Molecular Biology of the Cell
Vol. 20, 521–529, January 1, 2009
Localization of Double-stranded Small Interfering RNA to
Cytoplasmic Processing Bodies Is Ago2 Dependent and
Results in Up-Regulation of GW182 and Argonaute-2
Aarti Jagannath and Matthew J.A. Wood
Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom
Submitted August 4, 2008; Revised September 22, 2008; Accepted October 9, 2008
Monitoring Editor: Wendy Bickmore
Processing bodies (P-bodies) are cytoplasmic foci implicated in the regulation of mRNA translation, storage, and
degradation. Key effectors of microRNA (miRNA)-mediated RNA interference (RNAi), such as Argonaute-2 (Ago2),
miRNAs, and their cognate mRNAs, are localized to these structures; however, the precise role that P-bodies and their
component proteins play in small interfering RNA (siRNA)-mediated RNAi remains unclear. Here, we investigate the
relationship between siRNA-mediated RNAi, RNAi machinery proteins, and P-bodies. We show that upon transfection
into cells, siRNAs rapidly localize to P-bodies in their native double-stranded conformation, as indicated by fluorescence
resonance energy transfer imaging and that Ago2 is at least in part responsible for this siRNA localization pattern,
indicating RISC involvement. Furthermore, siRNA transfection induces up-regulated expression of both GW182, a key
P-body component, and Ago2, indicating that P-body localization and interaction with GW182 and Ago2 are important in
siRNA-mediated RNAi. By virtue of being centers where these proteins and siRNAs aggregate, we propose that the
P-body microenvironment, whether as microscopically visible foci or submicroscopic protein complexes, facilitates siRNA
processing and siRNA-mediated silencing through the action of its component proteins.
RNA interference (RNAi) is a powerful homology-based gene
silencing mechanism directed by small RNAs, including small
interfering RNAs (siRNAs) and microRNAs (miRNAs). RNAi
is a posttranscriptional process, leading to either transla-
tional repression of the target mRNA, typical of miRNA-
mediated silencing, or degradation of the target via siRNA-
mediated silencing (Hammond, 2005). Recently, several
components of the RNAi pathway, including Argonaute
proteins (Sen and Blau, 2005), miRNAs, and their targets
(Liu et al., 2005b; Pillai et al., 2005) have been localized to
processing bodies (P-bodies). P-bodies are recognized as
important cytoplasmic mRNA processing centers where
nontranslating mRNA is sorted and either stored, repressed,
or degraded (for review, see Eulalio et al., 2007a). Enzymes
associated with mRNA degradation, such the CCR4-CAF-1-
Not complex involved in deadenlyation (Cougot et al., 2004);
DCP1 and -2 (van Dijk et al., 2002) involved in decapping;
and XRN-1, a 5?-3? exonuclease (Ingelfinger et al., 2002), have
all been localized to P-bodies. These foci also contain pro-
teins involved in nonsense-mediated decay (Sheth and
Parker, 2006) and AU-rich element-mediated mRNA decay
pathways (Vasudevan and Steitz, 2007).
Although it is clear that RNAi and P-bodies are associ-
ated, the precise role of P-bodies in RNAi remains unclear.
There is evidence that P-body components are essential for
miRNA-based gene silencing. Depletion of certain P-body
components, including RCK/P54 (Chu and Rana, 2006) and
GW182 in human (Liu et al., 2005a) and Drosophila (Reh-
winkel et al., 2005) cells leads to a loss of silencing. Further-
more, the decay of miRNA targets seems to require the
deadenylation complex, decapping enzyme, and 5?-3? exo-
nuclease, all of which are P-body components (Rehwinkel et
al., 2005). Knockdown of Drosha and DGCR8, proteins in-
volved in miRNA production, leads to the loss of P-bodies in
human cells, indicating that miRNAs are crucial compo-
nents of P-bodies (Pauley et al., 2006). For silencing to occur,
it has been suggested that miRNAs and the RNAi proteins
direct the target mRNAs to P-bodies, where the general
degradation/repression machinery is localized (Eulalio et
al., 2007a,b) and that P-bodies are formed as a consequence
In siRNA-based gene silencing, the role of P-bodies is less
well understood. Because siRNAs direct Argonaute-2 (Ago2)
to cleave the homologous target RNA (Hammond et al., 2000;
Martinez et al., 2002), the close presence of mRNA degrading
machinery might not be essential to achieve silencing. In-
deed, depletion of P-body components such as LSm1 and
RCK/p54 (Chu and Rana, 2006) in human cells has no effect
on siRNA silencing, leading to a view that P-bodies are
dispensable for siRNA-mediated RNAi. However, depletion
of the P-body structural component GW182 has shown
mixed results; three reports show that silencing GW182 does
inhibit siRNA silencing to a certain extent (Jakymiw et al.,
2005; Liu et al., 2005a; Lian et al., 2007), whereas others show
no requirement of GW182 for siRNA function (Rehwinkel et
al., 2005; Chu and Rana, 2006; Eulalio et al., 2007b). However,
siRNAs have been found to localize to P-bodies (Jakymiw et
al., 2005), and the number and size of P-bodies were found
to increase upon siRNA transfection, in a target-dependant
manner (Lian et al., 2007). Moreover, after knockdown of
This article was published online ahead of print in MBC in Press
on October 22, 2008.
Address correspondence to: Matthew J.A. Wood (matthew.wood@
Abbreviations used: Ago2, Argonaute 2; P-body, processing body.
