articles
Distinct passenger strand and mRNA cleavage activities
of human Argonaute proteins
© 2009 Nature America, Inc. All rights reserved.
Bingbing Wang1,2, Shuqiang Li1,2, Hank H Qi2, Dipanjan Chowdhury3,4, Yang Shi2 & Carl D Novina1,2,5
Argonaute (AGO) proteins bind to small RNAs and mediate small RNA−induced silencing in eukaryotes. Using a minimal in vitro
system, we show that bacterially expressed human AGO1 and AGO2 but not AGO3 and AGO4 possess strand-dissociating activity
of microRNA (miRNA) duplexes. Both AGO1 and AGO2 function as RNA chaperones, capable of performing multiple rounds of
strand dissociation. Unexpectedly, both AGO1 and AGO2 demonstrate passenger strand cleavage activity of a small interfering
RNA (siRNA) duplex, but only AGO2 has target RNA cleavage activity. These observations indicate that passenger strand and
mRNA endonuclease activities are mechanistically distinct. We further validate these observations in mammalian extracts and
cultured mammalian cells, in which we demonstrate that AGO1 uses only miRNA duplexes when assembling translational
repression−competent complexes, whereas AGO2 can use both miRNA and siRNA duplexes. We show that passenger strand
cleavage and RNA chaperone activities that are intrinsic to both AGO1 and AGO2 are sufficient for RNA-induced silencing
complex (RISC) loading.
miRNAs are conserved 21−22-nucleotide (nt) noncoding RNAs
that downregulate gene expression post-transcriptionally (reviewed
in ref. 1). In the nucleus, Drosha processes long primary miRNA
(pri-miRNA) transcripts into precursor miRNAs (pre-miRNAs),
which are then exported from the nucleus by Exportin-5. In the
cytoplasm, Dicer processes pre-miRNAs into miRNA duplexes composed of a guide strand and a passenger strand. Typically, the guide
strand is incorporated into RISC, which, depending on the degree
of sequence complementarity between miRNAs and target mRNAs,
mediates either endonucleolytic cleavage or translational repression of
target mRNAs.
The AGO family of proteins is a core component of RISCs purified
from all species that are capable of small RNA−directed gene silencing (reviewed in ref. 2). The presence of PAZ and PIWI domains
structurally defines the AGO proteins. The PAZ domain binds to the
2-nt overhang at the 3′ end of small RNAs, and the PIWI domain
cleaves target mRNAs endonucleolytically2. The structural biology
of recombinant AGOs provides numerous insights into the functions
of AGOs3–7, but many questions remain. For example, it is unclear
why only AGO2 possesses endonucleolytic cleavage activity of target
mRNAs8,9. The PIWI domain of AGO2 has a conserved Asp-Asp-His
(DDH) motif that coordinates a divalent metal ion required for
target mRNA cleavage10. However, we know that the DDH motif is
not sufficient for AGO-mediated endonucleolytic ­cleavage of target
mRNAs because AGO3 possesses this motif but lacks ­target mRNA
cleavage activity. In another example, all ­crystallographic ­studies of
AGO proteins bound to guide strands analyze AGOs loaded with
oligonucleotides as single strands rather than as double-stranded
duplexes3–7. Therefore, the molecular details of guide strand loading
into AGO proteins are obscure.
Early data in vitro indicated that ATP-dependent RISC loading
precedes ATP-independent mRNA cleavage11. Subsequent data suggested that ATP-dependent helicase activity separates the strands of
siRNA and miRNA duplexes, followed by ATP-independent mRNA
cleavage12. In this ‘thermodynamic stability’ model of RISC loading, unwinding initiated at the 5′ end of the RNA duplex with lower
thermodynamic stability leads to preferential incorporation of the
strand with that 5′ end into RISC12. ATP-dependent helicases implicated in miRNA function13–16 include members of the DEAD-box
family of helicases (RNA helicase A14, MOV10 (ref. 15), RCK-p54
(ref. 16) and Gemin-3 (ref. 17)); however, the helicase activity of
these proteins has not been implicated in RISC function. In an
alternative ‘strand sampling’ model of RISC loading, AGO2 mediates ATP-independent cleavage of passenger strands of small RNA
duplexes; this is followed by dissociation of the fragments, resulting in maturation of active RISC13,18,19. Indeed, two studies have
identified ATP-independent RISC loading and function in AGO
proteins20,21. Still, the mechanism(s) and ATP dependence of RISC
loading are unclear.
To study assembly of functional RISCs, we developed a minimal
assay system composed only of small RNAs and recombinant human
AGO proteins. Using this system, and a complementary cell-based
system, we show that AGO1 and AGO2 can generate mature RISC by
separating the strands of miRNA duplexes.
1Department
of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 2Department of Pathology, Harvard Medical School,
Boston, Massachusetts, USA. 3Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 4Department of Medicine, Harvard
Medical School, Boston, Massachusetts, USA. 5Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA. Correspondence should be addressed to C.D.N.
([email protected]).
Received 25 March; accepted 30 September; published online 29 November 2009; doi:10.1038/nsmb.1712
nature structural & molecular biology advance online publication
articles
–
-3′
-5′ miR-21–miR-21*
–
+
–
+
–
1
–
+
–
+
–
2
–
+
–
+
3
–
–
+
–
+
Competitor
4
Free probe
G
G
ST
-A
G
ST O1
-A
G GO
ST
2
-A
G GO
ST
3
-A
G
O
4
5′3′-
32P
G
ST
-A
G
O
1
G
ST
-A
G
O
2
G
ST
-3′
-5′ miR-21P
G
ST
-A
G
O
1
G
ST
-A
G
O
2
G
ST
5′3′-
32P 32P
32P
G
ST
-A
G
O
1
G
ST
-A
G
O
2
G
ST
b
G
ST
-A
G
O
1
G
ST
-A
G
O
2
G
ST
a
150 kDa
100 kDa
75 kDa
Binding (%)
0.1
4.2
0.1
5
50 kDa
4.3
0.1
6
4.9
7
0.1
5.1
8
37 kDa
Free probe
Binding (%)
2.1
1.9
2.6
2.4
2.2
32P
2
22 nt
32P
–
G
– + – + Duplex
G
t
pu
– + – +
32P
ST
-A
G
+
ST
-A
G
G
O
ST
1
-A
G GO
ST
2
-A
G
O
1
G
ST
-A
G
ST O1
-A
G GO
ST
2
-A
G
O
G
1
ST
+
2
-A
G
O
G
2
ST
-A
G
G
O
ST
3
A
O
G
H
O
–
4
In
pu
t
G
ST
G
G
– + – + – +
+
–
+
– +
– +
– + – +
– + – +
21 nt
9 nt
Cleavage (%)
2.3
5.4
6.1
6.1
1.8
5.5
2.0
32P
32P
– + – + – + – +
2.1
O
1
ST
-A
G
O
G
2
ST
-A
G
O
1
G
+
ST
2
-A
G
G
ST O2
-A
G
G
O
ST
4
-A
O
G
H
O
4
In –
pu
t
G
ST
-A
G
O
G
1
ST
-A
G
O
G
2
ST
-A
G
O
O
H
–
1
+
2
32P
2.5
32P
32P
In
c
O
H
In –
pu
t
G
ST
–
+
Duplex
22 nt
9 nt
6.0
e
O
H
In –
p
G ut
S
G T-A
ST G
G -AGO1
ST O
-A 2
G GO
ST
-A 1
G
O
2
iR
(S -21
S
m )
iR
-2
1P
m
iR
-2
1*
G
S
G T-A
S G
G T-A O1
S G
G T-A O2
S G
G T-A O
S G 1
G T-A O
ST G 2
-A O1
G
O
2
d
Figure 1 AGO1 and AGO2 possess passenger strand cleavage activity toward miR-21P duplexes.
