Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

Molecular mechanisms that funnel RNA precursors into
endogenous small-interfering RNA and microRNA biogenesis
pathways in Drosophila
Keita Miyoshi, Tomohiro Miyoshi, Julia Verena Hartig, et al.
RNA 2010 16: 506-515 originally published online January 19, 2010
Access the most recent version at doi:10.1261/rna.1952110

Supplemental
Material
References
Email alerting
service

http://rnajournal.cshlp.org/content/suppl/2010/01/08/rna.1952110.DC1.html
This article cites 28 articles, 11 of which can be accessed free at:
http://rnajournal.cshlp.org/content/16/3/506.full.html#ref-list-1
Receive free email alerts when new articles cite this article - sign up in the box at the
top right corner of the article or click here

To subscribe to RNA go to:
http://rnajournal.cshlp.org/subscriptions

Copyright © 2010 RNA Society

Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

Molecular mechanisms that funnel RNA precursors
into endogenous small-interfering RNA and microRNAbiogenesis pathways in Drosophila
KEITA MIYOSHI,1,4 TOMOHIRO MIYOSHI,1,4 JULIA VERENA HARTIG,2 HARUHIKO SIOMI,1and MIKIKO C. SIOMI1,3
1

Keio University School of Medicine, Tokyo 160-8582, Japan
Gene Center, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Munich 81377, Germany
3
Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Saitama 332-0012, Japan
2

ABSTRACTIn Drosophila, three types of endogenous small RNAs—microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs or esiRNAs)—function as triggers in RNA silencing. Although piRNAs areproduced independently of Dicer, miRNA and esiRNA biogenesis pathways require Dicer1 and Dicer2, respectively. Recent
studies have shown that among the four isoforms of Loquacious (Loqs), Loqs-PB and Loqs-PD are involved in miRNA and esiRNA
processing pathways, respectively. However, how these Loqs isoforms function in their respective small RNA biogenesis
pathways remains elusive. Here, we show that Loqs-PD associates specifically with Dicer2 through its C-terminal domain. The
Dicer2–Loqs-PD complex contains R2D2, another known Dicer2 partner, and excises both exogenous siRNAs and esiRNAs fromtheir corresponding precursors in vitro. However, Loqs-PD, but not R2D2, enhanced Dicer2 activity. The Dicer2–Loqs-PD
complex processes esiRNA precursor hairpins with long stems, which results in the production of AGO2-associated small RNAs.
Interestingly, however, small RNAs derived from terminal hairpins of esiRNA precursors are loaded onto AGO1; thus, they areclassified as a new subset of miRNAs. These results suggest that the precursor RNA structure determines the biogenesis
mechanism of esiRNAs and miRNAs, thereby implicating hairpin structures with long stems as intermediates in the evolution of
Drosophila miRNA.
Keywords: esiRNA; Dicer2; Loquacious; miRNA; Drosophila

INTRODUCTIONIn RNA silencing, small RNAs of 20–30 nucleotides (nt)guide Argonaute proteins, the main catalytic factors involved
in RNA silencing, to the target RNAs to be silenced(Ghildiyal and Zamore 2009; Kim et al. 2009; Siomi andSiomi 2009). Endogenous small RNAs in animals canbe divided into three subgroups: microRNAs (miRNAs)(21–23 nt), PIWI-interacting RNAs (piRNAs) (24–30 nt),
and endogenous small interfering RNAs (endo-siRNAs or
esiRNAs) (z21 nt) (Ghildiyal and Zamore 2009; Kim et al.
2009; Siomi and Kuramochi-Miyagawa 2009; Siomi andSiomi 2009). miRNAs are encoded by their own genes and
4
These authors contributed equally to this work.
Reprint requests to: Mikiko C. Siomi, Keio University School of
Medicine, Tokyo 160-8582, Japan; e-mail: siomim@sc.itc.keio.ac.jp; fax: +813-5363-3266.
Article published online ahead of print. Article and publication date are at
http://www.rnajournal.org/cgi/doi/10.1261/rna.1952110.

506

are expressed ubiquitously (Ghildiyal and Zamore 2009;
Kim et al. 2009; Siomi and Siomi 2009). piRNAs are mainly
derived from intergenic repetitive regions of the genome,
including transposons and even non-transposon loci, which
are generically called piRNA clusters (Ghildiyal and Zamore
2009; Kim et al. 2009; Siomi and Kuramochi-Miyagawa
2009; Siomi and Siomi 2009). Unlike miRNAs, piRNAs areprimarily expressed in germline cells. esiRNAs also mainly
originate from transposons and repetitive genomic elements
but are not germline-specific. Thus, piRNAs and esiRNAs
are distinct from each other. They are also loaded onto
different Argonaute partners: piRNAs are associated with
germline-specific Argonaute members, the PIWI proteins,
whereas esiRNAs interact with ubiquitously expressed Argonaute (AGO) proteins. miRNAs are also loaded onto AGO
proteins (Ghildiyal and Zamore 2009; Kim et al. 2009; Siomi
and Kuramochi-Miyagawa 2009; Siomi and Siomi 2009).
The processing pathway of miRNAs has been extensively
studied (Bartel 2009; Ghildiyal and Zamore 2009; Kim et al.

RNA (2010), 16:506–515. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2010 RNA Society.

Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

esiRNA and miRNA biogenesis in Drosophila

2009; Siomi and Siomi 2009). In Drosophila, the primarytranscripts of miRNA genes, pri-miRNAs, are first processedin the nucleus into miRNA precursors, pre-miRNAs, by
a complex of Drosha, an RNase III enzyme, and Pasha,a double-stranded RNA-binding domain (dsRBD)-containing
protein (Denli et al. 2004). After export to the cytoplasm byExportin5 (Shibata et al. 2006; Okamura et al. 2007), premiRNAs are secondarily processed into mature miRNAs by
the Dicer1–Loqs complex (an RNase III enzyme and adsRBD-containing protein, respectively; Loqs is also known
as R3D1) (Lee et al. 2004; Förstemann et al. 2005; Jiang et al.
2005; Saito et al. 2005). Following this two-step maturation,
miRNAs are predominantly loaded onto AGO1 and exert
their function in regulating gene silencing (Okamura et al.
2004; Saito et al. 2005).
The esiRNAs in Drosophila are generated by Dicer2 andare loaded onto AGO2. In dicer2 mutants, the accumulation of esiRNAs is severely impaired, and some transposons
are abnormally overexpressed (Czech et al. 2008; Ghildiyal
et al. 2008; Kawamura et al. 2008; Okamura et al. 2008).
This strongly supports the idea that Dicer2 is involvedin the esiRNA processing pathway (Czech et al. 2008;
Ghildiyal et al. 2008; Kawamura et al. 2008; Okamura
et al. 2008). Dicer2, the other RNase III enzyme in
Drosophila, was originally shown to be required for the
production of exogenous siRNAs (exo-siRNAs) from long,completely annealing dsRNAs (Bernstein et al. 2001; Leeet al. 2004; Pham et al. 2004). During exo-siRNA processing, Dicer2 is associated with R2D2 (Liu et al. 2003), oneof the dsRBD-containing proteins. Without R2D2, Dicer2
is destabilized in vivo (Liu et al. 2003). However, using
in vitro assay systems, the exo-siRNA excising activity ofDicer2 was not affected by the addition or depletion of
R2D2 (Liu et al. 2003). Thus, R2D2 might only be necessary
for stabilizing Dicer2 in the exo-siRNA-producing pathway.
Like dicer2 mutants, loqs mutants fail to accumulate
esiRNAs in vivo, especially those originating from nontransposon-type precursors: long stem–loop structures containing many mismatch pairs in their stems (Ghildiyal et al.
2008; Okamura et al. 2008). This result suggests that Loqs isneeded for the esiRNA processing pathway, in addition to
its requirement in the miRNA processing pathway. The loqs
gene in Drosophila is known to give rise to four isoforms,
Loqs-PA to Loqs-PD (Drysdale et al. 2005; Förstemannet al. 2005; Hartig et al. 2009; Zhou et al. 2009). Loqs-PD isvery similar in size to Loqs-PC, and the sequences of LoqsPC and Loqs-PD differ only at the very C-terminal end.
Recent studies have indicated that Loqs-PB and Loq-PD are
involved in the miRNA and esiRNA processing pathways,
respectively (Hartig et al. 2009; Zhou et al. 2009). However,
how these two isoforms function in small RNA biogenesis
pathways remains elusive.
In this study, we found that the four Loqs isoforms,
Loqs-PA to Loqs-PD, associate differently with endogenous Dicer1 and Dicer2 and that the C-terminal region of

Loqs-PD determines the specific association with Dicer2.
The Dicer2–Loqs-PD complex is able to process esiRNAs,
but not miRNAs, from their corresponding precursors in in
vitro assay systems, while the Dicer1–Loqs-PA and Dicer1–
Loqs-PB complexes were able to process miRNA precursors, but not esiRNA precursors. The Dicer2–Loqs-PD
complex also contained R2D2 and thus showed an ability
to process long dsRNAs into siRNAs in vitro. However,Loqs-PD, but not R2D2, enhanced the esiRNA excision
activity of Dicer2. These results indicate that Dicer2 is able
to form a ternary complex with Loqs-PD and R2D2 in vivo
but that only Loqs-PD, associated with Dicer2, is required
to produce esiRNAs from their own precursors. Mutational
analyses of esiRNA long stem–loop-type precursors revealed that the Dicer1–Loqs-PB complex prefers to process
hairpins with stems of z22 nt, similar to miRNA precursors, whereas the Dicer2–Loqs-PD complex prefers toprocess precursors with longer stems. Indeed, small RNAs
derived from shorter esiRNA precursors, similar to the sizeof miRNA precursors, were found to be loaded onto AGO1;
thus, they are classified as an alternative source of miRNAtype regulatory RNAs, and we have termed them semiRNAs(siblings of esiRNA miRNAs). Together, these results suggest that the RNA precursor structure serves as the determinant for the esiRNA and miRNA processing pathways.
RESULTSInteraction of Loqs isoforms with Dicer1 and Dicer2
Loqs complexes were immunopurified from S2 cells using
anti-Loqs antibodies (Miyoshi et al. 2009). Silver stainingof the immunoprecipitates revealed that two prominent
proteins coimmunoprecipitated with Loqs isoforms Loqs-PA,
Loqs-PB, and Loqs-PC/PD (Fig. 1A; Förstemann et al.
2005; Hartig et al. 2009; Zhou et al. 2009). (Loqs-PB is also
known as R3D1-L, but hereafter we use Loqs-PB for
uniformity [Jiang et al. 2005].) Mass spectrometry analysis
revealed that the two Loqs-interacting proteins were Dicer1(z300 kDa) and Dicer2 (z200 kDa). Western blot analysis of the immunoprecipitates (Fig. 1A) confirmed theirassociations (Fig. 1B). While this work was in progress, twogroups reported that all Loqs isoforms interacted with exogenously expressed Dicer2 in S2 cells (Hartig et al. 2009;
Zhou et al. 2009). It was also found that Loqs-PB stronglyinteracted with Dicer1 whereas Loqs-PC and Loqs-PD did
not show such activity (Hartig et al. 2009; Zhou et al.
2009). Since we have monoclonal antibodies for Dicer1 andDicer2 (Miyoshi et al. 2009), we examined the binding
specificity of each Loqs isoform with endogenous Dicer1and Dicer2. myc-tagged Loqs-PA and Loqs-PB specificallyinteracted with endogenous Dicer1 (Fig. 1C). In contrast,
myc-tagged Loqs-PD showed a capacity to interact withendogenous Dicer2, but not with Dicer1 (Fig. 1C). Although there were some discrepancies between our results
www.rnajournal.org

507

Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

Miyoshi et al.

