Open access, freely available online

PLoS BIOLOGY

Processing of Pre-microRNAs
by the Dicer-1–Loquacious Complex
in Drosophila Cells
Kuniaki Saito[, Akira Ishizuka[, Haruhiko Siomi*, Mikiko C. Siomi*
Institute for Genome Research, University of Tokushima, Kuramoto, Tokushima, Japan

microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at
specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process fromprimary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs torelease hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm andfurther processed by Dicer to mature miRNAs. Here we show that Drosophila Dicer-1 interacts with Loquacious, adouble-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in
Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed thatalong with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the
specific pre-miRNA processing activity. These results support a model in which Loquacious mediates miRNA biogenesis
and, thereby, the expression of genes regulated by miRNAs.
Citation: Saito K, Ishizuka A, Siomi H, Siomi MC (2005) Processing of pre-microRNAs by the Dicer-1–loquacious complex in Drosophila cells. PLoS Biol 3(7): e235.

protein partner known as Pasha in ﬂies and Caenorhabditis
elegans, and its ortholog DGCR8 in mammals to convert primiRNAs to pre-miRNAs [49–52]. In plants, the predominantly
nuclear Dicer-like-1, equipped with two dsRBDs, is thought tocatalyze both pri-miRNA and pre-miRNA processing [53,54].
The HYL1 protein, which also contains a tandem dsRBD, isrequired for miRNA accumulation and may play the same
molecular role as Pasha/DGCR8 for Dicer-like-1 in plants[55,56]. In Drosophila, Dicer-2 is required for production ofsiRNAs [57,58], and forms a heterodimeric complex with thedsRNA-binding protein R2D2, which is required for its
function in RISC assembly, although Dicer-2 alone sufﬁces
to convert long dsRNA into siRNAs [59]. Drosophila Dicer-1 isassociated with the processing of pre-miRNAs [58,60].
However, if there is a dsRNA-binding protein partner for
Dicer-1, it has not been identiﬁed.
Here, we show that Drosophila Dicer-1 interacts with thedsRBD protein Loquacious (Loqs). Depletion of Loqs resultsin accumulation of pre-miRNAs in Drosophila S2 cells. Loqs is

Introduction
microRNAs (miRNAs) act as RNA guides by binding tocomplementary sites on target mRNAs to regulate gene
expression at the post-transcriptional level in plants andanimals [112], much as small interfering RNAs (siRNAs) doin the RNA interference (RNAi) pathway [1315]. The
expression of miRNAs is often developmentally regulated ina tissue-speciﬁc manner, suggesting an important role for
miRNAs in the regulation of endogenous gene expression[16–30]. The importance of miRNAs for development is also
highlighted by a recent computer-based analysis that predicted nearly a thousand miRNA genes in the human genome[31]. Furthermore, recent studies have revealed that miRNAs
regulate a large fraction of the protein-coding genes [32–34].
miRNAs are transcribed as long primary miRNA (primiRNA) transcripts by RNA polymerase II [35]. miRNAmaturation begins with cleavage of the pri-miRNAs by the
nuclear RNase III Drosha [36–38] to release approximately
70-nucleotide hairpin-shaped structures, called precursor
miRNAs (pre-miRNAs). Pre-miRNAs are then exported tothe cytoplasm by the protein Exportin 5, which recognizes thetwo-nucleotide 39 overhang that is a signature of RNase IIImediated cleavage [39–41]. In the cytoplasm, pre-miRNAs aresubsequently cleaved by a second RNase III enzyme, Dicer,
into approximately 22-nucleotide miRNA duplexes, with anend structure characteristic of RNase III cleavage [42–44].
Only one of the two strands is predominantly transferred to
the RNA-induced silencing complex (RISC) [45], whichmediates either cleavage of the target mRNA or translation
silencing, depending on the complementarity of the target[46] by a mechanism that remains unclear [47].
There is a growing list of double-stranded RNA (dsRNA)binding proteins that play important yet distinct roles in theRNAi pathway [48]. Both Drosha and Dicer contain dsRNAbinding domains (dsRBDs). Drosha requires a dsRNA-binding
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Received March 16, 2005; Accepted April 30, 2005; Published May 24, 2005
DOI: 10.1371/journal.pbio.0030235
Copyright: Ó 2005 Saito et al. This is an open-access article distributed under theterms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Abbreviations: dsRBD, double-stranded RNA-binding domain; dsRNA, doublestranded RNA; EGFP, enhanced green fluorescent protein; GST, glutathione Stransferase; IgG, immunoglobulin G; miRNA, microRNA; PACT, protein activator of
PKR; PKR, protein kinase dsRNA dependent; pre-miRNA, precursor miRNA; primiRNA, primary miRNA; RISC, RNA-induced silencing complex; RNAi, RNAinterference; siRNA, small interfering RNA; TRBP, TAR RNA binding protein.
Academic Editor: James C. Carrington, Oregon State University, United States ofAmerica*To whom correspondence should be addressed. E-mail: siomi@genome.
tokushima-u.ac.jp (HS), siomim@genome.tokushima-u.ac.jp (MCS)[These authors contributed equally to this work.

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Pre-miRNA Processing by Dicer-1–Loqs Complex

in Figure 2B), suggesting that the observed pre-miRNAaccumulation in Loqs-depleted cells is not simply due todestabilizing 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 Dicer1, Loqs is essential for efﬁcient pre-miRNA processing invivo.

predominantly cytoplasmic and is conserved in mammals.
Immuno-afﬁnity puriﬁcation experiments, together with theuse of recombinant Loqs, reveal that along with Dicer-1, Loqs
resides in a functional pre-miRNA processing complex, andstimulates and directs speciﬁc pre-miRNA processing activity.
These results support a model in which Loqs mediates
miRNA biogenesis and, thereby, the expression of genes
regulated by miRNAs.