(a) Schematic representation of the trigger miR-21 RNAs used in these studies. Bacterial expression
of purified, recombinant human AGO1 (GST-AGO1), AGO2 (GST-AGO2), AGO3 (GST-AGO3)
22 nt
22 nt
and AGO4 (GST-AGO4) is shown below. The Coomassie-stained gel of bacterially expressed
human AGO proteins indicates that these proteins were purified to apparent homogeneity. Input
9 nt
AGO proteins in these studies were normalized to the total protein amount and verified by western
9 nt
32
blotting with an anti-GST antibody. (b) EMSA using excess 5′ P-labeled ssRNAs, dsRNAs or
hairpin RNAs, as indicated, demonstrates that both AGO1 and AGO2 bind miR-21−miR-21*
T T SP SP
duplexes, but only AGO2 binds miR-21P duplexes efficiently. Specific RNA binding by AGO1
IP
and AGO2 was verified by addition of unlabeled competitor RNAs (+). The nonspecific
competitor RNA (−) in each experiment was tRNA used in an equimolar amount to the labeled RNA. (c) Passenger strand cleavage activity assays
using 5′ 32P-labeled miR-21P duplexes (left) or miR-21−miR-21* duplexes (right). GST alone, AGO1, AGO2, AGO1 plus AGO2, AGO3 or AGO4 were
incubated with each duplex as indicated. Passenger strand cleavage activity was identified by the production of 9-nt fragments. Single-nucleotide
ladders of the duplex RNA substrates generated by alkaline hydrolysis (OH−) are shown. (d) The mobility-shifted complexes composed of AGO1 or AGO2
and miR-21P, miR-21−miR-21* (miR-21*) or the guide strand of miR-21 (SS) identified by EMSA in a were excised from native gels and subjected
to denaturing gel electrophoresis. The 9-nt passenger strand cleavage fragments remain bound to AGO1 and AGO2. The 9-nt cleavage fragments were
visualized by nondenaturing gel electrophoresis, with each lane representing a pool of ten mobility-shifted complexes. (e) The association of AGO1 and
AGO2 with post-cleavage fragments of miR-21P is stable. Passenger strand cleavage activity assays were performed using 5′ 32P-labeled miR-21P
duplexes (input) with AGO1 and AGO2. Then, AGO1 and AGO2 were immunoprecipitated (IP) from total reactions (T) using anti-GST antibodies, and
the supernatants (S) and precipitates (P) were subjected to denaturing gel electrophoresis. Single-nucleotide ladders of the duplex RNA substrate
generated by alkaline hydrolysis (OH−) are shown.
m
© 2009 Nature America, Inc. All rights reserved.
25 kDa
RESULTS
AGO1 and AGO2 cleave passenger strands of siRNA duplexes
The exact mechanism of loading miRNAs and siRNAs into AGO proteins remains unclear, owing to the difficulty of expressing functional
AGOs in bacteria. Recombinant AGO proteins are notoriously difficult to express in Escherichia coli, partly because of their low solubility
and high toxicity4,22. To ascribe activities directly to AGO proteins
without the possibility of contamination from eukaryotic binding
partners, we expressed human AGO proteins in bacteria and purified
them to apparent homogeneity, as assessed by Coomassie blue staining
(Fig. 1a). Using western blotting analysis, we identified several
minor degradation fragments derived from each AGO protein, which
constituted less than 10% of each AGO preparation (Supplementary
Fig. 1a). MS analysis indicated that recombinant human AGO1 and
AGO2 proteins are highly pure and free from detectable contaminating RNA helicases and endonucleases (Supplementary Fig. 1b,c).
To test AGO function in RISC loading, we prepared several RNAs
representing different steps of biogenesis of miR-21, an abundant
advance online publication nature structural & molecular biology
articles
ST
-A
G
ST O1
-A
G GO
ST
2
-A
G
O
1
+
2
b
32P
G
G
G
G
ST
G
ST
-A
G
ST O1
-A
G GO
ST
-A 2
G
O
1
+
2
G
ST
ATP
a
DS
GST
32P
miR-21–miR-21*
miR-21 (comp.)
SS
DS
GST-AGO1
GST-AGO2
150 150 150 150 150 150 150 150 150 150
0
50 150 300 900 0
50 150 300 900
DS
SS
32P
SS
DS
SS/DS
DS
SS
0.95
SS/DS
1 0.38 0.41 0.24
0.53 0.59 0.68
1.4 1.5 2.8
0.98
c
1 0.23 0.25 0.15
0.71 0.68 0.81
3.0 2.7 5.5
32P
GST
Time (s) 10
DS
GST-AGO1
60 150 300
10
30
GST-AGO2
60 150 300
10
30
60 150 300
32P
0.96
G
G
DS
32P
SS
SS/DS
0.01 0.02 0.12 0.23 0.28 0.02 0.03 0.12 0.25 0.31
32P
d
GST
Time (min) 5
10
15 30
GST-AGO1
60
5
10
15 30
GST-AGO2
60
5
10
15 30
60
DS
DS
32P
SS
SS/DS
SS
1 0.27 0.25 0.18
0.65 0.72 0.74
2.4 2.8 4.1
ST
-A
G
O
2
ST
-A
G
O
G
3
ST
-A
G
O
4
1 0.53 0.52 0.28
0.42 0.58 0.67
0.8 1.1 2.5
ST
1.1
G
© 2009 Nature America, Inc. All rights reserved.
30
DS
SS
DS
SS
SS/DS
0.09 0.12 0.13 0.15 0.17 0.12 0.1 0.18 0.16 0.16
32P
SS
SS
SS/DS
0.30 0.70 1.30 1.80 2.20 0.25 0.60 1.10 2.01 2.42
1.4 0.05 0.08
Figure 2 AGO1 and AGO2 possess strand-dissociating activity toward miR-21−miR-21* duplexes. (a) Strand-dissociating activity assays using
approximately equimolar amounts of AGO1, AGO2, AGO1 plus AGO2, AGO3 or AGO4 and 5′ 32P-labeled miR-21P or miR-21−miR-21*, as indicated.