FIGURE 1. Interaction of Loqs isoforms with Dicer1 and Dicer2. (A) The Loqs-containing
complexes were immunoprecipitated from S2 cells using anti-Loqs antibodies and the proteins
contained in the immunoprecipitates were visualized by silver staining. The protein bands corresponding to Loqs-PA, Loqs-PB, and Loqs-PC/Loqs-PD are indicated. Two proteins coimmunopurified with Loqs were identified as Dicer1 and Dicer2 by mass spectrometry analyses. (n.i.)
Nonimmune IgG used as a negative control. (B) Western blot analyses using anti-Dicer1 and antiDicer2 antibodies confirmed that the immunoprecipitates obtained using anti-Loqs antibodies (panelA) contain both Dicer proteins, along with all Loqs isoforms. (C) Loqs-PA and Loqs-PB interact with
Dicer1, whereas Loqs-PD interacts with Dicer2. Each Loqs isoform was expressed in S2 cells, and theanti-myc immunoprecipitated complexes were probed with anti-Dicer1 and anti-Dicer2 antibodies.
(h.c.) Heavy chain of anti-c-myc antibody. (D) Deletion mutant analysis of Loqs-PD to determine thedomain necessary for interacting with Dicer2. Various deletion mutants of Loqs-PD (m1–m5) wereexpressed in S2 cells, and their interactions with endogenous Dicer2 were examined. The Loqs-PDm4 mutant interacted with Dicer2 as efficiently as wt Loqs-PD. The dsRNA-binding domains (lightgray boxes) and C-terminal region (dark gray boxes) found exclusively in Loqs-PD but not in other
Loqs isoforms, respectively, are indicated (upper diagram). The lower panel shows immunoblotting
results of the protein–protein interaction assays. EGFP was used as a negative control.

and the previous results of Hartig et al. (2009) and Zhou
et al. (2009), our findings are at least consistent with thefinding that only Loqs-PD is involved in the esiRNA processing pathway (see below). No Loqs-PC interaction with
508

RNA, Vol. 16, No. 3

Dicer1 or Dicer2 was detected, although
Loqs-PC and Loqs-PD differ only at their
C-terminal ends (Fig. 1C). Through immunofluorescence analyses using an antimyc antibody, we found that Loqs-PC
expressed in S2 cells localized predominantly in large foci in the cytoplasm(Supplemental Fig. 1), whereas other
Loqs isoforms, such as Loqs-PB, localized
evenly in the cytosol (Förstemann et al.
2005; Saito et al. 2005). This unique localization of Loqs-PC might be related tothe observation that little or no Loqs-PC
interacts with either Dicer1 or Dicer2.
We then determined the domain of
Loqs-PD that binds with Dicer2. A seriesof deletion mutants of Loqs-PD, m1 to
m5, was produced and expressed in
S2 cells as Flag-tagged peptides. LoqsPD-m2 to Loqs-PD-m5 were fused with
EGFP to attain a size similar to that ofwild-type (wt) Loqs-PD. Western blot
analysis using anti-Dicer2 antibodies
on anti-Flag immunoprecipitates showedthat the Loqs-PD-m4 mutant, which contains the second dsRBD and both flanking
regions, was able to interact with Dicer2
as efficiently as wt Loqs-PD (Fig. 1D).
Loqs-PD-m3, containing the second
dsRBD and the C-terminal flanking
region, associated weakly with Dicer2.
The C-terminus-lacking mutant (LoqsPD-m1) and the C-terminal end of
Loqs-PD per se showed little or no binding with Dicer2. These results demonstrate that the peptide covering aminoacid 206 to the end of Loqs-PD (Gly206–
Ile359) is the domain that interacts with
Dicer2 (Fig. 1D). Thus, the Loqs-PDspecific C-terminal sequence may help
the folding of the second dsRBD so that
Dicer2 can only bind Loqs-PD. A previous study showed that Dicer1 interacts
strongly with the third dsRNA-binding
domain of Loqs-PB (Ye et al. 2007).
Together, these data clarify the selectivity of Loqs isoforms for Dicer proteins.
The Dicer2–Loqs-PD complex
excises esiRNAs from precursors

It was recently shown in S2 cells that depletion of Loqs-PB
interfered with miRNA processing, whereas Loqs-PD depletion affected esiRNA processing (Hartig et al. 2009;
Zhou et al. 2009). By performing RNAi in S2 cells, followedDownloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

esiRNA and miRNA biogenesis in Drosophila

by Northern blot analyses for multiple miRNAs (bantam,
miR-2b, and miR-8) and non-transposon-derived esiRNAs(esiRNA-sl-1 and CG18854A), we confirmed that the requirement of Loqs isoforms in the miRNA and esiRNA
processing pathways indeed differ from one another (Supplemental Fig. 2). The level of an esiRNA derived fromtransposon DM1731, esiRNA-1731, was also significantly
lowered by Loqs-PD depletion (Supplemental Fig. 2). Thus,
Loqs-PD is also necessary for the production of transposon-derived esiRNAs.
We then assessed if the Dicer2–Loqs-PD complex wasable to process esiRNA precursors into mature esiRNAs in
vitro. A long stem–loop-type esiRNA precursor (preesiRNA), which contains the sequence of esiRNA-sl-1,
was designed (pre-sl) (Fig. 2A) and utilized in the assays.
We confirmed that small RNAs of z21 nt derived from theprecursor were loaded onto AGO2, as expected, but not
onto AGO1 in S2 lysates (Fig. 2A). All Loqs isoforms taggedwith a myc peptide were expressed in S2 cells following
transfection. Immunoprecipitation was performed 24 hpost-transfection using an anti-myc antibody to isolate
each complex. The Loqs-PA and Loqs-PB complexes that
contain Dicer1 (shown in Fig. 1C) showed no activity to
produce esiRNAs from the precursor (Fig. 2B). Loqs-PC,
whose interaction with Dicer1 and Dicer2 was under thelimit of detection by Western blot analysis (Fig. 1C), also
showed no such activity. However, the Loqs-PD complex
containing Dicer2 was able to process the esiRNA precursor
to the same extent as the complex immunoprecipitatedfrom S2 cells using anti-Loqs antibodies (Fig. 2C). When an
miRNA precursor, pre-let-7, was used as an alternative in
the assays, the Loqs-PD–Dicer2 complex produced only
a background signal (Fig. 2C). Pre-let-7 was efficiently
processed to mature let-7 by both of the Loqs-PA and
Loqs-PB complexes, as expected. The Loqs-PB complex
showed a relatively higher activity than the Loqs-PA complex, although both complexes contain Dicer1 at a similarlevel (Fig. 1C), suggesting that the Dicer1–Loqs-PB complex is more suited for miRNA processing. Loqs-PC
showed a slightly stronger signal than myc-EGFP, indicating that although the interaction with Dicer1 was notdetected by Western blot analysis (Fig. 1C), Loqs-PC might
be associated with Dicer1 at a very low level. It should be
noted that the expression levels of Loqs isoforms seemed tovary in tissues (Supplemental Fig. 3), suggesting a model inwhich changes in the ratio of these isoforms can affectprocessing of pre-miRNAs and pre-esiRNAs in vivo.
When the Loqs-PD complex was incubated with a long,completely complementary dsRNA (EGFP-dsRNA), which
serves as an exo-siRNA precursor, the long dsRNA wasprocessed to mature siRNAs of z22 nt (Fig. 2D). Therefore, we examined if Dicer2, when associated with Loqs-PD,
also contained R2D2. Western blot analysis using antiR2D2 antibodies revealed that R2D2 was detectable in
the Loqs-PD complex, albeit at low levels (Fig. 2E). The