Results

Loqs Associates with Dicer-1 In Vivo and In Vitro

We have used RNAi-based reverse-genetic methods [61] toscreen a list of Drosophila dsRBD proteins [62] for a protein(s)that has an effect on miRNA biogenesis in Drosophila S2 cells
and found a novel protein equipped with three dsRBDs (twocanonical dsRBDs at the N-terminal half, and one noncanonical dsRBD at the C-terminal), originally dubbed
CG6866 (candidate gene 6866), which has a role in premiRNA processing (data presented below). This protein bears
high similarity to R2D2 and to the C. elegans RNAi protein
RDE-4 (Figure 1), both of which contain dsRBDs and interact
with Dicer [59,63]. Thus the sequence data show that CG6866
is a paralog of R2D2. A parallel study presents genetic
evidence that several types of silencing are lost in CG6866
mutant ﬂies (Förstemann K, et al. DOI: 10.1371/journal.
pbio.0030236). Therefore, CG6866 was designated as Loquacious (‘‘very talkative’’).

This observation prompted us to ask if Loqs forms acomplex in vivo with Dicer-1. For these studies, we simultaneously expressed Dicer-1 tagged with the Flag epitope andLoqs tagged with the myc epitope in S2 cells. We then
immunoprecipitated Dicer-1 with anti-Flag antibodies, andLoqs 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 invivo, both proteins are localized predominantly in thecytoplasm 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.

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]. Depletionof 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

Dicer-1 and Loqs Are Present in a Functional Complex
That Mediates Pre-miRNA Processing
To examine the functional connection between the Dicer1–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

Figure 1. Loqs/CG6866 Is a Paralog of R2D2
A protein sequence alignment of Loqs, R2D2, and RDE-4 (C. elegans). The two canonical dsRBDs are boxed. Conserved residues are shaded in gray.
It is noted that the C-terminal region of Loqs contains a non-canonical dsRBD (see Figure 8).
DOI: 10.1371/journal.pbio.0030235.g001
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Figure 2. Depletion of Loqs and Dicer-1 Causes Pre-miRNA Accumulation(A) dsRNAs of Loqs, Dicer-1 (Dcr-1), Dicer-2 (Dcr-2), R2D2, or EGFP were introduced to S2 cells by soaking. Total RNA was isolated 4 d after initial
exposure of S2 cells to the indicated target genes. The RNA was separated on a 12% denaturing polyacrylamide gel, transferred to a nylon membrane,
and probed with 59-radiolabeled miR-bantam (miR-ban) antisense oligodeoxynucleotide (top panel) and re-probed for U6 snRNA (bottom panel). Afternormalization for loading, the relative ratios for pre-miRNA (pre-miR-ban) (left panel) and mature miRNA (miR-ban) (right panel) were calculated andnormalized to EGFP dsRNA experiments. Depletion of Dicer-1 (Dcr-1) by RNAi resulted in a marked accumulation of pre-miR-ban and a modest
reduction in levels of mature miR-ban.
(B) Upper panel: Analysis of transcript levels using RT-PCR for Loqs and Dicer-1 (Dcr-1) after treatment of Drosophila S2 cells with dsRNAs against eachprotein. AGO2 was used as control. Lower panel: Loqs dsRNA did not affect Dicer-1 protein levels. dsRNAs of Loqs, Dicer-1, or EGFP were introduced toS2 cells by soaking. After 4 d, cell lysates were prepared and the levels of Dicer-1 protein were measured by Western blotting.
(C) As in (A), total RNA was probed with 59-radiolabeled miR-8 antisense oligodeoxynucleotide (top panel) and re-probed for U6 snRNA (bottom panel).
DOI: 10.1371/journal.pbio.0030235.g002

tested if cytoplasmic lysates of S2 cells were capable ofprocessing synthetic Drosophila melanogaster let-7 precursor
RNA into functional mature let-7. In this experiment, thesynthetic let-7 precursor RNA was converted to mature let-7 inS2 cytoplasmic lysates (Figure 4A), as is the case in embryo
lysates [60]. In in vitro RNAi assay, target RNA harboring asequence perfectly complementary to mature let-7 wascleaved efﬁciently within the let-7 complementary sequence(Figure 4B), thus showing production of functional let-7 in S2cell lysates. Cytoplasmic lysates from Dicer-1- or Loqsdepleted cells were then subjected to the pre-let-7 processing
assay. Both Dicer-1 and Loqs depletion led to reductions ofmature let-7 compared with controls (Figure 4C), showing thatboth Dicer-1 and Loqs function in pre-miRNA processing.
We next used pre-miR-ban as a substrate for pre-miRNA
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processing assays. It was shown recently that S2 cell extracts
contained pri-miRNA processing activity that cleaved primiRNA 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 processingof 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 premiRNA with Dicer-1- and Loqs-depleted S2 cytoplasmic
extracts resulted in a marked reduction in mature miRNAlevels (Figure 5B). In contrast, depletion of Dicer-2 or R2D2
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Figure 3. Loqs Associates with Dicer-1(A) Loqs and Dicer-1 form a complex in vivo. Left panel: Protein extract was prepared from S2 cells that expressed both full-length myc-tagged Loqs
(myc-Loqs) and Flag-tagged Dicer-1 (Flag-Dcr-1). Protein extract containing only myc-Loqs was also prepared (control). Total extracts (input) and thematerials obtained after immunoprecipitation from the extracts with anti-Flag were run on an SDS-polyacrylamide gel. Western blots were preparedand immunostained with anti-myc (to detect myc-Loqs) or anti-Flag (to detect Flag–Dicer-1) antibodies. The protein band shown by an asterisk is anantibody used. Right panel: Protein extracts were prepared from S2 cells that expressed both myc-Loqs and Flag–Dicer-1, and immunoprecipitation was
performed with anti-myc antibody (a-myc). Non-specific antisera were also employed as a negative control (n.i.). Flag–Dicer-1 was specifically coimmunoprecipitated with myc-Loqs.
(B) Immunofluorescence using anti-Flag antibodies show that both Loqs and Dicer-1 are predominantly localized in the cytoplasm in Drosophila S2 cells(a-Flag). Flag–Dicer-1 (Dcr-1) and Flag–Loqs were transiently expressed in the cells by transfection. The nuclear DNA was stained with propidium iodide.
A DIC image of the same field is also shown (DIC).
(C) Loqs interacts with Dicer-1 in an RNase-resistant manner in vitro. 35S-labeled Dicer-1 was produced by an in vitro transcription and translation systemin the presence of [35S]methionine, treated with RNaseA, and incubated with either GST–Loqs or GST itself immobilized on glutathione-Sepharose resins.
After extensive washing, the bound fractions were resolved on an SDS-polyacrylamide gel and the protein labeled with 35S visualized byautoradiography. The Coomassie Blue stainings of GST and GST–Loqs used in this experiment are shown on the left.
DOI: 10.1371/journal.pbio.0030235.g003