Reactions without ATP (left) or with ATP (right) are shown. Strand-dissociating activity was quantified by the ratio of ssRNA (SS) produced from dsRNA
(DS), shown below each gel. (b) Quantification of AGO1- and AGO2-mediated strand-dissociating activity of a 5′-labeled miR-21−miR-21* duplex and
an unlabeled single-stranded competitor (comp.) corresponding to the labeled strand of the miR-21−miR-21* duplex, as indicated. The numbers above
the gel indicate the amount of RNA (fmol) added to each reaction. Increasing concentrations of cold competitor results in increased strand annealing
with newly dissociated strands (a property of RNA chaperones), which removes product and pulls the reaction to the right. However, this effect
(approximately two-fold increase) is limited because the newly annealed cold complementary strand−cold competitor strand is now duplexed RNA
and thus becomes a substrate for subsequent RNA strand-dissociating activity. (c,d) Time courses of strand-dissociating activity of AGO1 and AGO2
incubated with 5′ 32P-labeled miR-21−miR-21* duplexes in reactions without ATP. Substoichiometric ratios (1:3) of AGO proteins to 5′ 32P-labeled
RNAs were used.
and ubiquitous human miRNA: pre-miR-21, which represents
the product of Drosha processing; an imperfectly complementary
miR-21−miR-21* duplex, which represents the product of Dicer
processing; a perfectly complementary siRNA duplex ­ composed
of the miR-21 targeting strand and its reverse complement
(miR-21P); or each of these strands individually. For comparison,
we also prepared RNAs corresponding to an artificial miRNA,
CXCR4, which triggers translational repression or ­endonucleolytic
cleavage of target mRNAs with imperfect or perfect sequence
complementarity to the CXCR4 guide strand, respectively23,24:
an imperfectly complementary duplex of the CXCR4 siRNA
(CXCR4−CXCR4*); a perfectly complementary duplex (CXCR4P)
and the guide strand of CXCR4P.
First we tested RNA-binding affinities of AGO1 and AGO2 in an
electrophoretic mobility shift assay (EMSA; Fig. 1b). We incubated
the 5′ end−labeled single-stranded RNA (ssRNA) or double-stranded
RNA (dsRNA) with or without cold competitor and either AGO1 or
AGO2, and we resolved the RNA-protein complexes by ­nondenaturing
gel electrophoresis. Consistent with the observation that ssRNAs can
load RISC and mediate repression in cells25, both AGO1 and AGO2
bound to ssRNAs (panels 1, 2 and 6), although AGO2 was ­generally
more efficient in ssRNA binding than was AGO1 (panels 1 and 2).
Notably, AGO1 and AGO2 showed similar binding affinities for
pre−miR-21 and miR-21−miR-21* (panels 5, 7 and 8). In contrast,
AGO1 had a lower binding affinity for miR-21P than did AGO2
(Fig. 1b, panels 3 and 4). The binding affinities of AGO1 and AGO2
for dsRNAs is summarized in Supplementary Figures 2 and 3. The
binding affinity of AGO1 (Kd = ~5.9 µM) and AGO2 (Kd = ~4.6 µM)
for miR-21−miR-21* are approximately the same. In ­ contrast,
the binding affinity of AGO1 (Kd = ~32.3 µM) for miR-21P is
approximately seven times lower than the binding affinity of AGO2
(Kd = ~4.5 µM). Additionally, we confirmed that the interactions
between AGO1 or AGO2 and radiolabeled miR-21 ssRNAs or dsRNAs
were specific: unlabeled competitor RNAs inhibited RNA binding to
AGO1 and AGO2, but an unlabeled nonspecific competitor tRNA did
not affect small RNA binding to AGO1 and AGO2.
Passenger strand cleavage of siRNAs facilitates the guide strand loading
and maturation of RISC13,18,19. AGO2 cleaves both strands of an siRNA
nature structural & molecular biology advance online publication
a 152 PAZ 276
b
351 Mid 544 545 PIWI 770
32P
c
32P
Chi-A
Chi-B
Chi-C
G
ST
-A
G GO
ST
-A 1
G GO
ST
-C 2
h
G
ST i-A
-C
hi
G
ST B
-C
hi
G
ST C
-A
G
G
ST O1
-A
G GO
ST
-C 2
h
G
ST i-A
-C
h
G
ST i-B
-C
hi
-C
AGO2
G
ST
-C
h
G
ST i-A
G Chi
ST -B
-C
hi
-C
AGO1
– + – +– + – + – +
G
ST
-A
G GO
ST
-A 1
G GO
ST
-C 2
h
G
ST i-A
-C
h
G
ST i-B
-C
hi
-C
articles
– +– + – +– +– +
miR-21
– + – + – + – + – + Duplex
22 nt
Figure 3 AGO chimeras possess passenger strand
9 nt
cleavage activity of siRNA duplexes but lack
strand-dissociating activity of miRNA duplexes.
Cleavage (%) 2.2 7.5 7.8 2.3 7.7
2.5 7.4 7.4 1.9 7.5
(a) Schematic presentation of wild-type AGO1,
32P
wild-type AGO2, and AGO1-AGO2 chimeras
32P
(Chi) A, B and C. Each chimera was successfully
expressed in bacteria, and AGO chimeras were
detected by western blotting using an anti-GST
antibody. (b) Passenger strand cleavage activity
assays indicate that all AGO chimeras, A, B and C,
DS
possess endonucleolytic cleavage activity of the
SS
miR-21P duplex. Passenger strand cleavage is
Cleavage (%) 7.6 6.9
7.1
SS/DS
more robust in AGO chimeras A and C, which
possess the C-terminal PIWI domain of AGO2.
(c) Endonucleolytic cleavage activity of an miR-21 target RNA is present in AGO chimeras A and C but not in AGO chimera B, consistent with the known
role of the C-terminal PIWI domain of AGO2. (d) Strand-separating activity assays demonstrate that none of the AGO chimeras, A, B or C, has significant
strand-dissociating activity of the miR-21−miR-21* duplex.
© 2009 Nature America, Inc. All rights reserved.
duplex between nucleotides 9 and 10 from the 5′ end of the cleaved strand.