existence of R2D2 was unique to the Loqs-PD complex,
suggesting that Dicer2 might be able to associate with LoqsPD and R2D2 simultaneously in a dsRNA-independent
manner.
Loqs-PD, but not R2D2, enhances the esiRNA
processing activity of Dicer2 in vitro
Loqs-PB specifies miRNA precursors as a substrate ofDicer1 by physically associating with Dicer1 (Saito et al.
2005). In in vitro assay systems, Loqs-PB was also able topromote the miRNA processing activity of Dicer1 (Jiang
et al. 2005; Saito et al. 2005). To examine whether Loqs-PD
in the esiRNA processing pathway showed similar effects
to Loqs-PB in the miRNA processing pathway, esiRNA
processing assays were performed using a glutathione
S-transferase (GST)-tagged, recombinant Loqs-PD protein
produced in and purified from Escherichia coli (Fig. 3A). As
a control, GST-tagged R2D2 was also produced (Fig. 3A).
Flag-Dicer2 protein was expressed in S2 cells following
transfection and purified using an anti-Flag antibody underharsh conditions containing 1 M NaCl, which should disrupt most protein–protein interactions occurring in vivo.
Western blot analysis using anti-Loqs and anti-R2D2 antibodies revealed that Loqs-PC/PD and R2D2 were notdetected in the purified Dicer2 complex (Fig. 3B). The
purified Dicer2 alone did not show a significant activity toexcise esiRNAs from the precursors (Fig. 3C, lane 3). When
GST or GST-tagged R2D2 was added to the reaction mixture, the production of esiRNA was unchanged. However,when GST-tagged Loqs-PD was added instead, we foundthat the esiRNA production activity increased to a level similar to that of the Flag-Dicer2 complex immunoprecipitatedfrom S2 cells using anti-Flag antibodies (Fig. 3C, lane 2).
GST-Loqs-PD alone did not show this esiRNA processing
activity (Fig. 3C, lane 8). Both GST-Loqs-PD and GSTR2D2 interacted to an approximately equal degree with
Flag-Dicer2 immunoprecipitated in a buffer containing
1 M NaCl (Fig. 3D). We also examined whether the additionof GST-Loqs-PD affects the exo-siRNA processing ability ofDicer2 and revealed that GST-Loqs-PD but not GST-R2D2
enhances this activity (Fig. 3E). These results correlate wellwith the earlier observation that R2D2 is not necessary for
Dicer2 to process long dsRNAs into exo-siRNAs (Liu et al.
2003). Taken together, these results indicate that Loqs-PD
enhances Dicer2 activity to process esiRNAs from theirprecursors by associating with Dicer2.
Substrate preference of the Dicer1–Loqs-PB
and Dicer2–Loqs-PD complexes
How do the Dicer1–Loqs-PB and Dicer2–Loqs-PD complexes determine their substrates? To address the question,
we produced a series of deletion mutants of pre-sl (Fig.
2A; mutants are summarized in Fig. 4A) and performed
www.rnajournal.org

509

Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

Miyoshi et al.

FIGURE 2. Small RNA excision activities of the Dicer2–Loqs-PD complex immunopurified from S2 cells. (A) esiRNAs excised from theprecursor (pre-sl; the nucleotide sequence and a possible structural formation are shown at the top; the structural formation was predicted by
RNA & DNA folding applications; http://mfold.bioinfo.rpi.edu/) were found to be loaded onto AGO2, but not onto AGO1 in S2 cell lysates.
(Lysate ) esiRNA precursors incubated without S2 lysate, (lysate +) esiRNA precursors incubated with S2 lysate. (Lower panels) Western blot
results showing the specificity of immunoprecipitation. (B) The Dicer2–Loqs-PD complex was capable of excising esiRNAs from the precursor.
Loqs-containing complexes immunopurified from S2 cells using anti-Loqs antibodies (anti-Loqs) were employed as positive controls. (C) The
Dicer2–Loqs-PD complex failed to excise let-7 from the precursor (pre-let-7), whereas the Loqs-PA and Loqs-PB complexes specificallycontaining Dicer1 were able to process the precursor. (D) The Dicer2–Loqs-PD complex was also able to excise exo-siRNAs from the precursors(EGFP dsRNAs). (E) Western blot analysis using anti-R2D2 antibodies showed that the Dicer2–Loqs-PD complex also contained the R2D2
protein.

