicer-1 tagged with the Flag epitope and Loqs tagged with the myc epitope in S2 cells. We then immunoprecipitated Dicer-1 with anti-Flag antibodies, and Loqs with anti-myc antibody and then analyzed the precipitates by immunoblotting (Figure 3A). In reciprocal assays, Dicer-1 and Loqs were found to co-precipitate. Consistent with these findings that Dicer-1 and Loqs form a complex in vivo, both proteins are localized predominantly in the cytoplasm of S2 cells (Figure 3B).

We further investigated whether Loqs can bind to Dicer-1 in vitro. Dicer-1 was produced by an in vitro translation system and used in binding assays with recombinant Loqs fused to glutathione S-transferase (GST). GST–Loqs interacted with Dicer-1 even in the presence of RNase A, whereas GST itself showed no detectable binding (Figure 3C). These results demonstrate that the association of Loqs with Dicer-1 occurs both in vivo and in vitro, and that RNA molecules do not appear to mediate the association.

Dicer-1 and Loqs Are Present in a Functional Complex That Mediates Pre-miRNA Processing

To examine the functional connection between the Dicer-1–Loqs complex and pre-miRNA processing, we investigated if depletion of Dicer-1 or Loqs had any effect on the production of mature miRNA from the precursor. We first tested if cytoplasmic lysates of S2 cells were capable of processing synthetic Drosophila melanogaster let-7 precursor RNA into functional mature let-7. In this experiment, the synthetic let-7 precursor RNA was converted to mature let-7 in S2 cytoplasmic lysates (Figure 4A), as is the case in embryo lysates [60]. In in vitro RNAi assay, target RNA harboring a sequence perfectly complementary to mature let-7 was cleaved efficiently within the let-7 complementary sequence (Figure 4B), thus showing production of functional let-7 in S2 cell lysates. Cytoplasmic lysates from Dicer-1- or Loqs-depleted cells were then subjected to the pre-let-7 processing assay. Both Dicer-1 and Loqs depletion led to reductions of mature let-7 compared with controls (Figure 4C), showing that both Dicer-1 and Loqs function in pre-miRNA processing.

We next used pre-miR-ban as a substrate for pre-miRNA processing assays. It was shown recently that S2 cell extracts contained pri-miRNA processing activity that cleaved pri-miRNA into an approximately 60- to 70-bp pre-miRNA precursor [49]. This processing is known to occur in the nucleus; thus pre-miR-ban was prepared by in vitro processing of pri-miR-ban incubated with S2 nuclear extracts (Figure 5A). Uniformly labeled pre-miR-ban was then gel-purified and used as a substrate for analysis of pre-miRNA processing. Incubation of the pre-miRNA with S2 cytoplasmic extracts resulted in the appearance of a mature 21-nucleotide miR-ban (Figure 5B). We then examined the requirement of Dicer-1 and Loqs in pre-miR-ban processing. Incubation of pre-miRNA with Dicer-1- and Loqs-depleted S2 cytoplasmic extracts resulted in a marked reduction in mature miRNA levels (Figure 5B). In contrast, depletion of Dicer-2 or R2D2 showed no measurable reduction of mature miRNA levels (Figure 5B). We then assayed the pre-miRNA processing activity of the purified complexes (both Flag–Dicer-1 and Flag–Loqs complexes). That the Flag–Loqs complex contains Dicer-1 was confirmed by immunoblotting (data not shown). Both Dicer-1 and Loqs complexes were capable of generating maturemiR-ban from pre-miR-ban (Figure 5C). Several steps in the RNAi and miRNA pathways are known to require a divalent metal ion [64]. In addition, it is well known that RNase III-type enzymes require divalent metals for cleavage [65]. Flag–Dicer-1 complex was employed and the processing was performed in the presence of magnesium ions or EDTA in a buffer. As shown in Figure 5D, no pre-miRNA processing activity was detected at 10 mM EDTA. These results demonstrated that the Dicer-1–Loqs complex converts pre-miRNAs into mature miRNAs in a divalent metal ion–dependent manner.