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

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
mature miR-ban from pre-miR-ban (Figure 5C). Several steps in
the RNAi and miRNA pathways are known to require adivalent 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 premiRNAs into mature miRNAs in a divalent metal ion–
dependent manner.
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To further examine the requirement for Loqs in premiRNA processing, we puriﬁed Flag–Dicer-1 complex under aharsher condition (high salt), where Dicer-1 was stripped ofmost Loqs protein (Figure 6A), and used this Dicer-1 complex
in pre-miRNA processing assays with or without supplementof 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 theassay 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-miRNAprocessing activity (Figure 6C). These results show that Loqs isrequired for stimulating the processing of pre-miRNAs.
Interestingly, we found that the Dicer-1 complex puriﬁed
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Pre-miRNA Processing by Dicer-1–Loqs Complex

Figure 4. Synthetic D. melanogaster let-7 Precursor Processing in the Cytoplasmic Lysate of S2 Cells(A) S2 cytoplasmic lysate is capable of processing pre-let-7 into mature let-7. Pre-let-7 (the sequence shown on the top) was transcribed with T7 RNApolymerase in vitro (T7-pre-let-7). After gel purification, it was incubated with S2 cytoplasmic lysate, and resultant RNA was subjected to Northernblotting using an oligodeoxynucleotide recognizing both pre-let-7 and mature let-7. Total RNA prepared from pupa was also applied (pupa), whichshows where endogenous pre-let-7 (pre-let-7; ;70 nucleotides) and mature let-7 (let-7; 21 nucleotides) migrate on the blot. The cytoplasmic lysate (þ)
lane, but not () lane, shows a band corresponding to mature let-7, meaning that the synthetic T7-pre-let-7 was processed to the matured form in thelysate.
(B) Functional analysis of mature let-7 produced from synthetic T7-pre-let-7 in (A). Cap-labeled target RNA with let-7 target site (;500 nucleotides) was
incubated in S2 cytoplasmic lysate with or without T7-pre-let-7 for 3 h. let-7-directed cleavage product (;150 mucleotides) was observed only when T7pre-let-7 was included, indicating that let-7 produced in the lysate is functional.
(C) Dicer-1- or Loqs-depleted cytoplasmic lysate has less activity to produce mature let-7 from the precursor. Dicer-1 (Dcr-1) or Loqs was depleted from
S2 cells by RNAi, and the cytoplasmic lysate was assayed for pre-let-7 processing as in (A). "-" indicates no lysate.
DOI: 10.1371/journal.pbio.0030235.g004

possible that the Dicer-1 immunoprecipitates may containvery small amounts of Dicer-2 protein that can catalyze longdsRNA cleavage, and that addition of a large amount ofdsRBD-containing Loqs may block the activity of Dicer-2 inthis experiment.

under the harsh condition displayed considerable siRNAgenerating activity on the long dsRNA substrate in vitro(Figure 6D), although previous genetic studies have shownthat 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 in
Figure 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 ofrecombinant Loqs suppresses dsRNA processing activity
and enhances pre-miRNA processing activity. Our ﬁndings
thus imply that much of the apparent substrate speciﬁcity ofDicer-1 in vivo results from its association with Loqs.
Although very unlikely (Figure 6D), it is, however, formally
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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 fromeach complex were subjected to Northern blotting using anoligo probe recognizing both pre-miR-ban and mature miRban. The Dicer-1 complex contained both the pre- and matureform of miR-ban, and the complex seems to preferentially bind
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Pre-miRNA Processing by Dicer-1–Loqs Complex