510

RNA, Vol. 16, No. 3

Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

esiRNA and miRNA biogenesis in Drosophila

not by the Dicer2–Loqs-PD complex(Fig. 4F). Another mutant, pre-sl-M5,
which contains the same stem as presl-M4 but with the loop swapped withthat of pre-let-7, was also processed by
the Dicer1–Loqs-PB complex (Fig. 4G).
These data suggest that the length of
the stem, but not the loop, of the stem–
loop-structured, non-transposon-derived
esiRNA precursors largely determines recognition by different Dicer–Loqs complexes.
miRNAs are processed from corresponding precursors by the Dicer1–
Loqs-PB (or Dicer1–Loqs-PA) complex
and loaded onto AGO1 in vivo. We thenexamined if small RNAs derived fromprocessed intermediates of esiRNA precursors, similar to miRNA precursors(like pre-sl-M4 in Figure 4), are loaded
onto AGO2 in vivo. AGO1 and AGO2
were immunoprecipitated from S2 cells,
and RNA fractions associated with bothproteins were probed with DNA oligos
recognizing esiRNA-sl-1, esiRNA-sl-4,
and esiRNA-sl-4as (Fig. 5). esiRNA-sl-1
was detected as expected mostly in the
RNA pool immunoisolated with AGO2;
FIGURE 3. Loqs-PD, but not R2D2, enhanced the Dicer2 esiRNA processing activity in vitro. in contrast, both esiRNA-sl-4 and(A) Coomassie brilliant blue staining of GST-tagged, recombinant Loqs-PD and R2D2
esiRNA-sl-4as were found predomiproteins produced in and purified from E. coli. (B) Flag-tagged Dicer2 protein expressed in S2
cells following transfection was affinity-purified with an anti-Flag antibody in a buffer nantly with AGO1. b-elimination expercontaining 1 M NaCl. Western blot analysis revealed that the Dicer2 fraction isolated iments confirmed that esiRNA-sl-4 andunder such harsh conditions contained only traces of R2D2 and Loqs proteins. (C) In vitro esiRNA-sl-4as can be categorized as
esiRNA processing assays using the purified Dicer2 obtained in B. While Dicer2 alone did notexcise esiRNAs from the precursor (pre-esiRNA; lane 3, Fig. 2A), addition of GST-Loqs-PD AGO1 binders (data not shown). These(lane 5), but not of GST (lane 4) and GST-R2D2 (lane 6), increased the esiRNA production results suggest that esiRNA precursors
activity of Dicer2 to the same extent as that of Flag-Dicer2 immunoprecipitated from S2 are processed by the Dicer2–Loqs-PD
cells with an anti-Flag antibody (lane 2). GST-Loqs-PD alone did not show any esiRNA complex until the length of the stem isprocessing activity (lane 8). These results indicate that Loqs-PD appears to enhance theability of Dicer2 to process esiRNAs from their precursors by associating with Dicer2. long enough to be recognized by the par(D) Both GST-tagged Loqs-PD and R2D2, but not GST itself, were able to bind with Flag- ticular complex. However, once the stem
Dicer2 purified from S2 cells under a high-salt condition as in Figure 4B. Note that only becomes too short for Dicer2–Loqs-PDthe full-length proteins (black arrowheads) seemed to be bound with Dicer2. (*) Heavy dependent processing, as in miRNA prechains of anti-Flag antibody, (**) light chains of anti-Flag antibody. (E) GST-Loqs-PD (lane5) but not GST-R2D2 (lane 6) enhances the ability of Dicer2 to process long dsRNAs into cursors, the Dicer1–Loqs-PB (or PA)
complex comes into contact with it andexo-siRNAs.
processes it further into small RNAs.
We consider these final small RNA prodesiRNA processing assays using both Dicer–Loqs complexes
ucts as a unique subset of miRNAs, and suggest naming
immunoisolated from S2 cells. The anti-Loqs immunoprethem semiRNAs, or siblings of esiRNA miRNAs.
cipitates and the immunoisolated myc-tagged EGFP complex were also employed as controls in these assays. Pre-sl,
DISCUSSION
pre-sl-M1, pre-sl-M2, and even the pre-sl-M3 mutant,which contains a 37-nt-long stem, were specifically prosiRNAs in Drosophila are produced from completely
cessed by the Dicer2–Loqs-PD complex (Fig. 4B–E). On thematched dsRNA precursors by the Dicer2–R2D2 complex
other hand, the shorter pre-sl mutant, pre-sl-M4, which(Liu et al. 2003), whereas miRNAs are excised from precontains a 22-nt-long stem, as in most typical miRNA premiRNAs by Dicer1–Loqs (Förstemann et al. 2005; Jiang
cursors, was processed by the Dicer1–Loqs-PB complex but
et al. 2005; Saito et al. 2005). Therefore, it was believed thatwww.rnajournal.org

511

Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

Miyoshi et al.

siRNAs and miRNAs could be easily distinguished fromone another based on their origin and processing factors.
However, the discovery of esiRNAs made it difficult todistinguish these two classes of small
RNAs (Czech et al. 2008; Ghildiyal et al.
2008; Kawamura et al. 2008; Okamura
et al. 2008). This is because esiRNA
production requires Dicer2 and Loqs,
the processing factors required insiRNA and miRNA pathways, respectively (Czech et al. 2008; Ghildiyal et al.
2008; Kawamura et al. 2008; Okamura
et al. 2008). From these findings, it was
speculated that small RNA biogenesis
systems in Drosophila cells could intersect with each other and that they
are even more complex than previously
conceived. However, two groups recently showed that the requirement for
Loqs in the esiRNA and miRNA processing pathways differs in regard to
the isoforms: Loqs-PD is required for
esiRNA production, whereas Loqs-PB
functions in miRNA production (Hartig
et al. 2009; Zhou et al. 2009). In this study,
we not only confirmed the functional
differences of Loqs-PB and Loqs-PD,
but we also showed that Loqs-PA, whichis the most abundant Loqs isoform intestes, is involved in miRNA, but not in
esiRNA, production. We also showedthat Dicer2 was able to simultaneously
associate with Loqs-PD and R2D2 (Fig.
6), and that Loqs-PD, but not R2D2,
was able to stimulate esiRNA production from their precursors. This result
correlated well with a previous observation that depletion of R2D2 did not
significantly affect esiRNA production
in Drosophila cells (Czech et al. 2008;
Okamura et al. 2008). The associationof Dicer2 with two distinct factors,
Loqs-PD and R2D2, might be quite reasonable for its function because in thissituation Dicer2 does not have to selector change its partner (either Loqs-PD
or R2D2) for the processing of different
precursors (either esiRNA or exo-siRNA
precursors).
How do Dicer proteins distinguishLoqs-PA/Loqs-PB over Loqs-PD, orvice versa? A previous study clearly indicated that Dicer1 binds strongly withthe third dsRNA-binding domain of
Loqs-PB but not with other truncated512