Unexpectedly, in passenger strand cleavage assays using 32P-labeled miR-21
dsRNAs, both AGO1 and AGO2 generated 9-nt fragments from each
strand of miR-21P (Fig. 1c, left). Consistent with known AGO-mediated
endonuclease activities, passenger strand cleavage activity required magnesium ions because addition of EDTA abrogated this endonuclease activity (Supplementary Fig. 4). Also as expected, neither AGO1 nor AGO2
possessed passenger strand cleavage activity of miR-21−miR-21* (Fig. 1c,
right). Similarly, both AGO1 and AGO2 possessed passenger strand cleavage
activity of CXCR4P but not CXCR4−CXCR4* duplexes (Supplementary
Fig. 5). Neither AGO3 nor AGO4 demonstrated passenger strand cleavage
activity of the miR-21P duplex, indicating that this activity is specific to
AGO1 and AGO2.
Because target mRNA fragment release is rate limiting in the absence
of ATP26, we hypothesized that release of the cleaved ­pass­enger strand
fragment is rate limiting in RISC loading of siRNA duplexes in vitro.
To test whether the AGO1- and AGO2-bound siRNA duplexes contained passenger strand cleavage fragments, we performed EMSAs
using 32P-labeled probes as in Figure 1b, excised the mobility-shifted
complexes from the nondenaturing gels and subjected these complexes to denaturing gel electrophoresis (Fig. 1d). We identified 9-nt
fragments that are characteristic of passenger strand cleavage only in
the AGO1 and AGO2 mobility shifts of the miR-21P duplex but not in
mobility shifts of single-stranded miR-21 or miR-21−miR-21*. These
data indicate that the products of passenger strand cleavage remain
stably bound to AGOs and suggest that additional factors promote
the release of passenger strands to form functional RISC.
To further test the affinity of AGO proteins for passenger strand cleavage fragments, we incubated 32P-labeled miR-21P with AGO1 or AGO2,
precipitated each AGO protein with an anti-GST antibody and subjected
the resulting supernatants and pellets to denaturing gel electrophoresis
(Fig. 1e). We identified 9-nt passenger fragments only in AGO1 and AGO2
precipitates, further indicating that the products of passenger strand cleavage remain stably bound to AGO proteins in our minimal assay system.
AGO1 and AGO2 mediate multiple rounds of strand dissociation
miRNA duplexes cannot be loaded into AGOs by passenger strand
cleavage. However, guide strands of miRNA duplexes load onto AGO
G
ST
G AG
ST O
G -AG 1
ST O
G -Ch 2
ST i- A
G Ch
ST i-B
-C
hi
-C
1.
11
1.
21
0.
02
0.
06
0.
05
1.
00
1.
10
0.
05
0.
07
0.
12
G
ST
G -AG
ST O
G -AG 1
S
O
G T-C 2
ST hi
G -C -A
ST hi
-C -B
hi
-C
d
proteins. To test for AGO-intrinsic strand-dissociating activity, we
added each miR-21 dsRNA to AGO1, AGO2 or both proteins in
solution and assessed their strand-dissociating activity by nonde­
naturing gel electrophoresis. Consistent with previous observations
that miRNA biogenesis does not require ATP20,21, both AGO1 and
AGO2 dissociated the miR-21−miR-21* duplex into single strands
in the absence of ATP (Fig. 2a). Addition of ATP to these reactions
improved strand dissociation approximately two-fold. Quantitative
analysis indicated nearly identical strand-dissociating activity of AGO1
and AGO2 toward miR-21−miR-21* duplexes (Fig. 2a,b). Stranddissociating activity toward miR-21P was not observed for either
AGO1 or AGO2. Notably, neither AGO3 nor AGO4 showed stranddissociating activity toward any of the miR-21 dsRNAs, including the
miR-21−miR-21* duplex, indicating that strand-dissociating activity
is specific to AGO1 and AGO2.
In EMSA analyses (Fig. 1b), addition of a vast excess of labeled
miR-21−miR-21* duplexes relative to AGOs permits detection of
a mobility-shifted complex composed of an AGO and a dsRNA.
In strand-dissociating assays (Fig. 2a), stoichiometric amounts of
miR-21−miR-21* duplexes are incubated with AGOs. Expanded views
of the gels in Figure 2a do not show AGO2-bound miRNA duplexes or
ssRNAs (Supplementary Fig. 6), suggesting that only a minor fraction
of these RNAs are bound to AGO2 at a given time or that the binding
of AGOs to miRNA duplexes is transient in this assay. Under these
conditions, the strand-dissociating activity of AGOs predominates,
leaving little dsRNA from which a mobility-shifted complex might
be observed. Taken together, these data indicate that a small fraction
of AGOs bind to miR-21 duplex RNAs at any given time and suggest
that AGOs may sample the strands of the miR-21−miR-21* duplex
without loading a guide strand.
To show that a single AGO protein catalyzes multiple strand
­dissociations, we added substoichiometric amounts (1:3) of AGO1
and AGO2 to 5′ 32P-labeled miR-21−miR-21* duplexes and followed
strand dissociation as a function of time. The onset of single-strand
accumulation was rapid (Fig. 2c), and approximately 2.5 doublestranded duplexes per AGO were converted to single strands within
1 h (Fig. 2d), indicating catalytic AGO1- and AGO2-mediated strand
dissociation. Thus, AGO1 and AGO2 may dissociate the strands of
advance online publication nature structural & molecular biology
articles
a
an miRNA duplex without loading them. These data further imply
that other proteins stabilize AGO2-miRNA complexes after AGO2­mediated strand dissociation.
Distinct siRNA and target RNA cleavage activities of AGOs
To map domains of AGO1 and AGO2 required for strand-dissociating
and endonuclease activities, we expressed chimeras of the AGO1 and
AGO2 PAZ and PIWI domains5 (Fig. 3a). Consistent with current
models of AGO2 function, all chimeric proteins containing the PIWI
domain of AGO2 efficiently sampled miR-21P (Fig. 3b) and retained
the endonucleolytic cleavage activity of miR-21−targeted RNAs
(Fig. 3c). Additionally, wild-type AGO1 and chimera B also sampled
miR-21P, although neither AGO1 nor chimera B showed endonucleolytic cleavage activity of miR-21−targeted mRNAs. Unexpectedly,
all chimeras of AGO1 and AGO2 retained little strand-dissociating
activity toward miR-21−miR-21* (approximately one-tenth of the
activity of the wild-type proteins; Fig. 3d). Thus, efficient AGO1- and
AGO2-mediated strand dissociation of miR-21−miR-21* duplexes
requires matching the PAZ, Mid and PIWI domains. However, only
the PIWI domain of AGO2 can catalyze endonucleolytic cleavage of
target RNAs with perfectly complementary binding sites for miR-21,
as only wild-type AGO2 and chimeras A and C catalyzed target
RNA cleavage. A discussion of the structural determinants of strand
dissociation and RNA endonuclease activities is presented in the
Supplementary Discussion.
