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

Abstract

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 from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Here we show that Drosophila Dicer-1 interacts with Loquacious, a double-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 that along 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.

Full text

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 from
primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to
release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and
further processed by Dicer to mature miRNAs. Here we show that Drosophila Dicer-1 interacts with Loquacious, a
double-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 that
along 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.
Introduction
microRNAs (miRNAs) act as RNA guides by binding to
complementary sites on target mRNAs to regulate gene
expression at the post-transcriptional level in plants and
animals [112], much as small interfering RNAs (siRNAs) do
in the RNA interference (RNAi) pathway [1315]. The
expression of miRNAs is often developmentally regulated in
a 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 pre-
dicted 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 (pri-
miRNA) transcripts by RNA polymerase II [35]. miRNA
maturation 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 to
the cytoplasm by the protein Exportin 5, which recognizes the
two-nucleotide 39overhang that is a signature of RNase III-
mediated cleavage [39–41]. In the cytoplasm, pre-miRNAs are
subsequently cleaved by a second RNase III enzyme, Dicer,
into approximately 22-nucleotide miRNA duplexes, with an
end 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], which
mediates 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 the
RNAi pathway [48]. Both Drosha and Dicer contain dsRNA-
binding domains (dsRBDs). Drosha requires a dsRNA-binding
protein partner known as Pasha in flies and Caenorhabditis
elegans, and its ortholog DGCR8 in mammals to convert pri-
miRNAs to pre-miRNAs [49–52]. In plants, the predominantly
nuclear Dicer-like-1, equipped with two dsRBDs, is thought to
catalyze both pri-miRNA and pre-miRNA processing [53,54].
The HYL1 protein, which also contains a tandem dsRBD, is
required 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 of
siRNAs [57,58], and forms a heterodimeric complex with the
dsRNA-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 is
associated 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 the
dsRBD protein Loquacious (Loqs). Depletion of Loqs results
in accumulation of pre-miRNAs in Drosophila S2 cells. Loqs is
Received March 16, 2005; Accepted April 30, 2005; Published May 24, 2005
DOI: 10.1371/journal.pbio.0030235
Copyright: Ó2005 Saito et al. This is an open-access article distributed under the
terms 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, double-
stranded RNA; EGFP, enhanced green fluorescent protein; GST, glutathione S-
transferase; IgG, immunoglobulin G; miRNA, microRNA; PACT, protein activator of
PKR; PKR, protein kinase dsRNA dependent; pre-miRNA, precursor miRNA; pri-
miRNA, primary miRNA; RISC, RNA-induced silencing complex; RNAi, RNA
interference; siRNA, small interfering RNA; TRBP, TAR RNA binding protein.
Academic Editor: James C. Carrington, Oregon State University, United States of
America
*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.
PLoS Biology | www.plosbiology.org July 2005 | Volume 3 | Issue 7 | e2351202
Open access, freely available online
P
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BIOLOGY

predominantly cytoplasmic and is conserved in mammals.
Immuno-affinity purification experiments, together with the
use of recombinant Loqs, reveal that along with Dicer-1, Loqs
resides in a functional pre-miRNA processing complex, and
stimulates 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
We have used RNAi-based reverse-genetic methods [61] to
screen 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 (two
canonical dsRBDs at the N-terminal half, and one non-
canonical dsRBD at the C-terminal), originally dubbed
CG6866 (candidate gene 6866), which has a role in pre-
miRNA 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 (Fo
¨rstemann K, et al. DOI: 10.1371/journal.
pbio.0030236). Therefore, CG6866 was designated as Loqua-
cious (‘‘very talkative’’ ).
Depletion of Loqs and Dicer-1 by RNAi Results
in 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 predom-
inantly 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 simulta-
neously 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 precip-
itates 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 inter-
acted 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
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).
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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
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). After
normalization for loading, the relative ratios for pre-miRNA (pre-miR-ban) (left panel) and mature miRNA (miR-ban) (right panel) were calculated and
normalized 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 each
protein. 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 to
S2 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).
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Pre-miRNA Processing by Dicer-1–Loqs Complex

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 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
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 the
materials obtained after immunoprecipitation from the extracts with anti-Flag were run on an SDS-polyacrylamide gel. Western blots were prepared
and 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 an
antibody 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 co-
immunoprecipitated 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.
35
S-labeled Dicer-1 was produced by an in vitro transcription and translation system
in the presence of [
35
S]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
35
S visualized by
autoradiography. 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
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Pre-miRNA Processing by Dicer-1–Loqs Complex