Figure 5. In Vitro Processing Activities of Loqs and Dicer-1(A) Preparation of pre-miR-ban. Uniformly labeled pri-miR-ban was incubated with S2 nuclear lysate for the processing. The resultant pre-miR-ban
fragment (pre-miR-ban) was gel-purified and used as a substrate in the pre-miRNA processing assays.
(B) In vitro processing of pre-miR-ban using S2 cytoplasmic lysates. Cytoplasmic lysates were prepared 4 d after the initial exposure of S2 cells to theindicated target genes (as in Figure 2A) and used for in vitro processing. The gel-purified pre-miR-ban was incubated in cytoplasmic lysates for 1 h.
‘‘cyto. lysate’’ indicates parental S2 cytoplasmic lysate that shows activity for generating mature miR-ban from the gel-purified pre-miR-ban in (A).
(C) In vitro processing of pre-miR-ban using immunopurified Dicer-1 and Loqs complexes. Purified Flag–Dicer-1 (Flag-Dcr-1) and Flag–Loqs complexes
were incubated with pre-miR-ban for 2 h and tested for processing activity. ‘‘’’ shows the activity of a negative control prepared from parental S2 cells.
(D) The pre-miRNA processing activity of Flag–Dicer-1 complex in the presence and absence of magnesium ions. Purified Flag–Dicer-1 complex was
incubated with pre-miR-ban with or without magnesium ions in buffers. Addition of EDTA caused the abolition of the activity.
DOI: 10.1371/journal.pbio.0030235.g005

the precursor form of miR-ban (Figure 7A). In contrast, theprecursor 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 premiR-ban in the Loqs complex (Figure 7A). This may suggest
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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 associatewith both pre- and mature miRNAs in vivo.
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Figure 6. Loqs Stimulates the Specific Processing of Pre-miRNA by Dicer-1(A) Flag–Dicer-1 complex was purified under a harsh condition. Protein extract was prepared from S2 cells that expressed both full-length myc-tagged
Loqs (myc-Loqs) and Flag-tagged Dicer-1 (Flag-Dcr-1). The amounts of Loqs in Flag–Dicer-1 complexes prepared under high-salt condition and low-saltcondition were examined by Western blotting using anti-myc antibody, which show that less Loqs was co-purified with Dicer-1 in the high-saltcondition.
(B) The miRNA processing activities of Flag–Dicer-1 (Flag-Dcr-1) complexes in (A). Flag–Dicer-1 complex containing less Loqs showed a lower activity forthe processing.
(C) Recombinant Loqs stimulates the in vitro processing of pre-miR-ban by Flag–Dicer-1 complex purified in high-salt condition. 100 ng of purified GST
or GST–Loqs (see Figure 3C) were supplemented for the processing activity by Flag–Dicer-1 complex. GST–Loqs by itself does not show any pre-miRNAprocessing activity.
(D) GST–Loqs inhibits the siRNA-generating activity of Dicer-1. Uniformly labeled long dsRNA was incubated with Flag–Dicer-1 (Flag-Dcr-1) complex
purified in high-salt condition in (A) with or without GST–Loqs. The Flag–Dicer-1 complex by itself showed a considerable activity of generating siRNAfrom long dsRNA. Addition of GST–Loqs, but not GST, inhibited the processing. Note that the Flag–Dicer-1 (Flag-Dcr-1) complex does not contain Dicer2 (Dcr-2), judged by Western blot analysis using anti-Dicer-2 antibodies (right panel).
DOI: 10.1371/journal.pbio.0030235.g006

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

using anti-Dicer-1, anti-AGO1, or anti-Flag (for Loqs detection) antibodies. Not only Dicer-1 but also Loqs was detectedin 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 anothercomplex that contains AGO1 and Loqs but not Dicer-1. The
pre-miRNA processing activity of the AGO1 complex was thenexamined. 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,

We have previously shown that Argonaute protein AGO1 isrequired for stable production of mature miRNAs andassociates 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 premiRNA 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
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Figure 7. Dicer-1–Loqs Complexes Are Associated with Pre-miRNA and Mature miRNA In Vivo
(A) Northern blot analyses show that Flag–Dicer-1 (Flag-Dcr-1) complex contains both pre- and mature form of miR-ban. Notably, the precursor form of
miR-ban was accumulated in the Dicer-1 complex. In the case of the Loqs complex, the precursor apparently accumulated within the complex when the
Flag–Loqs complex was prepared in the presence of EDTA that was shown to inhibit the pre-miRNA processing activity in Figure 5D.
(B) AGO1 associates with Dicer-1 and Loqs. IgG-bound fractions prepared from S2 cells expressing AGO1–TAP, EGFP–TAP, or the parental S2 cells (),were subjected to Western blotting using antibodies against Dicer-1, AGO1, and Flag (for Loqs). Dicer-1 is not present in AGO2-associated complex.
(C) In vitro processing of pre-miR-ban using affinity-purified AGO1 complexes. Purified AGO1–TAP or AGO2–TAP complexes were incubated with premiR-ban (precursor) and tested for processing activity. ‘‘’’ shows the activity of a negative control prepared from parental S2 cells.
DOI: 10.1371/journal.pbio.0030235.g007

siRNA generating activity of Dicer-2 is not dependent upon
R2D2. However, the resultant siRNAs are not effectively
channeled into RISC in the absence of R2D2. The Dicer-2–
R2D2 complex, but not Dicer-2 alone, binds to siRNA, whichindicates that siRNA binding by the heterodimer is importantfor RISC entry [59,66]. In the case of Loqs, this protein alone is
not capable of converting pre-miRNAs into mature miRNAs,but it clearly stimulates and directs the speciﬁc pre-miRNAprocessing activity of Dicer-1. Furthermore, knocking down
Loqs markedly reduced the pre-miRNA processing activity incytoplasmic lysates in vitro (see Figures 4C and 5B), but did
not cause a signiﬁcant reduction of the level of Dicer-1
protein (see Figure 2B); implying that Dicer-1 may largely
depend on Loqs for its pre-miRNA processing activity. Thus,
the molecular role of Loqs for Dicer-1 is not simply similar tothat of R2D2 for Dicer-2.
It can be envisioned that Loqs may have one of several
roles in pre-miRNA processing. Dicer-1 contains only one
dsRBD, which may not be sufﬁcient for strong interaction
with and/or speciﬁc recognition of the pre-miRNA substrate(see Figure 6C and 6D). Loqs, containing three dsRBDs withno other identiﬁable domains being apparent, could provide

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, andsuggested that the resultant mature miRNAs are loaded onto
an AGO1-associated complex, which probably is miRNAassociated RISC [60], through speciﬁc interaction of AGO1
with Dicer-1 and Loqs.