AGO2 loads miRNAs into active RISC in vitro and in cells
Next we tested AGO activity in target RNA cleavage (Fig. 4). Consistent
with AGO2 function in vitro4 and in mammalian cells8,9, the minimal
assay system of AGO2 and a guide strand RNA was necessary and sufficient to mediate target mRNA cleavage at the predicted sites using
different single-stranded small RNAs (Fig. 4a,b, lane 6). This activity
required the presence of magnesium ions because addition of EDTA
abrogated this endonuclease activity (Supplementary Fig. 4b).
Additionally, AGO2 loaded with miRNA duplexes was sufficient to
direct endonucleolytic cleavage of target mRNAs (Fig. 4a,b, lane 12).
However, AGO2 loaded with siRNA duplexes did not lead directly to
endonucleolytic cleavage of target mRNAs (Fig. 4a,b, lane 9). Previous
c
FL6X/RL0X translation (%)
C
XC
R
4P
C
C XC
XC R
R 4–
4*
In
p
G ut
S
G T-A
ST G
G -A O
S G 1
G T-A O
S G 2
G T-A O
S G 1
G T-A O + 2
S G 1
G T-A O
S G 2
G T-A O
S
1
G T-A G O + 2
ST G 1
O
G A 2
S G
G T-A O 1
ST G +
-A O 1 2
G
O
2
C
XC
R
4AS
m
iR
-2
1
P
m
m iRiR 21
-2 –
1*
In
p
G ut
S
G T-A
S
G T-A G O
S
1
G T-A G O
ST G 2
G -A O
ST G 1
G -hA O1 + 2
S
G T-A go2
S G
G T-A O
ST G 1
G -A O 1 + 2
ST G
O
G -A 2
S G
G T-A O 1
ST G +
-A O 1 2
G
O
2
m
iR
-2
1
© 2009 Nature America, Inc. All rights reserved.
b
Figure 4 miRNA−miRNA* but not siRNA
Control
duplexes load into mRNA cleavage and
CXCR4-AS
translational repression−competent complexes
CXCR4P
CXCR4–CXCR4*
in vitro. (a) RNA with a perfectly complementary
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
32
miR-21−binding site was labeled with P at the
5′ end and incubated with GST, AGO1, AGO2
or both AGO1 and AGO2 without any miR-21
100
trigger RNA (lanes 2−4); with AGO1, AGO2 or
both AGO1 and AGO2 and mature miR-21
guide strands (lanes 5−7); AGO1, AGO2 or
75
both AGO1 and AGO2 and miR-21P trigger
RNA (lanes 8−10); or with AGO1or AGO2
and miR-21−miR-21* trigger RNA (lanes
50
11−12). (b) The same reactions as in a using
the 5′ 32P-labeled target RNA with a perfectly
25
complementary CXCR4 siRNA−binding site.
The bar on the left of each gel represents the
perfectly complementary site to miR-21 or
0
Cleavage (%)
7.1 6.8
3.1
4.2 4.1
2.7
CXCR4 in target substrates, respectively. A line
indicates the predicted cleavage products. These data demonstrate that AGO2 and a guide RNA strand are minimally necessary and sufficient for target
RNA cleavage, in vitro. Consistent with previously published data9, these data further indicate that the AGO1 is not necessary nor sufficient for target
mRNA cleavage, despite the presence of passenger strand cleavage activity. (c) Translational repression in RRL was measured by addition of a target
strand RNA with six imperfectly complementary binding sites for the guide strand of the CXCR4 siRNA and either the antisense strand of CXCR4 siRNA
(CXCR4-AS; gray bars), a CXCR4 siRNA (CXCR4P; white bars) or a CXCR4 miRNA mimic (CXCR4−CXCR4* duplex; dark gray bars) to the RRL. The
values were normalized to those from reactions lacking any small RNAs (black bars). Error bars indicate s.d., the average of three replicates.
reports suggested that bacterially expressed AGOs do not efficiently
load perfectly duplexed RNAs4. In contrast, our data indicate that
AGO1 and AGO2 bind to perfectly duplexed siRNAs and mediate passenger strand cleavage but do not efficiently release passenger strand
cleavage fragments.
Also consistent with previous observations8, our data demonstrate that AGO1 lacks endonucleolytic cleavage activity of target
RNAs (Fig. 4a,b, lane 5). The absence of this activity is not due to a
reduced ssRNA-binding affinity of AGO1 compared to AGO2, because
the Kd values of AGO1 and AGO2 for miRNA duplexes are similar
(Supplementary Figs. 2 and 3). Additionally, pre-annealing of the
ssRNA to the target mRNA followed by the addition of AGO2 but not
AGO1 resulted in cleavage of the target RNA (Supplementary Fig. 7a).
Thus, AGO1 possesses RISC-loading activities but lacks intrinsic target RNA cleavage activity.
We previously reported miRNA-directed translational repression
reactions in vitro using rabbit reticulocyte lysates (RRL)27,28. In these
reactions, addition of siRNAs to RRL did not trigger translational
repression in vitro27. However, merely separating the strands of the
siRNA by heating enabled translational repression, indicating weak
or absent siRNA RISC-loading activities in RRL. Therefore, RRL constitutes a natural assay system for analysis of RISC-loading activities
in translational repression.
To test loading of miRNAs into translational repression−competent
complexes, we used the CXCR4 reporter system23,24. The CXCR4 guide
strand has no homology to known miRNAs29, and therefore RISC
loading with CXCR4 trigger RNAs can be analyzed unambiguously
in extracts. In contrast, miR-21, a conserved and robustly expressed
miRNA9,30, is relatively abundant in RRL (data not shown); therefore,
use of miR-21 would not allow unambiguous analysis of dsRNA loading
into RISC. Indeed, immunoprecipitation of endogenous AGO2 but
not AGO1 from RRL led to cleavage at the correct position of a target
RNA that possessed a perfectly complementary binding site for
miR-21 (Supplementary Fig. 7b).
As expected, addition of a CXCR4 guide strand but not CXCR4specific siRNA to RRL led to the production of reporter mRNAs for
translational repression containing six imperfectly complementary
binding sites (FL6X). In contrast, addition of the miRNA mimic
nature structural & molecular biology advance online publication
articles
0.4
Actin
0
HEK 293T
HeLa
T
S
Relative PDCD4
mRNA level
0.8
12
P
miR-21P
CXCR4P
Relative PDCD4 mRNA level
c
b
H
AAG
O
H
1
AA
H GO
A2
A
H GO
A1
A
H GO
AAG 2
O
H
1
AAG
O
2
Relative miR-21 level
a 1.2
8
4
0
AGO1
d
AGO2
CXCR4P
CXCR4–CXCR4*
3
miR-21–miR-21*
CXCR4–CXCR4*
2
1
0
2.5
AGO1
AGO2
CXCR4P
CXCR4–CXCR4*
-A
G
C O1
on +
tro 2
l
AG
O
1
AG
O
H
2
A
H
A-
H
A-
-A
G
C O1
on +2
tro
l
AG
O
1
AG
O
H
2
A
H
A-
H
A-
CXCR4−CXCR4* led to translational repression of FL6X reporter
mRNAs to the same degree as achieved through addition of the CXCR4
guide strand alone (Fig. 4c and Supplementary Fig. 8). Notably, addition of 5′ 32P-labeled CXCR4P to RRL followed by immunoprecipitation of AGO2 indicated the presence of 9-nt cleavage fragments
associated with AGO2 precipitates (but not supernatants), characteristic of RISC loading by passenger strand cleavage (Supplementary
Fig. 7c). These data indicate that endogenous AGO2 in RRL possesses
passenger strand cleavage activity and that the inability to release
passenger strand cleavage products is probably responsible for the
absence of target mRNA cleavage triggered by an siRNA in vitro, as
we have previously hypothesized27.