© 2008 by The American Society for Cell Biology521
Lsm1 or RCK/p54, functional siRNAs are capable of induc-
ing P-body reassembly (Lian et al., 2007). This suggests that
although microscopic P-bodies are not necessarily required
for RNAi, silencing could occur in submicroscopic com-
plexes that might then trigger the assembly of larger, micro-
scopic structures. A recent study noted that P-body disas-
sembly was induced by a range of siRNAs, whose targets
were not related to mRNA metabolism and P-body compo-
nents (Serman et al., 2007), indicating that the structure and
function of P-bodies are more complex than currently be-
lieved. These conflicting results led us to further examine the
relationship between siRNA and P-bodies. It has been sug-
gested that it is more important to understand the individ-
ual contributions of P-body components to RNAi, rather
than the ability to aggregate/functions of the aggregate (Wu
and Belasco, 2008). Here, we show that in the presence of
microscopically visible P-bodies, double-stranded siRNAs
rapidly localize to these aggregates with a requirement for
Ago2 and that GW182 and Ago2, key RNAi machinery
components that localize to P-bodies, are up-regulated upon
siRNA transfection into cells.
MATERIALS AND METHODS
Cell Culture and Transfection
HeLa cells were cultured in DMEM (10% fetal bovine serum) in a 37°C
incubator with 5% CO2. Lipofectamine 2000 (Invitrogen, Paisley, United
Kingdom) was used for all transfections.
siRNAs. siAgo2 was obtained from Bioneer (Hørsholm, Denmark), siLuc was
from Dharmacon RNA Technologies (Lafayette, CO), and siMAPK was from
QIAGEN (Dorking, Surrey, United Kingdom). All other siRNAs were ob-
tained from Eurogentec (Seraing, Belgium). All sequences 5?-3?, sense strand
only, are as follows: siPPIB, GGA-AAG-ACU-GUU-CCA-AAA-AUU (Dhar-
macon RNA Technologies); siLA, GGU-GGU-GAC-GAU-CUG-GGC-UUU
(Dharmacon RNA Technologies); siAgo2, GCA-CGG-AAG-UCC-AUC-UGA-
AUU (Chu and Rana, 2006); siLuc, UAA-GGC-UAU-GAA-GAG-AUA-CUU
(Dharmacon RNA Technologies); siGW, GAA AUG CUC UGG UCC GCU
AUU (Lian et al., 2007); siRCK, GCA GAA ACC CUA UGA GAU UUU (Chu
and Rana, 2006); siSMN, GCA UGC UCU AAA GAA UGG UUU; and si-
MAPK (AllStars Positive control Mm/Hs MAPK1 control siRNA; QIAGEN).
DNA Constructs. pNEGFP-hDCP1a (van Dijk et al., 2002; Pillai et al., 2005)
was kind gift from Dr. Jens Lykke-Andersen (University of Colorado, Boul-
der, CO), transfected as 0.4 ?g/well (24-well plate).
Cells were fixed using 4% paraformaldehyde and permeabilized in 0.3%
Triton X-100. All blocking and incubation performed in 2% bovine serum
albumin, 2.5% goat serum, 0.3% Triton-X 100. Antibody concentrations were
1:5000 anti-GWB serum and 1:1000 Alexa 488 goat anti-human immunoglob-
ulin G (Invitrogen). Human anti-GW182 serum 18033 was a kind gift from Dr.
Marvin Fritzler (University of Calgary, Calgary, AB, Canada).
An LSM 510 laser scanning confocal microscope (Carl Zeiss, Jena, Germany)
was used. Images were taken with the 40? and 63? oil immersion objective
lenses. The following filter sets were used: MBS NT 80/20 or HFT UV
488/543/633 with DBS NFT 490 or NFT 545 or mirror or none. An argon laser
at 488 nm was used to excite enhanced green fluorescent protein (eGFP), with
emission collected using a 505- to 550-nm band pass filter. A helium-neon
laser was used to excite Cy3 at 543 nm, with emission collected using band
pass 560–615 nm; and to excite Cy5 at 633 nm, with emission collected using
long pass filter 650 nm. Fluorescence resonance energy transfer (FRET) signal
was collected by excitation at 543 nm and emission collection by using long
pass filter 650 nm. Wavelength spectra were collected by excitation at 543 nm
using the lambda scan setting, collecting images at approximately 10-nm
intervals between 560 and 732 nm. For all images, pixel time of 1.6 ms was
used. All lasers were used at 100% intensity. Constant levels of gain and offset
were maintained for slides imaged on the same day. Pix FRET software was
used to present images corrected for spectral bleed-through and background
elimination (Feige et al., 2005). For quantification of FRET, data were obtained
using the lambda spectrum setting and LSM 510 software. Fluorescence
emission intensity from the lambda scans for Cy3 (Fd, read at 560–570 nm)
and Cy5 (Fd, read at 660–670 nm) were recorded from approximately 20
P-bodies from a total of three images for each time point. Fa/Fd ratios were
calculated from these readings, as described in Raemdonck et al. (2006).
Quantification of Colocalization
Images were analyzed for colocalization using the Just Another Colocaliza-
tion Program (Jacop) plugin on ImageJ (National Institutes of Health, Be-
thesda, MD), and statistical data are reported from the Costes’ randomization
based colocalization module (Bolte and Cordelieres, 2006).
Quantitative Reverse Transcription-Polymerase Chain
RNA was harvested using the RNeasy kit (QIAGEN) and DNAse treated with
the RNase-free DNAse kit (Promega, Madison, WI). cDNA was prepared
from the RNA by using the High Capacity Reverse transcription kit (Applied
Biosystems, Foster City, CA). qPCRs were performed using the Power SYBR
Green master mix on the ABI7000 thermal cycler (Applied Biosystems). A
DDCt method of relative quantification was used for data analysis. Primer
sequences were as follows: human Ago2, forward 5?-CGCGTCCGAAGGCT-
GCTCTA-3? and reverse 5?-TGGCTGTGCCTTGTAAAACGCT-3?; human cy-
clophilin-B, forward 5?-AAAGTCACCGTCAAGGTGTATTT-3? and reverse
5?-TCACCGTAGATGCTCTTTCCTC-3?; human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), forward 5?-AAG-GTG-AAG-GTC-GGA-GTC-AA-3?
and reverse, 5?-GAA-GAT-GGT-GAT-GGG-ATT-TC-3?; human RCK/p54, for-
ward AGAGGCCCTGTGAAACCCA and reverse: CTTGCTGCTGAGTGC-
CATTA; and human GW182, forward CTCTGTGGATGCTCCTGAAAG and
Total cell protein was extracted with radioimmunoprecipitation assay buffer
(Sigma-Aldrich) and 10% Protease Inhibitor complex (Sigma Chemical, Poole,
Dorset, United Kingdom) and quantified using the Bradford method. Five, 15,
and 30 ?g of total protein was loaded onto an 8% polyacrylamide gel and run
at 100V. The protein was transferred to a nitrocellulose membrane overnight
at 50 V. Blocking and incubation with antibodies were in 5% milk in 0.1%
phosphate-buffered saline (PBS)-Tween.