Discussion
Our results indicate that Loqs and Dicer-1 form a complex
that converts pre-miRNAs into mature miRNAs; so how dothey act together in pre-miRNA processing? Sequence comparison reveals that Loqs is a paralog of R2D2 (see Figure 1).
Therefore, Loqs may play the molecular role of R2D2 for
Dicer-1. R2D2 forms a stable heterodimeric complex with
Dicer-2, while either protein alone seems to be unstable invivo [59]. In the absence of R2D2, Dicer-2 is still capable ofefﬁciently processing long dsRNA into siRNAs. Therefore, the
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Pre-miRNA Processing by Dicer-1–Loqs Complex
Hybond-Nþ membrane (Amersham Bioscience, Little Chalfont,
United Kingdom). After UV-crosslinking, the hybridization was
performed at 42 8C in 0.2 M sodium phosphate (pH 7.2), 7 % SDS,
and 1 mM EDTA with end-labeled antisense oligodeoxynucleotide,
and washed at 42 8C in 23 saline sodium citrate and 0.1% SDS.
Oligodeoxynucleotides used as probes were: bantam, 59-CAGCTTTCAAAATGATCTCAC-39; miR-8, 59-GACATCTTTACCTGACAGTATTA-39; U6 snRNA, 59-GGGCCATGCTAATCTTCTCTGTA-39;
and let-7, 59-AACTATACAACCTACTACCTCA-3.9 The blots were
exposed on BAS-MS2040 imaging plates, and signals were quantiﬁedusing BAS-2500 (Fuji, Tokyo, Japan).
RT-PCR analysis. One lg 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 theoligonucleotide primers for RT-PCR were: Dicer-1, 59-ACCAATGTACTGCGTTTG CA a nd 59-GTTTGCTGATCACAGAACTTAACGTT; Loqs, 59-ATGGACCAGGAGAATTTCCACGG-39 and 59CTACTTCTTGGTCATGATCTTCAAGTAC-39; and AGO2, 59-GCACAAGTGTGCGGTCTTGTATT-39 and 59-GTGAACTGCTTAATGCATTG-39.
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 lg/ml RNaseA for 30 min and thenstained with 0.4 lg/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 33 Flag-tagged Dicer-1 or Loqs, or myctagged-Loqs under the control of metallothionein promoter (originally from pRmHa-3 vector), were established. The expression of eachprotein was induced by adding copper ions into the medium. Afterovernight 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 lg/ml Leupeptin, 2 lg/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 8C for 1 h. Immunoprecipitated proteins were then recovered with
23SDS sample buffer or Elution buffer (Buffer A containing 400 lg/ml
of 33 Flag peptides, 10% glycerol, and 100 mM KOAc). For Northernblot analysis of RNAs co-puriﬁed with Flag–Dicer-1 or Flag–Loqs,
immunoprecipitates on beads were treated with ISOGEN andsubjected to RNA puriﬁcation.
Protein–protein interaction assays. To produce [35S]methioninelabeled proteins by a TNT in vitro transcription and translation kit(Promega, Madison, Wisconsin, United States), the Dicer-1 cDNA wasinserted 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 3
108 cells/ml into Hypotonic buffer (Buffer A without KOAc) and lysedby passing through a 30G needle. After centrifugation at 500 3 g for
20 min, the supernatant and the precipitate were separated. The
supernatant was centrifuged to obtain the supernatant as acytoplasmic 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: 59-CGCTCAGATGCAGATGTTGTTGAT-39 and 59-GATCGGTCGGCATAAGTTCAAAGC-39) and cloned into the SmaI site of pBluescriptSK
vector in the same direction with the T3 promoter. The plasmid wasdigested with ClaI, gel-isolated, and used as a template for in vitrotranscription reaction with MEGAscript T3 Kit (Ambion, Austin,
Texas, United States) in the presence of [a32P]GTP. In vitro
processing reaction of pri-miRNAs was performed with some

Figure 8. Domain Structure of Loqs and Its Homo sapiens and Xenopus
Homologs
The dsRNA-binding motif (dsRBD) is indicated as a black box. These
proteins contain three putative dsRBDs. Loqs shares ;34% amino acid
identity with TRBP and PACT and ;31% identity with Xlrbpa (Xenopus
laevis RNA-binding protein A). Sequence comparison between Loqs andits human and Xenopus homologs also showed a higher degree of aminoacid conservation in dsRBDs including C-terminal non-canonical dsRBDs.
The Xenopus homolog, Xlrbpa, of TRBP/PACT has been found toassociate with ribosomes in the cytoplasm [77], as is the case for many
RNAi factors including miRNAs [47,7882].
DOI: 10.1371/journal.pbio.0030235.g008