Last, we tested AGO loading of miR-21 dsRNAs in cells. HEK 293T
cells show robust miRNA activity31 and, notably, have ten-fold less
endogenous miR-21 compared to other cells with robust miRNA
activity, such as HeLa cells9, thus minimizing the chances of competition by endogenous miR-21 (Fig. 5a). To investigate the mechanism of miR-21 loading in cells, we cotransfected HEK 293T cells
with plasmids expressing epitope-tagged human AGO1 or AGO2 and
miR-21−miR-21* or miR-21P dsRNA duplexes. We then precipitated
the epitope-tagged AGOs (Fig. 5b) and performed reverse transcription PCR (RT-PCR) for a predicted mRNA target for miR-21
(Fig. 5c). This method to experimentally validate computationally predicted miRNA targets has been described32–35. One computationally
predicted36 and experimentally validated37–40 target of miR-21 is
programmed cell death 4 (PDCD4), a novel repressor of cellular transformation. Transfection of miR-21−miR-21* followed
by pull-down of either AGO1 or AGO2 led to an approximately
2.5-fold enrichment in PDCD4 mRNA compared to control RNA.
In contrast, transfection of miR-21P followed by AGO2 pull-down
enriched PDCD4 mRNA by approximately ten-fold, whereas
pull-down of AGO1 did not substantially enrich PDCD4 mRNA
FL6X/RL0X fold repression
Figure 5 AGO1 and AGO2 load miR-21−miR-21* duplexes into
2.0
functional RISC in cells. (a) The relative expression of endogenous
miR-21 in HeLa and HEK 293T human cell lines with robust
2
1.5
miRNA activities. (b) Ectopic expression followed by pull-down
of recombinant AGO1 and AGO2 was assessed in total lysates (T),
1.0
1
supernatants (S) or precipitates (P) from HEK 293T cells. The
amount of expressed AGO1 and AGO2 proteins was normalized
0.5
to β-actin (Actin) expression. (c) RT-PCR of the miR-21 target
0
0
PDCD4 mRNA demonstrated approximately 2.5-fold enrichment
AGO1 AGO2 AGO1+2 Control
AGO1 AGO2 AGO1+2 Control
in AGO1 and AGO2 precipitates from cells transfected with the
miR-21−miR-21* duplexes, as compared to precipitates from cells
transfected with the nonspecific control CXCR4−CXCR4* duplexes
(right). In contrast, RT-PCR of PDCD4 mRNA demonstrated an
approximately ten-fold enrichment in AGO2 precipitates from cells
transfected with the miR-21P trigger RNA, as compared to the
Actin
Actin
nonspecific control (left). (d) AGO1 does not potentiate AGO2mediated mRNA cleavage in cells. Limiting amounts of CXCR4P or CXCR4−CXCR4* were cotransfected into HEK 293T cells with plasmids
expressing recombinant AGO1, AGO2 or AGO1 and AGO2 (where AGO1 and AGO2 plasmid concentrations were one-half of the concentration of
each expression plasmid used alone) and with reporter plasmids as indicated. Target mRNA cleavage and translational repression were assessed by
luciferase assays (FL1P/RL0X and FL6X/RL0X, respectively). Each ratio was normalized to transfection of a scrambled control small RNA. Error bars
indicate s.d., corresponding to the average of two assays performed in duplicate. Western blots using an anti-GST antibody show the levels of ectopic
expression of AGOs.
FL1P/RL0X fold
repression
© 2009 Nature America, Inc. All rights reserved.
3
(less than 1.5-fold). These data indicate that, in vivo, both AGO1 and
AGO2 load the miR-21−miR-21* RNA duplexes into a repressioncompetent complex, whereas only AGO2 loads the miR-21P RNA duplexes
into a repression-competent complex, consistent with in vitro data.
To test passenger strand cleavage and strand-dissociating activities
of AGO1 and AGO2 in cells, we created a competition between AGO1
and AGO2 by limiting the amount of available dsRNAs (Fig. 5d). As
expected, ectopic expression of AGO1 alone did not stimulate mRNA
cleavage of a CXCR4 siRNA duplex or a CXCR4 miRNA duplex. In
contrast, ectopic expression of AGO2 did stimulate mRNA cleavage
using either dsRNA. Notably, coexpression of AGO1 with AGO2 (half
the amount of each protein alone) reduced AGO2-mediated mRNA
cleavage triggered by the CXCR4 siRNA but not the CXCR4 miRNA
mimic. Because AGO1 possesses passenger strand cleavage activity
but not target mRNA cleavage activity, these data suggest that AGO1
competes efficiently with AGO2 for limiting siRNA duplexes but
not for miRNA duplexes. It is likely that AGO1 cleaves both strands
of siRNA duplexes endonucleolytically, rendering them useless for
AGO2-mediated mRNA cleavage.
In contrast to the absence of mRNA cleavage activity of AGO1
in cells (Fig. 5d, left), ectopically expressed AGO1 stimulated translational repression triggered by the CXCR4 miRNA mimic but not
the CXCR4 siRNA (Fig. 5d, right). As in mRNA cleavage, ectopic
­expression of AGO2 stimulated translational repression equally
using either dsRNA, and ectopic coexpression of AGO1 with AGO2
did not potentiate translational repression by either protein alone
when triggered by the CXCR4 miRNA mimic. Unlike mRNA cleavage, however, ectopic coexpression of AGO1 with AGO2 did not
reduce the translational repression triggered by the CXCR4 siRNA
duplex. Previous observations imply that AGO1 and AGO2 perform
redundant functions in translational repression8; thus, competition
between AGO1 and AGO2 for limiting siRNAs would not exist. Still,
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articles
Endonuclease
Chaperone
siRNA
miRNA
AGO1
AGO1
AGO2
AGO2
Figure 6 RNA chaperone model of AGO1- and AGO2-mediated RISC
loading. Both siRNA and miRNA−miRNA* duplexes may be sampled
by AGO1 (purple ball) or AGO2 (blue ball). However, only AGO2 is
functionally complexed with target strands by endonucleolytic cleavage
of passenger strands (left). In contrast, both AGO1 or AGO2 may be
functionally complexed with the target strand through strand-separating
activity (right). In the RNA chaperone model, not all guide strands
complexed with an AGO protein are incorporated into functional RISCs,
as AGO proteins recycle to sample siRNA and miRNA−miRNA* duplexes.
to coprecipitate target mRNAs, and ssRNA is likely to be degraded
quickly when it is not bound by proteins.