RNA, Vol. 16, No. 3

versions containing the first and second dsRBD (Ye et al.
2007). Loqs-PD contains the first and second dsRBD but
lacks the third. Thus, these previous results support our

FIGURE 4. (Legend on next page)Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

esiRNA and miRNA biogenesis in Drosophila

findings that Loqs-PD did not bind to Dicer1. Deletion
analyses of Loqs-PD showed that truncation of the
C-terminal end (42 amino acids) caused a severe decreasein binding with Dicer2. However, the C-terminal region
itself was obviously insufficient for the association. In
addition to the C-terminal region, the second dsRBD and
its upstream region of z50 amino acids were also needed
for tight binding with Dicer2 (Fig. 1D). Loqs-PB shareswith Loqs-PD the second dsRBD and its two flanking
regions. Considering also that even Loqs-PC showed nobinding to Dicer2, the C-terminal end of Loqs-PD, which isabsent from Loqs-PC, is likely to determine the specificbinding with Dicer2.
Accumulated evidence suggests that several miRNAs in
animals and plants originate from genomic repetitive elements and transposons (Chapman and Carrington 2007).
A recent study also showed that unlike Saccharomyces
cerevisiae, other budding-yeast species, Saccharomyces
castellii and Candida albicans, have systems to generatesmall interfering RNAs, which mostly correspond to transposable elements and to Y9 subtelomeric repeats residingin their genomes; however, they do not have miRNAs(Drinnenberg et al. 2009). These data together suggest thatmiRNAs may have arisen much later in evolution thanesiRNAs. This assumption further implies that in Drosophila, dicer1 may have arisen as a result of duplication ofa proto dicer2-like gene and that such gene duplication
might have helped the production of miRNAs originatingfrom genomic repetitive elements. Drosophila has maintained miRNA and esiRNA pathways independently fromeach other, probably because both pathways are essential;
the miRNA system is used for regulating gene expression
and the esiRNA system functions as an innate immune
system. However, in higher animals, like mammals, only
one Dicer, which is likely to be dedicated to the miRNA
pathway, is maintained. This is because through evolution,
mammals have acquired a more elaborate immune system,
namely, ’’adaptive immunity’’ to protect themselves from
pathogenic invaders, such as viruses.
In addition, it can be speculated that in parallel with
miRNA evolution and dicer gene duplication in Drosophila,
the loqs gene has evolved to give rise to various isoforms thatdifferentially associate with Dicer1 and Dicer2 and function
independently in processing miRNAs and esiRNAs because
both pathways are indispensable for Drosophila.

FIGURE 5. Small RNAs (esiRNA-sl-4 and esiRNA-sl-4as) arisingfrom the region most proximal to the loop in the hairpin esiRNA
precursor are loaded predominantly onto AGO1 in S2 cells. esiRNAsl-1 was loaded onto AGO2 as expected. RNA pools immunoisolatedwith AGO1 and AGO2 from S2 cells were probed with DNA oligos for
esiRNA-sl-1, esiRNA-sl-4, and esiRNA-sl-4as.

MATERIALS AND METHODS
Plasmid constructs

The myc-tagged constructs for Loqs-PB, Loqs-PA and EGFP havebeen described previously (Förstemann et al. 2005). The construct
for myc-tagged Loqs-PD (Hartig et al. 2009) was produced using
the strategy described in Förstemann et al. 2005 with the primers
59-AGCGGATCCATGGAACAAAAACTTATTTCTGAAGAAGACTTGGAACAAAAACTTATTTCTGAAGAAGACTTGGCCGTGAGTATCATTCAAGACATC-39 and 59-CAAAGCGGCCGCTTAGATCTTGATGAACTC-39 and the construct for myc-tagged Loqs-PC
was produced with the primers 59-CAAGGATCCAACGAATCTGTAAAGCACCT-39 and 59-CAAAGCGGCCGCCTACTGCGG
GGCTGTAAATAAG-39. All constructs for Loqs-PD deletionmutants were generated by PCR using full-length myc-tagged
Loqs-PD plasmid as a template. PCR products were subcloned into
pRH33Flag (Saito et al. 2005) or pRH33Flag-EGFP (Saito et al.
2005). To obtain a cDNA encoding Dicer2, poly(A)+ RNAs were
purified from S2 cells and reverse transcription polymerase chain
reaction (RT-PCR) was carried out using a ProSTAR Ultra-HF
RT-PCR system (Stratagene). To produce a 33Flag-tagged Dicer2
protein, full-length Dicer2 cDNA was subcloned into pRH33Flag(Saito et al. 2005). To produce GST-tagged proteins, Loqs-PD and
R2D2 cDNAs were subcloned into the expression vector, pGEX6p-2 (GE Healthcare). The fusion proteins
were induced and purified from E. coli, as
FIGURE 4. esiRNA processing using pre-sl deletion mutants. (A) The nucleotide sequences and described by the manufacturer.

possible structural formation of various pre-sl deletion mutants (pre-sl-M1 to pre-sl-M5) are
shown. Pre-sl-M5 contains the loop of pre-let-7 (gray) instead of the pre-sl loop. (B) esiRNA
processing was performed with pre-sl as in Figure 2B. The anti-Loqs immunoprecipitates andboth Dicer1–Loqs-PB and Dicer2–Loqs-PD complexes immunoprecipitated with an anti-myc
antibody, as in Figure 2B, were utilized as the enzymatic sources. The myc-EGFP complex was
employed as a negative control. (C) esiRNA processing with pre-sl-M1, as in B. (D) esiRNA
processing with pre-sl-M2, as in B. (E) esiRNA processing with pre-sl-M3, as in B. (F) pre-sl-M4
processing, as in B. Note that the precursor was now processed by myc-Loqs-PB but not by themyc-Loqs-PD complex. (G) pre-sl-M5 processing, as in B.