the additional RNA-binding modules required for speciﬁc
recognition of the pre-miRNA, and thereby stabilize premiRNA binding for Dicer-1. Loqs could also organize bindingof Dicer-1 on the pre-miRNA, contributing to the speciﬁc
positioning of the Dicer-1 cleavage site. Alternatively, since
dsRBDs are known to not only bind dsRNAs but also mediate
protein–protein interactions [67], Loqs may directly bind
Dicer-1 through its dsRBDs. This protein–protein interaction
may trigger a conformational change of Dicer-1 thatfacilitates either the formation of an intramolecular dimerof its two RNase III domains [50,68], which creates a pair ofcatalytic sites, or the handover of the Dicer-1 cleaved mature
miRNAs to the RISC.
Sequence analysis revealed that protein activator ofprotein kinase dsRNA dependent (PKR) (PACT) [69] andHIV TAR RNA binding protein (TRBP) [70] in mammals bear
34% identity to Loqs, and share a highly similar domain
structure with it (Figure 8). Both PACT and TRBP are thought
to play a role in the regulation of translation throughmodulating PKR that also contains two dsRBDs [71–73].
PACT interacts with PKR and enhances the autophosphorylation of PKR [67], which in turn, phosphorylates the asubunit of eukaryotic translation initiation factor 2 (eIF2a)and leads to an inhibition of mRNA translation in response toviral infection and other stimuli. TRBP prevents PKRmediated inhibition of protein synthesis through binding to
PKR [74]. Considered together, it will be important to ﬁnd
out Loqs’ partners other than Dicer-1 for possible involvement of Loqs in miRNA-mediated translational regulation
in Drosophila.

Materials and Methods
RNAi. dsRNAs were introduced to S2 cells by soaking essentially as
described [75]. Brieﬂy, approximately 5 3 106 cells were soaked in
1 ml of serum-free medium containing 15 lg of dsRNA for 30 min atroom 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 lg of total RNA wasseparated on 12% acrylamide-denaturing gel and transferred onto
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Pre-miRNA Processing by Dicer-1–Loqs Complex
modiﬁcations to previously reported method [36]. Brieﬂy, in 1 ml
reaction, 10 mM creatine phosphate, 0.5 mM ATP, 30 lg/ml creatine
kinase, 0.1 U/ul RNasin, 0.1 lg yeast RNA, and 500 ll nuclear lysate
were added, and pri-miR-bantam in 0.53 Buffer A with 100 mM KOAc
was further added to the mixture. After 2 h incubation at 26 8C, 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 invitro pre-miRNA processing with cytoplasmic lysates was the same asthat for the in vitro pri-miR-bantam processing. Cytoplasmic lysate
used in this assay was 5 ll in a 10-ll 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 Mgþþ-depletion assay, 10 mM EDTA
was added instead of Mgþþ. For the processing by Flag–Dicer-1, highsalt puriﬁed (800 mM KOAc) Flag–Dicer-1 was added in the presenceor 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 wasincubated 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 lg/ml creatine kinase, and 0.1 U/ul RNasin. Reactions were allowed
to proceed for 3 h at 26 8C. Cleavage products of the RNAi reaction
were analyzed by electrophoresis on 4% denaturing polyacrylamidegels.
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 andassociated 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 purchasedfrom Abcam (Cambridge, United Kingdom).

Supporting Information
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).

Acknowledgments
We thank members of the Siomi laboratory for discussions andcomments on the manuscript. KS was supported in part by afellowship from the Fragile X Research Foundation (FRAXA). AI is a
predoctoral fellow (DC1) of the Japan Society for the Promotion of
Science (JSPS). This work was supported by grants to MCS and HS
from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan (MEXT) and the JSPS.
Competing interests. The authors have declared that no competinginterests exist.
Author contributions. KS, AI, HS, and MCS conceived anddesigned the experiments. KS, AI, and MCS performed the experiments. KS, AI, HS, and MCS analyzed the data. HS, KS, AI, and MCS&
wrote the paper.

References
1. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic genelin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:
843–854.
2. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, et al. (2000) The
21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis
elegans. Nature 403: 901–906.
3. Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrowlike mRNA targets directed by a class of Arabidopsis miRNA. Science 297:
2053–2056.
4. Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM (2003) bantam
encodes a developmentally regulated microRNA that controls cellproliferation and regulates the proapoptotic gene hid in Drosophila. Cell
113: 25–36.
5. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, et al. (2003) Control ofleaf morphogenesis by microRNAs. Nature 425: 257–263.
6. Xu P, Vernooy SY, Guo M, Hay BA (2003) The Drosophila microRNA Mir-14
suppresses cell death and is required for normal fat metabolism. Curr Biol
13: 790–795.
7. Johnston RJ, Hobert O (2003) A microRNA controlling left/right neuronal
asymmetry in Caenorhabditis elegans. Nature 426: 845–849.
8. Chen CZ, Li L, Lodish HF, Bartel DP (2004) MicroRNAs modulate
hematopoietic lineage differentiation. Science 303: 83–86.
9. Yekta S, Shih IH, Bartel DP (2004) MicroRNA-directed cleavage of HOXB8
mRNA. Science 304: 594–596.
10. Carrington JC, Ambros V (2003) Role of microRNAs in plant and animal
development. Science 301: 336–338.
11. Bartel DP (2004) MicroRNAs: Genomics, biogenesis, mechanism, andfunction. Cell 116: 281–297.
12. Baulcombe D (2004) RNA silencing in plants. Nature 431: 356–363.
13. Meister G, Tuschl T (2004) Mechanisms of gene silencing by doublestranded RNA. Nature 431: 343–349.
14. Sontheimer EJ (2005) Assembly and function of RNA silencing complexes.
Nat Rev Mol Cell Biol 6: 127–138.
15. Tomari Y, Zamore PD (2005) Perspective: Machines for RNAi. Genes Dev
19: 517–529.
16. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, et al.
(2000) Conservation of the sequence and temporal expression of let-7
heterochronic regulatory RNA. Nature 408: 86–89.
17. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identiﬁcationof novel genes coding for small expressed RNAs. Science 294: 853–858.
18. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T(2002) Identiﬁcation of tissue-speciﬁc microRNAs from mouse. Curr Biol
12: 735–739.