Antibodies used were as follows: polyclonal rabbit anti-cyclophilin-B (Abcam,
Cambridge, United Kingdom) used at 1:100,000 overnight 4°C; monoclonal
mouse anti-GAPDH (Abcam) used at 1:1,000,000 dilution overnight 4°C; and
horseradish peroxidase-conjugated goat anti-rabbit or mouse secondary antibod-
ies (Millipore Bioscience Research Reagents, Temecula, CA) were used at 1:5000
dilution at room temperature for 1 h.
The data sets for the microarrays listed in Supplemental Table 1 were down-
loaded from the National Center for Biotechnology Information (NCBI) GEO
website, consolidated on Excel (Microsoft, Redmond, WA) and then analyzed
on Theiler’s murine encephalomyelitis virus (TMEV) to detect genes that were
significantly changed in expression levels across all arrays. Expression levels
corresponding to each gene of interest were recorded for all the arrays, and
we used p ? 0.05 for significance.
Student’s t test (two-tailed) in Excel (Microsoft) or GraphPad Prism (GraphPad
Software, San Diego, CA) used for all statistical analyses. Statistical analyses
from microarrays were generated using TMEV and also from the original
data set analysis available on NCBI GEO. For siRNA knockdown and
real-time PCR experiments, three biological replicates were tested in trip-
licate. For quantification of FRET, 20 P-bodies from a total of three images
siRNAs Localize Rapidly to P-Bodies
To study the pattern of intracellular siRNA localization, we
visualized siRNAs targeted against cyclophilin-B (siPPIB,
with Cy3 label on 5? antisense strand), SMN (siSMN with
Cy3 label on 5? sense strand), or firefly luciferase (siLuc with
Cy3 label on 5? sense strand) transfected into HeLa cells. The
cells were fixed after 4 or 24 h and immunostained with
human anti-GW182 serum 18033, and colocalization of the
siRNAs with P-bodies was studied. Significant colocaliza-
tion of the antisense-labeled siRNA was observed (Fig. 1A).
Colocalization of siPPIB to P-bodies was statistically
quantified using Costes’ randomization-based colocaliza-
tion, with a highly significant (p ? 0.01) colocalization
coefficient of 0.268 ? 0.038 (SE) (Figure 1B) (Costes et al.,
A. Jagannath and M.J.A. Wood
Molecular Biology of the Cell522
2004; Bolte and Cordelieres, 2006). Quantification of siLuc
and siSMN colocalization to P-bodies showed a lower but
again highly significant colocalization coefficient (siLuc,
0.159 ? 0.09; siSMN, 0.15 ? 0.03, p ? 0.01). On monitoring
siRNA localization in live cells expressing the P-body
decapping enzyme component hDcp1a tagged with eGFP
(eGFP-hDcp1a) to visualize P-bodies (van Dijk et al., 2002;
Pillai et al., 2005), we saw that siRNAs could be found in
P-bodies within 30 min of transfection (Supplemental Fig-
ure 1). Some large bodies containing intense siRNA sig-
nals were also present; these have been described previ-
ously to be vesicles/endosomes that remained from
transfection (Jakymiw et al., 2005).
If siRNAs were recruited to P-bodies simply because they
were already bound to the target in the RISC complex, only
the antisense strand should have been present in P-bodies.
However, not only the antisense siRNA but also sense
strand-labeled siSMN, sense strand-labeled siPPIB, and
siRNA with no complementary target, siLuc (Figure 1A),
localized to P-bodies, confirming a similar previous report
(Jakymiw et al., 2005). This led us to investigate whether the
reason a complementary target was not required for siRNA
localization to P-bodies is because the siRNA localized to
P-bodies in double-stranded form, before binding the target
in the RISC complex.
Imaging of siRNA Labeled with FRET Dyes Reveals That
Double-stranded siRNA Localizes to P-Bodies
FRET technology has been shown to be a powerful real-time
methodology to follow the intracellular fate and function of
siRNAs where FRET has been successfully applied to dis-
criminate between labeled siRNAs in single- or double-
stranded conformation (Raemdonck et al., 2006; Jarve et al.,
2007). Therefore, to test our hypothesis that double-stranded
siRNA localizes to P-bodies, we decided to use FRET-la-
beled siRNA, which was siPPIB siRNA with 5? sense strand
Cy3 label and 5? antisense strand Cy5 label (FRET siPPIB).
When these fluorophores are ?10 nm apart, upon excitation
of the donor Cy3, due to energy transfer, emission from the
acceptor, Cy5, is detected (Massey et al., 2006). In FRET
siPPIB, the fluorophores are ?6 nm apart in the native
double-stranded siRNA and should lead to a FRET signal.