Loqs Stimulates and Confers upon Dicer-1 the Specific Processing of Pre-miRNAs

To further examine the requirement for Loqs in pre-miRNA processing, we purified Flag–Dicer-1 complex under a harsher condition (high salt), where Dicer-1 was stripped of most Loqs protein (Figure 6A), and used this Dicer-1 complex in pre-miRNA processing assays with or without supplement of recombinant GST–Loqs (see left panel in Figure 3C). Without any supplement, the Flag–Dicer-1 complex purified under the harsh condition showed less activity than that under mild condition (Figure 6B). Then we added GST–Loqs in the assay mixture. The addition of GST–Loqs to the Dicer-1 complex stimulated the processing of pre-miRNA (Figure 6C). GST–Loqs alone did not show any significant pre-miRNA processing activity (Figure 6C). These results show that Loqs is required for stimulating the processing of pre-miRNAs. Interestingly, we found that the Dicer-1 complex purified under the harsh condition displayed considerable siRNA-generating activity on the long dsRNA substrate in vitro (Figure 6D), although previous genetic studies have shown that Dicer-1 is not required for siRNA production [58]. The addition of GST–Loqs inhibited this effect (Figure 6D). Western blot analysis failed to show that the Dicer-1 complex used in this experiment contains Dicer-2 (right panel inFigure 6D). GST–Loqs alone showed no activity for generating siRNAs from long dsRNAs. These results suggested that Dicer-1 stripped of much of its bound Loqs processes both dsRNA and pre-miRNA substrates, but re-addition of recombinant Loqs suppresses dsRNA processing activity and enhances pre-miRNA processing activity. Our findings thus imply that much of the apparent substrate specificity of Dicer-1 in vivo results from its association with Loqs. Although very unlikely (Figure 6D), it is, however, formally possible that the Dicer-1 immunoprecipitates may contain very small amounts of Dicer-2 protein that can catalyze long dsRNA cleavage, and that addition of a large amount of dsRBD-containing Loqs may block the activity of Dicer-2 in this experiment.

Dicer-1–Loqs Complexes Associate with Pre- and Mature miRNAs In Vivo

We examined the presence of endogenous miRNA in RNA preparations from Flag–Dicer-1 and Flag–Loqs complexes obtained from S2 cells using anti-Flag antibodies. Complexes were prepared as in Figure 3A, and RNA preparations from each complex were subjected to Northern blotting using an oligo probe recognizing both pre-miR-ban and mature miR-ban. The Dicer-1 complex contained both the pre- and mature form of miR-ban, and the complex seems to preferentially bind the precursor form of miR-ban (Figure 7A). In contrast, the precursor form of miR-ban was barely detectable in the Loqs complex, though it contained mature miR-ban. However, EDTA treatment, which inhibits pre-miRNA processing activity (see Figure 5D), resulted in an accumulation of pre-miR-ban in the Loqs complex (Figure 7A). This may suggest that part of Flag-tagged Loqs protein interacts with Dicer-1 or pre-miRNAs or both. Alternatively, Flag–Loqs complexes may rapidly process pre-miRNAs into mature miRNAs and, therefore, may only transiently interact with them. Nonetheless, these results suggest that Dicer-1–Loqs complexes associate with both pre- and mature miRNAs in vivo.

An AGO1-Associated Complex Contains Dicer-1 and Loqs, and Is Capable of Pre-miRNA Processing

We have previously shown that Argonaute protein AGO1 is required for stable production of mature miRNAs and associates with Dicer-1 [60]. Thus, we sought to ascertain if Loqs was also present in an AGO1-associated complex, and if so, if the AGO1 complex was capable of processing pre-miRNA in vitro. We simultaneously expressed Flag–Loqs and AGO1 tagged with TAP in S2 cells and purified the AGO1–TAP complex through immunoglobulin G (IgG) bead-binding. The IgG bound was then subjected to Western blot analysis using anti-Dicer-1, anti-AGO1, or anti-Flag (for Loqs detection) antibodies. Not only Dicer-1 but also Loqs was detected in the AGO1 complex (Figure 7B). These results indicated that all three proteins are present in the same complex, although they cannot exclude the possibility that there is one complex that contains AGO1 and Dicer-1 but not Loqs, and another complex that contains AGO1 and Loqs but not Dicer-1. The pre-miRNA processing activity of the AGO1 complex was then examined. As in Figure 5, pre-miR-ban was utilized as a substrate. The AGO1 complex was able to efficiently process pre-miR-ban into the mature form (Figure 7C). In contrast, another Argonaute protein AGO2-associated complex showed no such activity, which is consistent with our previous finding that the AGO2-associated complex does not contain Dicer-1 [60]. Considered together, these results showed that Dicer-1 and Loqs form a functional complex that mediates the genesis of mature miRNAs from pre-miRNAs, and suggested that the resultant mature miRNAs are loaded onto an AGO1-associated complex, which probably is miRNA-associated RISC [60], through specific interaction of AGO1 with Dicer-1 and Loqs.

RNAi

dsRNAs were introduced to S2 cells by soaking essentially as described [75]. Briefly, approximately 5 × 10⁶ cells were soaked in 1 ml of serum-free medium containing 15 μg of dsRNA for 30 min at room temperature followed by addition of 2 ml of the medium containing 15% serum, 3 mM glutamine, and penicillin-streptomycin. After 4 d, cells were harvested and subjected to total RNA preparation for Northern blot analysis, or cytoplasmic lysate preparation for in vitro processing assays. dsRNAs used in RNAi were: double-stranded RNA for enhanced green fluorescent protein (EGFP), homologous to nucleotides 11–717 of the EGFP coding sequence; dsDcr-2, 4091–4888 of the Dicer-2 coding; dsR2D2, 1–936; dsDcr-1, 10–950 of the Dicer-2 coding; dsLoqs, 330-1342.