PLoS Biology | www.plosbiology.org

19. Lagos-Quintana M, Rauhut R, Meyer J, Borkhardt A, Tuschl T (2003) New
microRNAs from mouse and human. RNA 9: 175–179.
20. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny
RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:
858–862.
21. Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis
elegans. Science 294: 862–864.
22. Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, et al. (2002)
miRNPs: A novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 16: 720–728.
23. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002)
MicroRNAs in plants. Genes Dev 16: 1616–1626.
24. Ambros V, Lee RC, Lavanway A, Williams PT, Jewell D (2003) MicroRNAs
and other tiny endogenous RNAs in C. elegans. Curr Biol 13: 807–818.
25. Aravin AA, Lagos-Quintana M, Yalcin A, Zavolan M, Marks D, et al. (2003)
The small RNA proﬁle during Drosophila melanogaster development. Dev Cell
5: 337–350.
26. Dostie J, Mourelatos Z, Yang M, Sharma A, Dreyfuss G (2003) Numerous
microRNPs in neuronal cells containing novel microRNAs. RNA 9: 180–
186.
27. Grad Y, Aach J, Hayes GD, Reinhart BJ, Church GM, et al. (2003)
Computational and experimental identiﬁcation of C. elegans microRNAs.
Mol Cell 11: 1253–1263.
28. Houbaviy HB, Murray MF, Sharp PA (2003) Embryonic stem cell-speciﬁc
MicroRNAs. Dev Cell 5: 351–358.
29. Lai EC, Tomancak P, Williams RW, Rubin GM (2003) Computational
identiﬁcation of Drosophila microRNA genes. Genome Biol 4: R42.
30. Lim LP, Lau NC, Weinstein EG, Abdelhakim A, Yekta S, et al. (2003) The
microRNAs of Caenorhabditis elegans. Genes Dev 17: 991–1008.
31. Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RHA, et al.
(2005) Phylogenetic shadowing and computational identiﬁcation of humanmicroRNA genes. Cell 120: 21–24.
32. John B, Enright AJ, Aravin A, Tuschl T, Sander C, et al. (2004) Human
microRNAs targets. PLoS Biol 2: e363.
33. Brennecke J, Stark A, Russell RB, Cohen SM (2005) Principles of
microRNA-target recognition. PLoS Biol 3: e85.
34. Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, et al. (2005)
Microarray analysis shows that some microRNAs downregulate large
numbers of target mRNAs. Nature 433: 769–773.
35. Lee Y, Kim M, Han J, Yeom KH, Lee S, et al. (2004) MicroRNA genes aretranscribed by RNA polymerase II. EMBO J 23: 4051–4060.
36. Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation:
Stepwise processing and subcellular localization. EMBO J 21: 4663–4670.
37. Lee Y, Ahn C, Han J, Choi H, Kim J, et al. (2003) The nuclear RNase III
Drosha initiates microRNA processing. Nature 425: 415–419.

0010

July 2005 | Volume 3 | Issue 7 | e235

Pre-miRNA Processing by Dicer-1–Loqs Complex
Argonaute proteins in small RNA-directed cleavage pathways. Genes Dev
18: 1655–1666.
61. Boutros M, Kiger AA, Armknecht S, Kerr K, Hild M, et al. (2004) Genomewide RNAi analysis of growth and viability in Drosophila cells. Science 303:
832–835.
62. Lasko P (2000) The Drosophila melanogaster genome: Translation factors andRNA binding proteins. J Cell Biol 150: F51–F56.
63. Tabara H, Yigit E, Siomi H, Mello CC (2002) The dsRNA binding protein
RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct
RNAi in C. elegans. Cell 109: 861–871.
64. Schwarz DS, Tomari Y, Zamore PD (2004) The RNA-induced silencing
complex is a Mg2þ-dependent endonuclease. Curr Biol 14: 787–791.
65. Nicholson AW (2003) The ribonuclease III superfamily: Forms andfunctions in RNA, maturation, decay, and gene silencing. In: Hannon
GJeditor. RNAi: A guide to gene silencing. Cold Spring Harbor Laboratory
Press, Plainview (New York): pp. 149–174.
66. Tomari Y, Matranga C, Haley B, Martinez N, Zamore PD (2004) A protein
sensor for siRNA asymmetry. Science 306: 1377–1380.
67. Peters GA, Hartmann R, Qin J, Sen GC (2001) Modular structure of PACT:
Distinct domains for binding and activating PKR. Mol Cell Biol 21: 1908–
1920.
68. Zhang H, Kolb F, Jaskiewicz L, Westhof E, Filipowicz W (2004) Single
processing center models for human Dicer and bacterial RNase III. Cell
118: 57–68.
69. Patel RC, Sen GC (1998) PACT, a protein activator for the interferoninduced protein kinase, PKR. EMBO J 17: 4379–4390.
70. Gatignol A, Buckler-White A, Berkhout B, Jeang KT (1991) Characterization of a human TAR RNA-binding protein that activates the HIV-1
LTR. Science 251: 1597–1600.
71. Fierro-Monti I, Mathews MB (2000) Proteins binding to duplexed RNA:One motif, multiple functions. Trends Biochem Sci 25: 241–246.
72. Kaempfer R (2003) RNA sensors: Novel regulators of gene expression.
EMBO Rep 4: 1043–1047.
73. Saunders LR, Barber GN (2003) The dsRNA binding protein family: Criticalroles, diverse cellular functions. FASEB J 17: 961–983.
74. Park H, Davies MV, Langland JO, Chang HW, Nam YS, et al. (1994) TAR
RNA-binding protein is an inhibitor of the interferon-induced protein
kinase PKR. Proc Natl Acad Sci U S A 91: 4713–4717.
75. Clemens JC, Worby CA, Simonson-Leff N, Muda M, Maehama T, et al.
(2000) Use of double-stranded RNA interference in Drosophila cell lines todissect signal transduction pathways. Proc Natl Acad Sci U S A 97: 6499–
6503.
76. Kataoka Y, Takeichi M, Uemura T (2001) Developmental roles andmolecular characterization of a Drosophila homologue of Arabidopsis
Argonaute1, the founder of a novel gene superfamily. Genes Cells 6: 313–325.
77. Eckmann CR, Jantsch MF (1997) Xlrbpa, a double-stranded RNA-binding
protein associated with ribosomes and heterogeneous nuclear RNPs. J Cell
Biol 138: 239–253.
78. Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ (2001)
Argonaute2, a link between genetic and biochemical analyses of RNAi.
Science 293: 1146–1150.
79. Ishizuka A, Siomi MC, Siomi H (2002) A Drosophila fragile X protein
interacts with components of RNAi and ribosomal proteins. Genes Dev 16:
2497–2508.
80. Djikeng A, Shi H, Tschudi C, Shen S, Ullu E (2003) An siRNAribonucleoprotein is found associated with polyribosomes in Trypanosoma
brucei. RNA 9: 802–808.
81. Kim J, Krichevsky A, Grad Y, Hayes GD, Kosik KS, et al. (2004)
Identiﬁcation of many microRNAs that copurify with polyribosomes inmammalian neurons. Proc Natl Acad Sci U S A 101: 360–365.
82. Pham JW, Pellino JL, Lee YS, Carthew RW, Sontheimer EJ (2004) A Dicer-2dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila.
Cell 117: 83–94.