We first tested this siRNA in solution in vitro by using a
fluorescent spectrophotometer, and we found upon excita-
tion of the donor Cy3 that a reliable FRET signal could be
detected from Cy5 at 670 nm. A much lower signal was
obtained from siRNA tagged with Cy3 alone, showing that
the bleed-through of Cy3 into the Cy5 emission region was
much lower than any FRET signal collected (Supplemental
Figure 2A). We transfected FRET siPPIB into HeLa cells,
which were then fixed and immunostained with human
(A) HeLa cells grown on poly-l-lysine–coated cover-
slips were transfected with 50 nM Cy3 siPPIB, Cy3
siSMN, or Cy3 siLuc (Red). At the given time points
after transfection, cells were fixed and immunostained
with anti-GWB serum (green). Overlays of the red and
green images show colocalization of siRNA and P-
bodies, indicated by arrows. Bar, 10 ?m. (B) Colocal-
ization of Cy3 siPPIB to P-bodies was statistically
quantified using the Jacop program from six images,
and three containing ?10 cells per image. Significant
colocalization was observed, with the average Pear-
value for statistically significant colocalization ob-
tained with Costes randomization based colocalization
for each individual image ?0.01.
Rapid localization of siRNA to P-bodies.
siRNA Localizes to P-Bodies
Vol. 20, January 1, 2009523
transfected with 50 nM FRET siPPIB. After 24 h, the cells were fixed and immunostained with human anti-GWB serum. Both siRNA strands colocalized
in P-bodies (antisense strand image: Cy3, FRET siPPIB, excitation 543 nm, emission 560–615 nm [red]; sense strand image: Cy5, FRET siPPIB, excitation
643 nm, emission above 660 nm [yellow]; and anti-GWB [green]). Arrows indicate colocalization of siPPIB and P-bodies. (B) Cy3 (donor) and Cy5
(acceptor) are a FRET pair, and the proximity of these dyes on the double-stranded siRNA results in FRET (FRET siPPIB) (images: FRET, FRET siPPIB,
excitation 543 nm, emission above 660 nm [yellow]). Colocalization with P-bodies (green) indicated with arrow. Bar, 20 ?m. (C) Typical emission
wavelength spectrum (lambda spectrum) obtained from a single P-body colocalizing with FRET PPIB (blue). Also included is a spectrum from P-body
in cells transfected with control siRNAs (Cy3 siLuc and Cy5 siPPIB) showing little FRET signal (red). Excitation at 543 nm and emission collected every
10 nm after 550 nm. Cy3 emission peak, ?570 nm; Cy5 emission peak, ?670 nm. (D) FRET siPPIB is potent at silencing PPIB; Western blot indicating
knockdown of PPIB at 72 h after transfection of FRET siPPIB into HeLa cells. Three lanes indicate samples in triplicate. GAPDH used as loading control.
(Fa is emission from acceptor on excitation of donor; Fd is emission from donor upon excitation of donor) from FRET siPPIB is indicated by light bars,
as indicated by Student’s t test). At 72 h, there is no longer a significant difference. (F) Quantification of target mRNA silencing timeline correlates with
72 h (*p ? 0.05, **p ? 0.01; Student’s t test); however, maximum silencing occurs at 48 h, with levels rising again at 72 h.
Double-stranded siRNA is detected in P-bodies using FRET technology. (A) HeLa cells were grown on poly-l-lysine–coated coverslips and
A. Jagannath and M.J.A. Wood
Molecular Biology of the Cell524
anti-GWB serum after 24 h. It was possible to detect both
sense and antisense strands using the respective Cy3 and
Cy5 labels (Figure 2A) in P-bodies. In addition, we detected
a strong FRET signal (Figure 2B), indicating that these dyes
were in proximity, i.e., that the two siRNA strands were
associated together. On examining the emission wavelength
spectrum from a single P-body, we detected both the donor
(Cy3 at ?570 nm) and acceptor (Cy5 at ?670 nm) spectra,
arising only from donor excitation (543 nm) (Figure 2C). Sev-
eral studies have shown that labeling the 5? end of siRNAs
with fluorescent dyes has a minimal effect on gene silencing
activity; we therefore performed a Western blot assay on
cells transfected with 50 nM FRET siPPIB to confirm its
efficacy. FRET siPPIB produced robust silencing of its target
mRNA (Figure 2D).
To measure the amount of double-stranded siRNA in the
P-bodies over time, we measured the FRET signal obtained
from siRNA localized to P-bodies in live cells, where P-
bodies were visualized with eGFP-hDcp1a (Supplemental
Figure 2B). We quantified the FRET signal from the sensi-
tized acceptor intensity relationships, i.e., from the ratio of
acceptor Cy5 emission (Fa) to donor Cy3 emission (Fd) upon
donor excitation only. This method has been used to quan-
tify FRET signals from double-labeled siRNAs previously, to
measure stability of the siRNA as a function of time (Raem-
donck et al., 2006). Because microscopically visible P-bodies
are structures ?100–300 nm in diameter (Eystathioy et al.,
2002) (a small diameter relative to the distance required
between two siRNA strands for a FRET signal), it is possible
that a FRET signal may arise from the proximity of the two
dyes on individual, separated siRNA strands in the P-body
oriented at random such that they are ?10 nm apart. Hence,
we controlled for this by transfecting equal amounts of two
separate siRNAs, siPPIB with 5? antisense strand Cy5 label
and siLuc with 5? sense strand Cy3 label. Any FRET signal
collected from cells transfected with these siRNAs would
therefore be the result of the two dyes in the P-body being in
sufficient proximity at random.
The Fa/Fd from the control was significantly lower than
Fa/Fd from the double-labeled FRET siPPIB (Figure 2E),
indicating the FRET signal we captured was indeed from
double-stranded siRNA. We found that the difference in
Fa/Fd between the FRET siPPIB and control was highest at 4 h
after transfection, reducing over time such that no difference
was observed at 72 h. Furthermore, we found that although
Fa/Fd from the FRET siPPIB reduced over time, levels from
the control experiments showed no significant change.