Northern blot analysis

Total RNA was isolated from S2 cells with ISOGEN (Nippon Gene, Toyama, Japan). 20 μg of total RNA was separated on 12% acrylamide-denaturing gel and transferred onto Hybond-N+ membrane (Amersham Bioscience, Little Chalfont, United Kingdom). After UV-crosslinking, the hybridization was performed at 42 °C in 0.2 M sodium phosphate (pH 7.2), 7 % SDS, and 1 mM EDTA with end-labeled antisense oligodeoxynucleotide, and washed at 42 °C in 2× saline sodium citrate and 0.1% SDS. Oligodeoxynucleotides used as probes were: bantam, 5′- CAGCTTTCAAAATGATCTCAC-3′; miR-8, 5′- GACATCTTTACCTGACAGTATTA-3′; U6 snRNA, 5′- GGGCCATGCTAATCTTCTCTGTA-3′; and let-7, 5′- AACTATACAACCTACTACCTCA-3.′ The blots were exposed on BAS-MS2040 imaging plates, and signals were quantified using BAS-2500 (Fuji, Tokyo, Japan).

RT-PCR analysis

One μg of total RNA was used for the first-strand cDNA synthesis with Stratascript RT and random primers (Stratagene, La Jolla, California, United States). Sequences of the oligonucleotide primers for RT-PCR were: Dicer-1, 5′- ACCAATGTACTGCGTTTGCA and 5′- GTTTGCTGATCACAGAACTTAACGTT; Loqs, 5′- ATGGACCAGGAGAATTTCCACGG-3′ and 5′- CTACTTCTTGGTCATGATCTTCAAGTAC-3′; and AGO2, 5′- GCACAAGTGTGCGGTCTTGTATT-3′ and 5′- GTGAACTGCTTAATGCATTG-3′.

Immunofluorescence analysis

Immunofluorescence analysis was performed by fixing S2 cells with 2% formaldehyde for 15 min. Cells were permeabilized using 0.1% Triton X-100. Flag-tagged proteins were stained for 30 min with anti-Flag M2 (1:1,000 dilution) antibody (Sigma, St. Louis, Missouri, United States). After extensive wash in PBS, cells were treated with 100 μg/ml RNaseA for 30 min and then stained with 0.4 μg/ml propidium iodide. Alexa-488 anti-mouse IgG was used as secondary antibody. All images were collected using a Zeiss (Oberkochen, Germany) LSM510 laser scanning microscope.

Immunoprecipitation of Flag–Dicer-1, Flag–Loqs, and myc-Loqs

S2 cell lines, stably expressed 3× Flag-tagged Dicer-1 or Loqs, or myc-tagged-Loqs under the control of metallothionein promoter (originally from pRmHa-3 vector), were established. The expression of each protein was induced by adding copper ions into the medium. After overnight incubation, the whole cell extract was prepared in Buffer A (30 mM HEPES pH 7.4, 150 mM KOAc, 2 mM MgOAc, 5 mM DTT, 2 μg/ml Leupeptin, 2 μg/ml Pepstatin, 0.5% Aprotinin) containing 0.1% NP-40 by sonication, and followed by centrifugation. Flag–Dicer-1 and Flag–Loqs were bound to anti-Flag M2 agarose beads at 4 °C for 1 h. Immunoprecipitated proteins were then recovered with 2× SDS sample buffer or Elution buffer (Buffer A containing 400 μg/ml of 3× Flag peptides, 10% glycerol, and 100 mM KOAc). For Northern blot analysis of RNAs co-purified with Flag–Dicer-1 or Flag–Loqs, immunoprecipitates on beads were treated with ISOGEN and subjected to RNA purification.

Protein–protein interaction assays

To produce [³⁵S]methionine-labeled proteins by a TNT in vitro transcription and translation kit (Promega, Madison, Wisconsin, United States), the Dicer-1 cDNA was inserted into an expression vector, pET-28 (Novagen, Madison, Wisconsin, United States). GST pull-down assays were carried out using GST–Loqs and GST itself that were bound to glutathione Sepharose 4B resins (Amersham Biosciences) in Buffer A containing 0.1% NP-40. After incubation with TNT products and extensive washing, the bound proteins were separated by SDS-PAGE. RNaseA treatment was carried out by adding the enzyme to the binding mixture. To produce GST fusion protein, Loqs cDNA was subcloned into a pGEX-5X expression vector (Amersham Biosciences). The fusion proteins, as well as GST itself, were induced and purified as described by the manufacturer.