In addition to facilitating RNA strand dissociation, RNA chaperones also facilitate RNA strand annealing. Although we show that
AGO2 binds to miRNAs first and is then recruited to target mRNAs
(Supplementary Fig. 7d), we have also demonstrated that endo­genous
AGOs may be recruited to miRNAs that are pre-annealed to target
mRNAs27,28. The low affinity of AGO1 for ssRNAs compared to that of
AGO2 (Fig. 1 and Supplementary Figs. 2 and 3) suggests that AGO1
may facilitate miRNA duplex strand dissociation for subsequent
loading onto AGO2. It is also possible that AGO1 facilitates guide
strand association with the target mRNA followed by AGO2 recruitment to guide strand−target mRNA duplexes. Thus, AGO1 and
AGO2 might be considered RNA chaperones, because they facilitate
dissociation of strands of miRNA duplexes and guide strand ­annealing
to mRNA targets.
the exact reason(s) that AGO1 does not compete with AGO2 for limiting siRNA duplexes in translational repression is not clear.
Taken together, these data indicate that AGO1 uses strand­dissociating activity more efficiently than it does passenger strand
cleavage activity in stimulating translational repression. In contrast,
AGO2 does not demonstrate a preference for strand-dissociating
activity or passenger strand cleavage activity in stimulating mRNA
cleavage or translational repression.
DISCUSSION
AGO1 and AGO2 dissociate strands of miRNA duplexes
AGO1 and AGO2 have been identified in multiple mammalian RISC
complexes15,17,41,42, but the exact functions of these proteins in RISC
loading have not been determined. Many models of miRNA function
imply direct coupling of miRNA biogenesis and RISC loading20,43,44.
However, many knockdown experiments using siRNAs and miRNA
mimics indicate that RISC loading may bypass the miRNA biogenesis
machinery. Our data demonstrate that AGO1 and AGO2 have ­intrinsic
RISC-loading activity that is independent of the miRNA biogenesis
machinery. Consistent with the view that human RISC assembly does
not require ATP21, we demonstrate that human AGO1 and AGO2 possess ATP-independent strand-dissociating activity of an miRNA duplex
but not an siRNA duplex (Fig. 2). Indeed, protein motif searches
did not identify any known RNA helicase or ATP-­binding motifs in
the primary sequence data of the human AGOs (data not shown).
The lack of ATP dependence and the increasing strand-dissociating
activity over time using substoichiometric amounts of AGO proteins
suggest that AGO proteins may function not as RNA helicases but,
instead, as RNA chaperones45.
In this RNA chaperone model (Fig. 6), AGO1 and AGO2 dissociate
the strands of more than one miRNA duplex, leading to free miR-21
and AGO-bound miR-21. This model implies that, in a subpopulation
of RISC, the AGO−guide strand interaction is weak and/or that, in
cells, other RISC-interacting proteins stabilize the AGO−guide strand
interaction. Indeed, this must be the case for at least a subpopulation
of RISCs, because the AGO-miRNA interaction is sufficiently stable
Passenger strand cleavage by AGO2 activates RISC in cells
Here we report that AGO1 and AGO2 possess siRNA endonuclease
(strand sampling) activity, but only AGO2 possesses target RNA cleavage activity. However, siRNA duplexes do not lead directly to AGO2mediated target RNA cleavage in our minimal assay system (Fig. 4a,b,
lane 9). One explanation may be that AGO2 samples both strands of
an siRNA duplex but a strand-selectivity factor (absent from minimal reactions) is required to selectively inhibit sampling of the guide
strand. However, we show that the AGO2 remains stably bound to
passenger strand cleavage fragments (Fig. 1d,e), implying that passenger strand release from AGO2 (maturation of active RISC) is rate
limiting. These results are analogous to kinetic analysis of RISC cleavage of target mRNAs26. In that study26, in the absence of ATP, the rate
of multiple rounds of target mRNA cleavage was limited by the rate
of release of the mRNA cleavage fragments from RISC. It is possible
that a factor that is absent from minimal reactions may be required to
promote release of the cleavage products to generate active RISC.
It is not clear why AGO1 demonstrates passenger strand cleavage
activity in vitro but only slightly enriches miR-21 target mRNAs in
cells when the miR-21P duplex is transfected into cells. AGO1 may
be used preferentially for strand dissociation, whereas AGO2 may
be used preferentially for passenger strand cleavage for RISC loading. The exact mechanism(s) of RISC loading will require further
structure-function analyses of AGOs bound to different trigger RNAs
assessed in vitro and in cells.
Methods
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/nsmb/.
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
Acknowledgments
We thank M. Janas and J. Doench for insightful recommendations and critical
reading of the manuscript. We also thank Y. Pan for assistance with the AGO
nature structural & molecular biology advance online publication
articles
pull-down assays. We thank E. Gagnon for creating Figure 6. This work was
supported by a Distinguished Young Scholars Award from the W.M. Keck
Foundation to C.D.N.
AUTHOR CONTRIBUTIONS
B.W. designed and performed experiments, analyzed data and co-wrote the
manuscript; S.L. and H.H.Q. performed experiments; D.C. and Y.S. analyzed data;
C.D.N. designed experiments, analyzed data and co-wrote the manuscript.
© 2009 Nature America, Inc. All rights reserved.
Published online at http://www.nature.com/nsmb/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
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ONLINE METHODS
© 2009 Nature America, Inc. All rights reserved.
Expression and purification of AGO proteins. We cloned full-length human
AGO1−4 into XhoI/NotI sites of pGEX-4T-1 and expressed the N-terminal GST
fusion AGO proteins in Rosetta strains. We cultured 500 ml of these bacteria at
37 °C until the cultures reached an absorbance at 600 nm (A600nm) of 0.8. We
then induced protein expression with 0.5 mM IPTG for 5 h at room temperature
(23–25 °C). We washed the bacteria with 1× PBS buffer, lysed cells for 20 min on
ice in STE lysis buffer (19 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl,
0.2% (v/v) Triton X-100, 2 mM PMSF, 1 mM DTT and 1× Protease Inhibitor
(Roche)), froze the lysates at −80 °C and then sonicated the lysates for 30 s six
times with 1 min intervals, on ice. We added 250 µl glutathione agarose resin
and incubated the samples for 2 h at 4 °C with rotation, washed the resins five
times with 1× PBS, loaded the resins onto Bio-spin disposable chromatography
columns (BioRad) and eluted the recombinant AGO proteins with 50 mM TrisHCl, pH 8.0, and 20 mM glutathione at room temperature. We then concentrated and purified the eluted proteins with Microcon (Millipore, YM-100).