Two possible reasons for the decline over time in the
Fa/Fd of the FRET siPPIB relative to the control are that the
siRNA was being actively used for target gene silencing or
that it was being degraded. If degradation alone occurred,
Fa and Fd would both decrease proportionately over time,
but the ratio would remain constant. This is the case for the
control. However with FRET siPPIB, Fa reduces dispropor-
tionately faster than Fd, which indicates increasing distance
between the dyes without degradation leading to a decreas-
ing ratio (possibly indicative of strand separation. We went
on to measure the levels of the target mRNA (cyclophilin-B)
at 4, 24, 48, and 72 h after transfection of 50 nM siPPIB into
HeLa cells, by using real-time RT PCR. We found that max-
imum silencing occurred at 48 h (cyclophilin-B mRNA
?20% of the control) after transfection. At 72 h, silencing of
the target mRNA is relieved, with cyclophilin-B mRNA
levels reaching ?45% of control (Figure 2F). This pattern of
mRNA silencing correlates closely with the findings in Fig-
ure 2E, where the double-stranded siRNA FRET signal in
P-bodies was found to decrease with time, to reach a level no
different from the control at 72 h.
This pattern is a correlation only and does not show that
the siRNA fraction in P-bodies is the only active fraction.
bodies. (A) RCK/p54, GW182, and Ago2 were knocked down by
administration of 100 nM siRNA against each target individually in
HeLa cells. Knockdown of respective targets was measured using
real-time PCR at 72 h after transfection. (B) HeLa cells were grown on
poly-l-lysine–coated coverslips and transfected with 100 nM siRNA
targeting RCK/p54, GW182, or Ago2. After 48 h, the cells were trans-
fected with Cy3 siPPIB. At 72 h, the cells were fixed and immuno-
stained with human anti-GWB serum. Colocalization of siRNA with
P-bodies is observed in the case of control. P-bodies are lost upon
knockdown of GW182 and RCK/p54 and no corresponding colocal-
ization of siPPIB and P-bodies is seen. In Ago2 knockdown, marginally
reduced localization of siRNA to P-bodies is seen. Bar, 10 ?m. (C)
Colocalization of siPPIB to P-bodies was statistically quantified using
the Jacop program. Six images containing between three and six cells
per image were analyzed for both control and Ago2 knockdown, and
average Pearson’s colocalization coefficient was calculated for both,
using Costes’ randomization based colocalization. The Pearson’s coef-
ficient is significantly lower in Ago2 knockdown (p ? 0.035; n ? 6).
Knockdown of Ago2 limits localization of siRNA to P-
siRNA Localizes to P-Bodies
Vol. 20, January 1, 2009 525
and the level of GW182 was measured after 72 h by using real-time PCR. Significant up-regulation of GW182 was seen in all targeting siRNAs
(between 35 and 222%; average 106%; *p ? 0.05). siLuc, which did not have a complementary target, did not induce any increase. (B) From
the NCBI GEO website, 18 microarray data sets were chosen based on satisfying the following conditions: 1) 100 nM siRNA against any
target. 2) Transfected into HeLa cells. 3) RNA harvested at 24 h. 4) Microarrays done in duplicates in dye reversal. 5) Microarrays done on
either of two platforms, GPL3991 or GPL3992, for easy comparison between samples. 6) p values available to indicate significance in ratios.
Of the 18, 13 showed significant up-regulation of GW182, *p ? 0.05. (C) In a similar experiment as A, levels of Ago2 were measured using
real-time PCR, and results indicate three of four siRNAs cause a significant up-regulation of Ago2 * ? p ? 0.05. (D) Ago2 up-regulation as
indicated by the same microarray analysis as (B), * ? p ? 0.05. (E) In a similar experiment as (A), levels of RCK/p54 were measured using
real-time PCR, results indicate none of the siRNAs tested cause a significant up-regulation of RCK/p54. (F) Confirmation of lack of RCK/p54
Transfection of siRNA induces up-regulation of GW182. (A) Seven different siRNAs (100 nM) were transfected into HeLa cells,
A. Jagannath and M.J.A. Wood
Molecular Biology of the Cell526
Cytoplasmic Ago2 that does not localize to P-bodies has
been shown to be involved in silencing (Leung et al., 2006).
However, our data does indicate that the siRNA fraction in
P-bodies is probably active. One way to test whether this is
indeed the case is to see whether Ago2, the main component
of RISC, was associated in any way with the siRNA fraction
localizing to P-bodies. This Ago2 requirement would indi-
cate that the siRNA fraction localizing to P-bodies does
interact with RISC.
Ago2 Is Required for Localization and/or Retention of
siRNAs in P-Bodies
To test the idea that siRNA localization to P-bodies required
Ago2, we studied the effect of knocking down Ago2 and
other P-body components on both P-body integrity and
siRNA localization to P-bodies. We knocked down Ago2,
GW182, and RCK/p54 in HeLa cells by using siRNA trans-
fection (Chu and Rana, 2006; Lian et al., 2007), and we found
high levels of knockdown in all cases (?80%) (Figure 3A).
We confirmed the level of Ago2 protein knockdown at 90%
by measuring knockdown on an eGFP reporter (data not
shown). It has been reported previously that Ago2 knock-
down has no noticeable effect on P-body integrity or number
in mammalian cells, whereas knockdown of GW182 and
RCK/p54 leads to the disappearance of P-bodies, and our
observations confirmed the same (Figure 3B) (Lian et al.,
2007). We then transfected Cy3-siPPIB into these cells and
observed the localization pattern of siRNAs. In the case of
GW182 and RCK/p54 knockdown, we found a diffuse pat-
tern of siRNA localization in the cytoplasm, consistent with
the fact that microscopically visible P-body aggregates were
no longer present. In the Ago2 knockdown experiments, we
found that compared with the control, Cy3-siPPIB in Ago2
knockdown cells showed reduced siRNA colocalization
with P-bodies (Figure 3B). This observation was quantified
using Costes’ randomization-based colocalization and a sig-
nificant difference was observed in the colocalization coeffi-
cient between Ago2 knockdown and control (Figure 3C). We
therefore conclude that Ago2 facilitates siRNA localization
to and/or retention in P-bodies.