Immunoprecipitation, silver
staining, and Western blot analysis
Immunoprecipitation from S2 cell lysates
was performed using a modified protocolof a previously reported method (Miyoshi
et al. 2005). Antibodies were cross-linked towww.rnajournal.org

513

Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

Miyoshi et al.

FIGURE 6. A model for esiRNA, exo-siRNA, and miRNA biogenesis
pathways in Drosophila. The Dicer1–Loqs-PB (and the Dicer1–Loqs-PA)
complex processes miRNA precursors, whereas the Dicer2–Loqs-PD
complex processes esiRNA precursors. The Dicer2–Loqs-PD complex
also contains R2D2 and thus processes long dsRNAs into siRNAs. The
shorter esiRNA precursor intermediates, which are structurally similar
to miRNA precursors, are processed by the Dicer1–Loqs-PB complex;
this process gives rise to semiRNAs, a new subset of miRNAs.

Gammabind sepharose beads (GE Healthcare) with 20 mM dimethyl pimelimide. To obtain myc-tagged Loqs proteins, immunoprecipitation was performed using a monoclonal anti-c-myc antibody (9E10, catalog number M4439, Sigma-Aldrich). To purify
Flag-Dicer2, immunoprecipitation was performed using anti-Flag
M2 agarose beads (Sigma-Aldrich). After immunoprecipitation,
the beads were washed extensively with a buffer containing 30 mM
HEPES (pH 7.4), 150 mM potassium acetate, 5 mM magnesium
acetate, 5 mM DTT, 0.1% NP40, 2 mg/mL pepstatin, 2 mg/mL
leupeptin, and 0.5% aprotinin. Silver staining and Western blotting
were performed as described (Miyoshi et al. 2005). Anti-Loqs, antiDicer1, and anti-Dicer2 antibodies were used as described (Miyoshi
et al. 2009).

Northern blot analysis
Northern blot analysis was carried out essentially as described
previously (Saito et al. 2005). Oligo DNAs used as probes were:esiR-sl-4, 59-GAAAATCAAAGCCAGCGGAAG-39; esiRNA-sl-4as,
59-GACTTCCGGCGGTTAAGATTT-39.

G-39 and 59-CTGGAAAATCAAAGCCAGCGGAAGTGACTTCC
GGCGGTTAAGATTTTCCTATAGTGAGTCGTATTA-39 for presl-M4; 59-TAATACGACTCACTATAGGAAAATCTTAACCGCCGGAAGGTAATTACACATCACTTCCGCTGGCTTTGATTTTCCA
G-39 and 59-CTGGAAAATCAAAGCCAGCGGAAGTGATGTGTAATTACCTTCCGGCGGTTAAGATTTTCCTATAGTGAGTCGTA
TTA-39 for pre-sl-M5. A DNA fragment coding for dsRNA (EGFP
dsRNA) was amplified from epEGFP-C1 (Clontech) by PCR using
the primers: 59-TAATACGACTCACTATAGGGATGGTGAGCAAGGGCGAGGA-39 and 59-TAATACGACTCACTATAGGGTAGG
TCAGGGTGGTCACGAG-39. The PCR products and annealed
oligo DNAs were used as templates for an in vitro transcription
reaction using MEGAscript T7 kits (Ambion) in the presence of
32
P-UTP. These transcripts and 59-32P end-labeled pre-let-7(Miyoshi et al. 2009) were self-annealed. In vitro processing reactions of pre-miRNA (pre-let-7), pre-esiRNAs, and long dsRNA(EGFP dsRNA) with purified myc-Loqs proteins or Flag-Dicer2
were performed as described (Saito et al. 2005). For processing
using Flag-Dicer2, high-salt (1 M NaCl) purified Flag-Dicer-2 wasadded in the presence or absence of bacterially produced GSTLoqs-PD or GST-R2D2. After 90 min incubation at 26°C, RNAs
were purified with ISOGEN LS (Nippon Gene) and separated on
8% acrylamide denaturing gels containing 13 TBE buffer, 8 M
urea, and 25% formamide.

SUPPLEMENTAL MATERIAL
Supplemental material can be found at http://www.rnajournal.org.

ACKNOWLEDGMENTS
We thank K. Förstemann for sharing unpublished data and for
helpful discussions. We also thank Q. Liu and K. Saito for antibodies, technical assistance, advice, and discussion; T.N. Okada,T. Sunohara, A. Ishizuka, and H. Tsukumo for technical assistanceand reagents; and other members of the Siomi laboratory fordiscussions and comments on the manuscript. This work was
supported by a Boehringer Ingelheim Fonds fellowship to J.V.H.,
MEXT grants to H.S., and NEDO (New Energy and Industrial
Technology Development Organization) grants to M.C.S. M.C.S. is
supported by CREST from JST.
Received October 6, 2009; accepted November 25, 2009.

In vitro processing assays
DNA fragments coding pre-esiRNA, pre-sl, and the pre-sl mutants, pre-sl-M1 to pre-sl-M5, were obtained from PCR reactions.
The primers used were: 59-TAATACGACTCACTATAGGGATTG
AATGTAGCGCCCTGG-39 and 59-TTCTGTTCTTAAGGGCCCC
CCAGTAT-39 for pre-sl; 59-TAATACGACTCACTATAGGGCGC
CCTGGTAGCCTGTAGTT-39 and 59-GCCCCCCAGTATCCTGT
AGTTGG-39 for pre-sl-M1; 59-TAATACGACTCACTATAGACTCCAACAAGTTCGCTCCCGG-39 and 59-TGGACTCAAACAAGTC
CCTC-39 for pre-sl-M2; 59-TAATACGACTCACTATAGGCGCTTCACAGGCGCTGGAAAA-39 and 59-CTTCCGCTCGACAGACG
CTG-39 for pre-sl-M3. The template DNAs were produced byannealing sets of oligo DNAs for pre-sl-M4 and pre-sl-M5. The
oligo DNAs used were: 59-TAATACGACTCACTATAGGAAAATCTTAACCGCCGGAAGTCACTTCCGCTGGCTTTGATTTTCCA