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 in
Figure 6D). GST–Loqs alone showed no activity for generat-
ing 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
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 RNA
polymerase in vitro (T7-pre-let-7). After gel purification, it was incubated with S2 cytoplasmic lysate, and resultant RNA was subjected to Northern
blotting using an oligodeoxynucleotide recognizing both pre-let-7 and mature let-7. Total RNA prepared from pupa was also applied (pupa), which
shows 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 the
lysate.
(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 T7-
pre-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.
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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, there-
fore, may only transiently interact with them. Nonetheless,
these results suggest that Dicer-1–Loqs complexes associate
with both pre- and mature miRNAs in vivo.
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 the
indicated 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.
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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 detec-
tion) 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,
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-salt
condition were examined by Western blotting using anti-myc antibody, which show that less Loqs was co-purified with Dicer-1 in the high-salt
condition.
(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 for
the 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-miRNA
processing 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 siRNA
from 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 Dicer-
2 (Dcr-2), judged by Western blot analysis using anti-Dicer-2 antibodies (right panel).
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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.
Discussion
Our results indicate that Loqs and Dicer-1 form a complex
that converts pre-miRNAs into mature miRNAs; so how do
they act together in pre-miRNA processing? Sequence com-
parison 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 in
vivo [59]. In the absence of R2D2, Dicer-2 is still capable of
efficiently processing long dsRNA into siRNAs. Therefore, the
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, which
indicates that siRNA binding by the heterodimer is important
for 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-miRNA
processing activity of Dicer-1. Furthermore, knocking down
Loqs markedly reduced the pre-miRNA processing activity in
cytoplasmic 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 to
that 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 with
no other identifiable domains being apparent, could provide
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 pre-
miR-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
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Pre-miRNA Processing by Dicer-1–Loqs Complex

the additional RNA-binding modules required for specific
recognition of the pre-miRNA, and thereby stabilize pre-
miRNA binding for Dicer-1. Loqs could also organize binding
of 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 that
facilitates either the formation of an intramolecular dimer
of its two RNase III domains [50,68], which creates a pair of
catalytic sites, or the handover of the Dicer-1 cleaved mature
miRNAs to the RISC.
Sequence analysis revealed that protein activator of
protein kinase dsRNA dependent (PKR) (PACT) [69] and
HIV 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 through
modulating PKR that also contains two dsRBDs [71–73].
PACT interacts with PKR and enhances the autophosphor-
ylation of PKR [67], which in turn, phosphorylates the a
subunit of eukaryotic translation initiation factor 2 (eIF2a)
and leads to an inhibition of mRNA translation in response to
viral infection and other stimuli. TRBP prevents PKR-
mediated 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 involve-
ment 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 5 310
6
cells were soaked in
1 ml of serum-free medium containing 15 lg 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 lg of total RNA was
separated on 12% acrylamide-denaturing gel and transferred onto
Hybond-Nþmembrane (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 8Cin23saline sodium citrate and 0.1% SDS.
Oligodeoxynucleotides used as probes were: bantam, 59-CAGCTTT-
CAAAATGATCTCAC-39;miR-8,59-GACATCTTTACCTGACAG-
TATTA-39;U6snRNA,59-GGGCCATGCTAATCTTCTCTGTA-39;
and let-7, 59-AACTATACAACCTACTACCTCA-3.9The blots were
exposed on BAS-MS2040 imaging plates, and signals were quantified
using 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 (Stra-
tagene, La Jolla, California, United States). Sequences of the
oligonucleotide primers for RT-PCR were: Dicer-1, 59-ACCAATG-
TACTGCGTTTGCA and 59-GTTTGCTGATCACAGAACT-
TAACGTT; Loqs, 59-ATGGACCAGGAGAATTTCCACGG-39and 59-
CTACTTCTTGGTCATGATCTTCAAGTAC-39; and AGO2, 59-GCA-
CAAGTGTGCGGTCTTGTATT-39and 59-GTGAACTGCTTAATG-
CATTG-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 then
stained 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 33Flag-tagged Dicer-1 or Loqs, or myc-
tagged-Loqs under the control of metallothionein promoter (origi-
nally 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,
2lg/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
48C for 1 h. Immunoprecipitated proteins were then recovered with
23SDS sample buffer or Elution buffer (Buffer A containing 400 lg/ml
of 33Flag 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 [
35
S]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 3
10
8
cells/ml into Hypotonic buffer (Buffer A without KOAc) and lysed
by passing through a 30G needle. After centrifugation at 500 3g 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 super-
natant 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-CGCTCA-
GATGCAGATGTTGTTGAT-39and 59-GATCGGTCGGCATAAG
TTCAAAGC-39) 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 [a
32
P]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 and
its human and Xenopus homologs also showed a higher degree of amino
acid conservation in dsRBDs including C-terminal non-canonical dsRBDs.
The Xenopus homolog, Xlrbpa, of TRBP/PACT has been found to
associate with ribosomes in the cytoplasm [77], as is the case for many
RNAi factors including miRNAs [47,7882].
DOI: 10.1371/journal.pbio.0030235.g008
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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 0.53Buffer 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 nucleo-
tides 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 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 concen-
tration of buffer adjusted. For Mg
þþ
-depletion assay, 10 mM EDTA
was added instead of Mg
þþ
. 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, 10
4
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 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 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).
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 and
comments on the manuscript. KS was supported in part by a
fellowship 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, and
Technology of Japan (MEXT) and the JSPS.
Competing interests. The authors have declared that no competing
interests exist.
Author contributions. KS, AI, HS, and MCS conceived and
designed the experiments. KS, AI, and MCS performed the experi-
ments. KS, AI, HS, and MCS analyzed the data. HS, KS, AI, and MCS
wrote the paper. &
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PLoS Biology | www.plosbiology.org July 2005 | Volume 3 | Issue 7 | e2351212
Pre-miRNA Processing by Dicer-1–Loqs Complex