GW182 Is Up-Regulated upon siRNA Transfection
GW182 is one P-body component that has been shown to
directly interact with RISC; an interaction required for si-
lencing of miRNA targets in Drosophila (Behm-Ansmant et
al., 2006; Eulalio et al., 2008b). The effect of GW182 knock-
down in human cells on the capacity for effective siRNA-
mediated silencing has yielded mixed data. However,
GW182 has three human paralogues, which may have re-
dundant function making it difficult to assess the importance
of GW182 alone in siRNA-mediated silencing. To study
whether GW182 (and therefore P-bodies) was associated
with siRNA-mediated RNAi, we investigated if siRNA
transfection had any effect on GW182 (trinucleotide repeat
containing 6A, TNRC6A) expression. Transfection of six dif-
ferent siRNAs with complementary targets all caused an
up-regulation of TNRC6A in 72 h (Figure 4A) compared
with a control siRNA against luciferase (lacking a comple-
mentary target) that did not. To see whether this finding is
applicable to all siRNAs, we interrogated publicly available
microarray data from the NCBI GEO website. Eighteen sets
of arrays were chosen using the following criteria: compa-
rable microarray platform, 100 nM siRNA used on HeLa
cells and RNA extracted 24 h after transfection. These con-
stituted arrays from the following studies (Jackson et al.,
2006a,b; Schwarz et al., 2006). See Supplemental Table 1 for
list of target genes/siRNAs. Analysis of the entire data set
showed highly significant up-regulation of TNRC6A by 70%
(p ? 0.0001). Because this analysis was across all arrays, it
indicates this up-regulation occurs in response to transfec-
tion of most siRNAs. Individually, of the 18 array sets, 13
showed significant up-regulation of TNRC6A (Figure 4B),
confirming our experimental findings in Figure 4A and
showing that up-regulation of TNRC6A upon siRNA trans-
fection is a general phenomenon. From our study, we found
a smaller but significant up-regulation of Ago2 (eukaryotic
initiation factor 2C, EIF2C2) (50%; p ? 0.001) (Figure 4C),
which we confirmed by quantifying the level of Ago2
mRNA in samples transfected with four different siRNAs.
Three of the four showed significant up-regulation of Ago2,
and this required the presence of a target mRNA (Figure
4D). The microarray data were taken at a 24-h time point,
and PCR analysis was conducted at a 72-h time point. At
72 h, a greater degree of up-regulation can be observed both
in the case of GW182 and Ago2. We also investigated the
expression levels of other characterized P-body components
by using the microarray data sets and real-time PCR, but we
found no significant changes; the case of RCK/p54 (Figure 4,
E and F) is given as an example.
To investigate whether these components were required
for siRNA-mediated silencing, we knocked down GW182,
Ago2, and RCK/p54, and 48 h later we transfected the cells
with 100 nM siPPIB. After 24 h, levels of PPIB knockdown
were measured using real-time PCR. We found that knock-
down of GW182 and PCK/p54 had no effect on PPIB silenc-
ing, whereas knockdown of Ago2 relieved PPIB silencing
(Figure 4G), thereby confirming a previous report on the
requirement of Ago2 for siRNA-mediated silencing (Lian et
al., 2007). The lack of an effect on silencing after GW182
knockdown can be either due to redundancy of GW182
paralogues or/and because it may share silencing function
with Ago2 (Behm-Ansmant et al., 2006). The presence of
nonfunctional siRNA alone is not sufficient to cause this
effect, as seen with the case of siLuc, which lacks a comple-
We have shown that siRNAs rapidly localize to P-bodies in
double-stranded form. Furthermore, effective localization
requires Ago2 and transfection of functional siRNAs in gen-
eral causes an up-regulation of the key P-body component
GW182 and the important RNAi effector Ago2. Together,
our results suggest an important role for P-bodies in siRNA-
mediated RNAi as cytoplasmic microenvironments that fa-
cilitate the interaction of double-stranded siRNAs with other
RNAi elements. It is known that a functional RNAi pathway
is required for the formation of P-bodies and furthermore,
that Ago2 is concentrated in P-bodies even in the absence of
miRNAs, as seen in miRNA-deficient dicer knockout cell
lines (Leung et al., 2006). This indicates that RNAi and
P-bodies are intimately linked; here, we show that this rela-
tionship extends to siRNA-mediated RNAi.
Figure 4 (cont).
as described in B. (G) Silencing of GW182 does not affect siRNA-
mediated silencing. HeLa cells were transfected with 100 nM siRCK,
siGW, or siAgo2 and after 48 h with siPPIB. Levels of siPPIB mRNA
were measured after 24 h by using real time PCR. Knockdown of
RCK/p54 and GW182 did not affect PPIB silencing, whereas knock-
down of Ago2 significantly relieved silencing. (H) Knockdown of
RCK/p54, GW182, and Ago2 at the same time point as PPIB mea-
surement in G.
up-regulation using the same microarray analysis
siRNA Localizes to P-Bodies
Vol. 20, January 1, 2009527
Our results indicate that siRNAs rapidly localize to P-
bodies in double-stranded conformation and interact with
core P-body components. However knockdown of GW182
does not impair the silencing of PPIB by siRNAs; therefore,
we conclude siRNAs do not require the presence of micro-
scopically visible P-bodies for efficient gene silencing to
occur. The complete structure of P-bodies is yet to be de-
fined; moreover, we do not yet know whether the P-body
population displays structural and/or functional heteroge-
neity. Together, our data suggest that there may be a mini-
mum P-body composition for effective siRNA-mediated
RNAi. Thus, submicroscopic aggregates or “minimal” P-
bodies produced as a result of knockdown of P-body com-
ponents such as Lsm or RCK/p54 may retain the minimal
P-body components necessary for siRNA-mediated RNAi.
That knockdown of these two proteins does not prevent the
reassembly of visible P-bodies formed due to siRNA func-
tion lends support to this idea (Lian et al., 2007). Our data
from FRET imaging of double-stranded siRNA in P-bodies
show an intriguing pattern of decay, whereby there is a
significant difference between the rates at which the signal
decays from the FRET siRNA compared with the control.