514

RNA, Vol. 16, No. 3

REFERENCES
Bartel DP. 2009. MicroRNAs: Target recognition and regulatory
functions. Cell 136: 215–233.
Bernstein E, Caudy AA, Hammond SM, Hannon GJ. 2001. Role fora bidentate ribonuclease in the initiation step of RNA interference.
Nature 409: 363–366.
Chapman EJ, Carrington JC. 2007. Specialization and evolution ofendogenous small RNA pathways. Nat Rev Genet 8: 884–896.
Czech B, Malone CD, Zhou R, Stark A, Schlingeheyde C, Dus M,
Perrimon N, Kellis M, Wohlschlegel JA, Sachidanandam R, et al.
2008. An endogenous small interfering RNA pathway in Drosophila. Nature 453: 798–802.
Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ. 2004.
Processing of primary microRNAs by the Microprocessor complex. Nature 432: 231–235.

Downloaded from rnajournal.cshlp.org on February 19, 2010 - Published by Cold Spring Harbor Laboratory Press

esiRNA and miRNA biogenesis in Drosophila

Drinnenberg IA, Weinberg DE, Xie KT, Mower JP, Wolfe KH,
Fink GR, Bartel DP. 2009. RNAi in budding yeast. Science 326:
544–550.
Drysdale RA, Crosby MA, FlyBase Consortium. 2005. FlyBase: Genesand gene models. Nucleic Acids Res 33: D390–D395.
Förstemann K, Tomari Y, Du T, Vagin VV, Denli AM, Bratu DP,
Klattenhoff C, Theurkauf WE, Zamore PD. 2005. Normal microRNA
maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol 3:
e236. doi: 10.1371/journal.pbio.0030236.
Ghildiyal M, Zamore PD. 2009. Small silencing RNAs: An expanding
universe. Nat Rev Genet 10: 94–108.
Ghildiyal M, Seitz H, Horwich MD, Li C, Du T, Lee S, Xu J, Kittler EL,
Zapp ML, Weng Z, et al. 2008. Endogenous siRNAs derived fromtransposons and mRNAs in Drosophila somatic cells. Science 320:
1077–1081.
Hartig JV, Esslinger S, Böttcher R, Saito K, Förstemann K. 2009.
Endo-siRNAs depend on a new isoform of loquacious and target
artificially introduced, high-copy sequences. EMBO J 28: 2932–
2944.
Jiang F, Ye X, Liu X, Fincher L, McKearin D, Liu Q. 2005. Dicer-1 andR3D1-L catalyze microRNA maturation in Drosophila. Genes &Dev 19: 1674–1679.
Kawamura Y, Saito K, Kin T, Ono Y, Asai K, Sunohara T, Okada TN,
Siomi MC, Siomi H. 2008. Drosophila endogenous small RNAsbind to Argonaute 2 in somatic cells. Nature 453: 793–797.
Kim VN, Han J, Siomi MC. 2009. Biogenesis of small RNAs in
animals. Nat Rev Mol Cell Biol 10: 126–139.
Lee YS, Nakahara K, Pham JW, Kim K, He Z, Sontheimer EJ,
Carthew RW. 2004. Distinct roles for Drosophila Dicer-1 andDicer-2 in the siRNA/miRNA silencing pathways. Cell 117: 69–81.
Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X. 2003.
R2D2, a bridge between the initiation and effector steps of the
Drosophila RNAi pathway. Science 301: 1921–1925.

Miyoshi K, Tsukumo H, Nagami T, Siomi H, Siomi MC. 2005. Slicer
function of Drosophila Argonautes and its involvement in RISC
formation. Genes & Dev 19: 2837–2848.
Miyoshi K, Okada TN, Siomi H, Siomi MC. 2009. Characterization of
the miRNA-RISC loading complex and miRNA-RISC formed in
the Drosophila miRNA pathway. RNA 15: 1282–1291.
Okamura K, Ishizuka A, Siomi H, Siomi MC. 2004. Distinct roles for
Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes & Dev 18: 1655–1666.
Okamura K, Hagen JW, Duan H, Tyler DM, Lai EC. 2007. The
mirtron pathway generates microRNA-class regulatory RNAs in
Drosophila. Cell 130: 89–100.
Okamura K, Chung WJ, Ruby JG, Guo H, Bartel DP, Lai EC. 2008.
The Drosophila hairpin RNA pathway generates endogenous shortinterfering RNAs. Nature 453: 803–806.
Pham JW, Pellino JL, Lee YS, Carthew RW, Sontheimer EJ. 2004.
Dicer-2-dependent 80s complex cleaves targeted mRNAs during
RNAi in Drosophila. Cell 117: 83–94.
Saito K, Ishizuka A, Siomi H, Siomi MC. 2005. Processing of premicroRNAs by the Dicer-1-Loquacious complex in Drosophila
cells. PLoS Biol 3: e235. doi: 10.1371/journal.pbio.0030235.
Shibata S, Sasaki M, Miki T, Shimamoto A, Furuichi Y, Katahira J,
Yoneda Y. 2006. Exportin-5 orthologues are functionally divergent
among species. Nucleic Acids Res 34: 4711–4721.
Siomi MC, Kuramochi-Miyagawa S. 2009. RNA silencing in germlines—
exquisite collaboration of Argonaute proteins with small RNAs for
germline survival. Curr Opin Cell Biol 21: 426–434.
Siomi H, Siomi MC. 2009. On the road to reading the RNAinterference code. Nature 457: 396–404.
Ye X, Paroo Z, Liu Q. 2007. Functional anatomy of the Drosophila
microRNA- generating enzyme. J Biol Chem 282: 28373–28378.
Zhou R, Czech B, Brennecke J, Sachidanandam R, Wohlschlegel JA,
Perrimon N, Hannon GJ. 2009. Processing of Drosophila endo-siRNAs
depends on a specific Loquacious isoform. RNA 15: 1886–1895.

www.rnajournal.org

515