Preparation of nuclear and cytoplasmic lysate for in vitro processing assays

S2 cells were suspended at approximately 1 × 10⁸ cells/ml into Hypotonic buffer (Buffer A without KOAc) and lysed by passing through a 30G needle. After centrifugation at 500 × g for 20 min, the supernatant and the precipitate were separated. The supernatant was centrifuged to obtain the supernatant as a cytoplasmic lysate. The pellet was washed twice with Hypotonic buffer and lysed by sonication in Buffer A containing 100 mM KOAc and 20% glycerol, followed by centrifugation to obtain the supernatant as a nuclear lysate. Total protein concentration in each lysate was determined with Protein assay (Bio-Rad, Hercules, California, United States) and adjusted to be equal.

Preparation of pre-miR-ban

A DNA fragment coding pri-miR-ban was obtained from PCR reaction (primers used are: 5′- CGCTCAGATGCAGATGTTGTTGAT-3′ and 5′- GATCGGTCGGCATAAG TTCAAAGC-3′) and cloned into the SmaI site of pBluescriptSK vector in the same direction with the T3 promoter. The plasmid was digested with ClaI, gel-isolated, and used as a template for in vitro transcription reaction with MEGAscript T3 Kit (Ambion, Austin, Texas, United States) in the presence of [α³²P]GTP. In vitro processing reaction of pri-miRNAs was performed with some modifications to previously reported method [36]. Briefly, in 1 ml reaction, 10 mM creatine phosphate, 0.5 mM ATP, 30 μg/ml creatine kinase, 0.1 U/ul RNasin, 0.1 μg yeast RNA, and 500 μl nuclear lysate were added, and pri-miR-bantam in 0.5× Buffer A with 100 mM KOAc was further added to the mixture. After 2 h incubation at 26 °C, RNAs were purified with ISOGEN LS (Nippon Gene) and separated on 7.5% acrylamide denaturing gel, from which pre-miR-ban (about 60 nucleotides in length) was recovered.

In Vitro pre-miRNA processing assays

The condition used for in vitro pre-miRNA processing with cytoplasmic lysates was the same as that for the in vitro pri-miR-bantam processing. Cytoplasmic lysate used in this assay was 5 μl in a 10-μl reaction. For processing assays with purified complexes, immuno-purified Flag–Dicer-1 or Flag–Loqs was used instead of crude cytoplasmic lysate and the final concentration of buffer adjusted. For Mg⁺⁺-depletion assay, 10 mM EDTA was added instead of Mg⁺⁺. For the processing by Flag–Dicer-1, high-salt purified (800 mM KOAc) Flag–Dicer-1 was added in the presence or absence of bacterially produced GST–Loqs and the final concentration of buffer adjusted.

In Vitro cleavage assay

Preparation of cap-labeled ftz RNA with a let-7 target site and RNAi reaction were carried out essentially as described [60]. In brief, 10⁴ cpm of cap-labeled let-7 target RNA was incubated with 200 nM in vitro transcribed pre-let-7 RNA in Buffer A containing 100 mM KOAc, 10 mM creatine phosphate, 0.5 mM ATP, 30 μg/ml creatine kinase, and 0.1 U/ul RNasin. Reactions were allowed to proceed for 3 h at 26 °C. Cleavage products of the RNAi reaction were analyzed by electrophoresis on 4% denaturing polyacrylamide gels.

TAP purification

The expression of AGO1–TAP or AGO2–TAP in S2 cells was induced by adding copper ion into the medium [60]. After overnight incubation, the cytoplasmic lysate was prepared in a Buffer A containing 150 mM KOAc. AGO1–TAP or AGO2–TAP and associated materials to the TAP-tagged fusion protein were bound to IgG Sepharose (Amersham Biosciences). Bound proteins on IgG beads were directly used for in vitro pre-miRNA processing assay, or eluted with SDS sample buffer for Western blotting analysis. The polyclonal antibodies against AGO1 were a kind gift from T. Uemura (Kyoto University) [76]. The anti-Dicer-1 antibody (AB4735) was purchased from Abcam (Cambridge, United Kingdom).

Accession Numbers

The GenBank (http://www.ncbi.nih.gov/Genbank/) accession numbers for the genes and gene products discussed in this paper are: ago-1 (NM_079010), ago-2 (NM_140518), dicer-1 (NM_079729), dicer-2 (NM_079054), loqs/cg6866 (NM_135802), and r2d2 (NM_135308).

The Rfam (http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml) accession numbers for the genes and gene products discussed in this paper are: bantam (MI0000387), let-7 (MI0000416), and miR-8 (MI0000128).