This indicates a greater loss of FRET than that which occurs
simply due to decay, indicating strand separation may con-
tribute to this. If this is the case, P-bodies may be centers
where double-stranded siRNA interacts with the RNAi ma-
chinery and processed to single-stranded form.
Importantly, both our experimental work and interroga-
tion of publically available microarray data show that up-
regulation of GW182 and Ago2 is a general response to
siRNA function. In both cases, there are few instances where
statistically significant up-regulation is not seen. This could
be due to any of the following reasons: experimental varia-
tion and noise, certain siRNAs causing a higher up-regula-
tion than other siRNAs, or off-target effects of the siRNA and
further downstream effects that affect the levels of these
proteins. That Ago2 is up-regulated in response to the pres-
ence of functional siRNA is not surprising, because it is the
main effector of siRNA-mediated silencing. The up-regulation
of GW182 is more intriguing, because GW182’s exact function
is yet to be determined. In Drosophila, Ago1–GW182 complexes
are essential for miRNA-mediated silencing (Eulalio et al.,
2008b), although how this complex achieves silencing is still
debated (Eulalio et al., 2008a; Wu and Belasco, 2008). Teth-
ering of GW182 to target mRNA shows that GW182 may
facilitate silencing through a mode that bypasses the re-
quirement for Ago2 (Behm-Ansmant et al., 2006). Other
work has shown that the GW/WG repeat domains in
GW182 form evolutionarily and functionally conserved
binding platforms for RNAi machinery (El-Shami et al.,
2007). Our results show that a general phenomenon upon
transfecting siRNA is that both GW182 and Ago2 are up-
regulated. Increases in their levels may be the cellular sys-
tem’s response to large increases in RNAi activity. It is
possible that these increases ensure that endogenous RNAi
function is maintained. siRNA against luciferase that lacks a
complementary target does not cause this effect, indicating
that it is not simply the presence of siRNA but rather siRNA
function against a complementary target mRNA that in-
duces up-regulation. These results are consistent with previ-
ous studies that showed siRNA-mediated silencing induced
increases in P-body size and number in a target-dependent
manner (Lian et al., 2007) and that GW182 is an important
player in the RNAi pathway and is required for silencing (Liu
et al., 2005a; Eulalio et al., 2008b). Our data, in the light of these
studies, confirms that siRNAs do interact with P-bodies and
their RNAi-related components.
In summary, our results show that P-bodies are cytoplas-
mic sites where siRNAs aggregate, in addition to being
centers where nontranslating mRNA and RNAi components
are found. Moreover, siRNA transfection induces the up-
regulation of two important P-body components involved in
RNAi, Ago2 and GW182. This suggests that although mi-
croscopically visible P-bodies are not absolutely required for
siRNA silencing, the interaction of siRNAs with core P-body
components, whether in visible P-bodies or in submicro-
scopic minimal P-bodies, provides a microenvironment that
facilitates siRNA-mediated silencing (Jakymiw et al., 2005).
We thank Dr. Sridhar Vasudevan, Prof. Edward Chan, Dr. Andrew Jakymiw,
Dr. Grant Churchill, Dr. Raman Parkesh, and Dr. Shankar Srinivas for tech-
nical assistance and helpful discussions. We also thank Drs. Lykke-Andersen
and Marvin Fritzler for providing reagents. This work was supported by
grants from the UK Medical Research Council (to M. W.) and a Clarendon
Scholarship (to A. J.).
Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P., and Izaurralde,
E. (2006). mRNA degradation by miRNAs and GW182 requires both CCR 4,
NOT deadenylase and DCP 1, DCP2 decapping complexes. Genes Dev. 20,
Bolte, S., and Cordelieres, F. P. (2006). A guided tour into subcellular colo-
calization analysis in light microscopy. J. Microsc. 224, 213–232.
Chu, C. Y., and Rana, T. M. (2006). Translation repression in human cells by
microRNA-induced gene silencing requires RCK/p54. PLoS Biol. 4, e210.
Costes, S. V., Daelemans, D., Cho, E. H., Dobbin, Z., Pavlakis, G., and Lockett,
S. (2004). Automatic and quantitative measurement of protein-protein colo-
calization in live cells. Biophys. J. 86, 3993–4003.
Cougot, N., Babajko, S., and Seraphin, B. (2004). Cytoplasmic foci are sites of
mRNA decay in human cells. J. Cell Biol. 165, 31–40.
El-Shami, M., Pontier, D., Lahmy, S., Braun, L., Picart, C., Vega, D., Hakimi,
M. A., Jacobsen, S. E., Cooke, R., and Lagrange, T. (2007). Reiterated WG/GW
motifs form functionally and evolutionarily conserved ARGONAUTE-bind-
ing platforms in RNAi-related components. Genes Dev. 21, 2539–2544.
Eulalio, A., Behm-Ansmant, I., and Izaurralde, E. (2007a). P bodies: at the
crossroads of post-transcriptional pathways. Nat. Rev. Mol. Cell Biol. 8, 9–22.
Eulalio, A., Behm-Ansmant, I., Schweizer, D., and Izaurralde, E. (2007b).
P-body formation is a consequence, not the cause, of RNA-mediated gene
silencing. Mol. Cell. Biol. 27, 3970–3981.
Eulalio, A., Huntzinger, E., and Izaurralde, E. (2008a). Getting to the root of
miRNA-mediated gene silencing. Cell 132, 9–14.
Eulalio, A., Huntzinger, E., and Izaurralde, E. (2008b). GW182 interaction
with Argonaute is essential for miRNA-mediated translational repression and
mRNA decay. Nat. Struct. Mol. Biol. 15, 346–353.