38. Basyuk E, Suavet F, Doglio A, Bordonne R, Bertrand E (2003) Human let-7
stem-loop precursors harbor features of RNase III cleavage products.
Nucleic Acids Res 31: 6593–6597.
39. Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear
export of pre-microRNAs and short hairpin RNAs. Genes Dev 17: 3011–
3016.
40. Bohnsack MT, Czaplinski K, Gorlich D (2004) Exportin 5 is a RanGTPdependent dsRNA-binding protein that mediates nuclear export of premiRNAs. RNA 10: 185–191.
41. Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U (2004) Nuclear exportof microRNA precursors. Science 303: 95–98.
42. Hutvagner G, McLachlan J, Pasquinelli AE, Balint E, Tuschl T, et al. (2001) A
cellular function for the RNA-interference enzyme Dicer in the maturationof the let-7 small temporal RNA. Science 293: 834–838.
43. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, et al. (2001) Genes andmechanisms related to RNA interference regulate expression of the small
temporal RNAs that control C. elegans developmental timing. Cell 106: 23–
34.
44. Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, et al. (2001) Dicer
functions in RNA interference and in synthesis of small RNA involved indevelopmental timing in C. elegans. Genes Dev 15: 2654–2659.
45. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, et al. (2003) Asymmetry in
the assembly of the RNAi enzyme complex. Cell 115: 199–208.
46. Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi
enzyme complex. Science 297: 2056–2060.
47. Olsen PH, Ambros V (1999) The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein
synthesis after the initiation of translation. Dev Biol 216: 671–680.
48. Tomari Y, Zamore PD (2005) MicroRNA biogenesis: Drosha can’t cut it
without a partner. Curr Biol 15: R61–64.
49. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processingof primary microRNAs by the Microprocessor complex. Nature 432: 231–
235.
50. Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, et al. (2004)
The Microprocessor complex mediates the genesis of microRNAs. Nature
432: 235–240.
51. Han J, Lee Y, Yeom KH, Kim YK, Jin H, et al. (2005) The Drosh-DGCR8complex in primary microRNA processing. Genes Dev 18: 3016–3027.
52. Landthaler M, Yalcin A, Tuschl T (2004) The human DiGeorge syndrome
critical region gene 8 and its D. melanogaster homolog are required formiRNA biogenesis. Curr Biol 15: 2162–2167.
53. Papp I, Mette MF, Aufsatz W, Daxinger L, Schauer SE, et al. (2003) Evidence
for nuclear processing of plant micro RNA and short interfering RNA
precursors. Plant Physiol 132: 1382–1390.
54. Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis throughDicer-like 1 protein functions. Proc Natl Acad Sci U S A 101: 12753–12758.
55. Vazquez F, Gasciolli V, Crete P, Vaucheret H (2004) The nuclear dsRNA
binding protein HYL1 is required for microRNA accumulation and plantdevelopment, but not posttranscriptional transgene silencing. Curr Biol 14:
346–351.
56. Han MH, Goud S, Song L, Fedoroff N (2004) The Arabidopsis doublestranded RNA-binding protein HYL1 plays a role in microRNA-mediatedgene regulation. Proc Natl Acad Sci U S A 101: 1093–1098.
57. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for abidentate ribonuclease in the initiation step of RNA interference. Nature
409: 363–366.
58. Lee YS, Nakahara K, Pham JW, Kim K, He Z, et al. (2004) Distinct roles for
Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways.
Cell 117: 69–81.
59. Liu Q, Rand TA, Kalidas S, Du F, Kim HE, et al. (2003) R2D2, a bridge
between the initiation and effector steps of the Drosophila RNAi pathway.
Science 301: 1921–1925.
60. Okamura K, Ishizuka A, Siomi H, Siomi MC (2004) Distinct roles for

PLoS Biology | www.plosbiology.org

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