Eystathioy, T., Chan, E. K., Tenenbaum, S. A., Keene, J. D., Griffith, K., and
Fritzler, M. J. (2002). A phosphorylated cytoplasmic autoantigen, GW182,
associates with a unique population of human mRNAs within novel cyto-
plasmic speckles. Mol. Biol. Cell 13, 1338–1351.
Feige, J. N., Sage, D., Wahli, W., Desvergne, B., and Gelman, L. (2005).
PixFRET, an ImageJ plug-in for FRET calculation that can accommodate
variations in spectral bleed-throughs. Microsc. Res. Tech. 68, 51–58.
Hammond, S. M. (2005). Dicing and slicing–the core machinery of the RNA
interference pathway. FEBS Lett. 579, 5822–5829.
Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J. (2000). An
RNA-directed nuclease mediates post-transcriptional gene silencing in Dro-
sophila cells. Nature 404, 293–296.
Ingelfinger, D., Arndt-Jovin, D. J., Luhrmann, R., and Achsel, T. (2002). The
human LSm1-7 proteins colocalize with the mRNA-degrading enzymes
Dcp1/2 and Xrnl in distinct cytoplasmic foci. RNA 8, 1489–1501.
Jackson, A. L. et al. (2006a). Position-specific chemical modification of siRNAs
reduces “off-target” transcript silencing. RNA 12, 1197–1205.
Jackson, A. L., Burchard, J., Schelter, J., Chau, B. N., Cleary, M., Lim, L., and
Linsley, P. S. (2006b). Widespread siRNA “off-target” transcript silencing
mediated by seed region sequence complementarity. RNA 12, 1179–1187.
A. Jagannath and M.J.A. Wood
Molecular Biology of the Cell528
Jakymiw, A., Lian, S., Eystathioy, T., Li, S., Satoh, M., Hamel, J. C., Fritzler, Download full-text
M. J., and Chan, E. K. (2005). Disruption of GW bodies impairs mammalian
RNA interference. Nat. Cell Biol. 7, 1267–1274.
Jarve, A. et al. (2007). Surveillance of siRNA integrity by FRET imaging.
Nucleic Acids Res. 35, e124.
Leung, A. K., Calabrese, J. M., and Sharp, P. A. (2006). Quantitative analysis
of Argonaute protein reveals microRNA-dependent localization to stress
granules. Proc. Natl. Acad. Sci. USA 103, 18125–18130.
Lian, S., Fritzler, M. J., Katz, J., Hamazaki, T., Terada, N., Satoh, M., and Chan,
E. K. (2007). Small interfering RNA-mediated silencing induces target-depen-
dent assembly of GW/P bodies. Mol. Biol. Cell 18, 3375–3387.
Liu, J., Rivas, F. V., Wohlschlegel, J., Yates, J. R., 3rd, Parker, R., and Hannon,
G. J. (2005a). A role for the P-body component GW182 in microRNA function.
Nat. Cell Biol. 7, 1261–1266.
Liu, J., Valencia-Sanchez, M. A., Hannon, G. J., and Parker, R. (2005b).
MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies.
Nat. Cell Biol. 7, 719–723.
Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T.
(2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.
Cell 110, 563–574.
Massey, M., Algar, W. R., and Krull, U. J. (2006). Fluorescence resonance
energy transfer (FRET) for DNA biosensors: FRET pairs and Forster distances
for various dye-DNA conjugates. Anal. Chim. Acta 568, 181–189.
Pauley, K. M., Eystathioy, T., Jakymiw, A., Hamel, J. C., Fritzler, M. J., and
Chan, E. K. (2006). Formation of GW bodies is a consequence of microRNA
genesis. EMBO Rep. 7, 904–910.
Pillai, R. S., Bhattacharyya, S. N., Artus, C. G., Zoller, T., Cougot, N., Basyuk,
E., Bertrand, E., and Filipowicz, W. (2005). Inhibition of translational initiation
by Let-7 MicroRNA in human cells. Science 309, 1573–1576.
Raemdonck, K., Remaut, K., Lucas, B., Sanders, N. N., Demeester, J., and De
Smedt, S. C. (2006). In situ analysis of single-stranded and duplex siRNA
integrity in living cells. Biochemistry 45, 10614–10623.
Rehwinkel, J., Behm-Ansmant, I., Gatfield, D., and Izaurralde, E. (2005). A
crucial role for GW182 and the DCP 1, DCP2 decapping complex in miRNA-
mediated gene silencing. RNA 11, 1640–1647.
Schwarz, D. S., Ding, H., Kennington, L., Moore, J. T., Schelter, J., Burchard,
J., Linsley, P. S., Aronin, N., Xu, Z., and Zamore, P. D. (2006). Designing
siRNA that distinguish between genes that differ by a single nucleotide. PLoS
Genet. 2, e140.
Sen, G. L., and Blau, H. M. (2005). Argonaute 2/RISC resides in sites of
mammalian mRNA decay known as cytoplasmic bodies. Nat. Cell Biol. 7,
Serman, A., Le Roy, F., Aigueperse, C., Kress, M., Dautry, F., and Weil, D.
(2007). GW body disassembly triggered by siRNAs independently of their
silencing activity. Nucleic Acids Res. 35, 4715–4727.
Sheth, U., and Parker, R. (2006). Targeting of aberrant mRNAs to cytoplasmic
processing bodies. Cell 125, 1095–1109.
van Dijk, E., Cougot, N., Meyer, S., Babajko, S., Wahle, E., and Seraphin, B.
(2002). Human Dcp 2, a catalytically active mRNA decapping enzyme located
in specific cytoplasmic structures. EMBO J. 21, 6915–6924.
Vasudevan, S., and Steitz, J. A. (2007). AU-rich-element-mediated upregula-
tion of translation by FXR1 and Argonaute 2. Cell 128, 1105–1118.
Wu, L., and Belasco, J. G. (2008). Let me count the ways: mechanisms of gene
regulation by miRNAs and siRNAs. Mol. Cell 29, 1–7.
siRNA Localizes to P-Bodies
Vol. 20, January 1, 2009529