Small RNAs. We obtained the following miRNA sequences from
mirBaseSequence (http://microrna.sanger.ac.uk/sequences/): human miR-21
(5′-UAGCUUAUCAGACUGAUGUUGA-3′); miR-21* (5′-CAACACCAGU
CGAUGGGCUGU-3′) and pre−miR-21 (5′-UGUCGGGUAGCUUAUCAGACU
GAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCU
GACA-3′). The miR-21-P sequence is 5′-AACAUCAGUCUGAUAAGCUAGU-3′.
The sequences of the CXCR4 siRNA are 5′-GUUUUCACUCCAGCUAACACA-3′
(sense strand) and 5′-UGUUAGCUGGAGUGAAAACUU-3′ (antisense strand).
The CXCR4* sequence is 5′-GUUUUCACAAAGCUAACACA-3′. Integrated DNA
Technologies (IDT) synthesized all RNA molecules and modified all RNAs with
or without 5′ monophosphate. We resuspended all RNA molecules in diethylpyrocarbonate (DEPC)-treated water and quantified the RNA oligonucleotides
using NanoDrop UV spectrometry.
5′ end labeling of RNA molecules and formation of RNA duplexes. We labeled
each non−5′-phosphorylated RNA strand using T4 polynucleotide kinase (PNK,
New England Biolabs). We incubated 20 pmol of RNA strand in 20-µl reactions
containing 1× PNK buffer and 100 µCi of γ-32P-ATP at 37 °C for 60 min and
removed the unincorporated nucleotides using ProbeQuant G-50 micro columns (GE Healthcare). We gel purified and mixed each labeled RNA strand with
unlabeled reverse complementary strand in equimolar amounts to form the
duplexes miR-21−miR-21*, miR-21P, CXCR4−CXCR4* or CXCR4P by denaturing at 75 °C for 3 min and cooling to room temperature for 5 min. We also
denatured and cooled the labeled pre−miR-21 to form a hairpin structure.
Strand-dissociating activity assay. We performed RNA duplex stranddissociating activity as described46 in 20-µl reactions containing 3 µg of yeast
tRNAs, 20 mM HEPES-KOH, pH 7.5, 2 mM DTT, 3 mM MgCl2, 0.1 mg ml−1
BSA, 1 U RNase Out (Invitrogen), 150 fmol of RNA duplex and 50 fmol of purified recombinant human Ago with or without 1 mM ATP at 37 °C for 30 min.
We terminated these reactions with 5 µl of loading buffer (0.1 M Tris, pH 7.5,
20 mM EDTA, 0.5% (w/v) SDS, 0.1% (w/v) NP-40, 0.1% (w/v) bromophenol
blue, 0.1% (w/v) xylene cyanol and 50% (v/v) glycerol and separated the reaction products by native 10% PAGE, run at 500 V for 2 h at 4 °C. We quantified
the gels by Phosphorimager analysis (Molecular Dynamics). We performed
time course analysis of strand-dissociating activity using a constant amount
of AGO protein and separated 10 µl of reaction products by native 10% PAGE
run at 500 V for 2 h at 4 °C. We dried the gels and quantified the bands by
Phosphorimager analysis.
Electrophoretic mobility shift assay. To test RNA-binding activity of AGOs, we
incubated 150 fmol of radiolabled ssRNAs or duplex RNAs with 50 fmol purified
AGOs in 20-µl reactions containing 3 µg of yeast tRNAs, 20 mM HEPES-KOH,
pH 7.5, 3 mM MgCl2, 10% (v/v) glycerol, 1 mM DTT and 0.1 mg ml−1 BSA, with
or without 25 pmol of cold ssRNAs or duplex RNAs for 10 min. We stopped these
reactions by placing them on ice and adding 3 µl of loading buffer (50% (v/v) glycerol, 1% (w/v) bromophenol blue and 1% (w/v) xylene cyanol), and 10-µl aliquots
were electrophoresed by 8% PAGE at 500V for 2 h at 4 °C46. We dried the gels and
quantified the bands by Phosphorimager analysis. To assess the association between
doi:10.1038/nsmb.1712
passenger strand cleavage fragments and AGO proteins, we subjected the mobilityshifted RNA-protein complexes to autoradiography and excised and eluted the
mobility-shifted bands in 0.3 M NaCl overnight at 4 °C. We extracted the eluate
with phenol:chloroform:isoamy-alcohol (25:24:1, pH 6.0), ethanol-precipitated the
protein and separated the resuspended pellet by 20% PAGE containing 7M urea.
Quantitative analysis of AGO1 and AGO2 affinities to dsRNAs. To test the time
course of RNA-binding activity of AGOs, we incubated 150 fmol of radiolabled
ssRNAs or duplex RNAs with 50 fmol of purified AGOs in 20-µl reactions containing 3 µg of yeast tRNAs, 20 mM HEPES-KOH, pH 7.5, 3 mM MgCl2, 10%
(v/v) glycerol, 1 mM DTT and 0.1 mg ml−1 BSA for the indicated time periods. To determine the Kd of AGO1 or AGO2, we used either constant dsRNAs
(miR-21P or miR-21−miR-21*) or AGOs in affinity reactions for 45 min. We
stopped these reactions by placing them on ice, adding 3 µl of 6× loading buffer
(50% (v/v) glycerol, 1% (w/v) bromophenol blue and 1% (w/v) xylene cyanol).
We electrophoresed a 10-µl aliquot by 8% PAGE at 500 V for 2 h at 4 °C and
quantified the bands by Phosphorimager analysis.
AGO1 and AGO2 precipitation in vitro. To immunoprecipitate AGO proteins,
we conjugated anti-GST monoclonal antibody to Protein G beads (CalBiochem)
as specified by the manufacturer. We added these beads to reactions, incubated
them for 1 h at 4 °C with bidirectional mixing, washed the beads three times with
IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl and 0.01% (w/v) Triton X-100) and
subjected the washed beads to western blotting or RNA extraction.
To immunoprecipitate AGO2 from RRL, we incubated reactions with AGO2
antibody−conjugated (Wako) or anti-ICAM antibody−conjugated (AMAC)
Protein G beads (CalBiochem) for 1 h at 4 °C. We washed the beads three times
with IP buffer and subjected the precipitates to western blotting or RNA extraction. We separated the labeled RNA products by 8% PAGE containing 7 M urea.
RNA cleavage assay. We prepared the miR-21 target substrate as described28. The
sequences for miR-21 and CXCR4 target substrates are 5′-GAACAAUUGCUU
UUACAGAUGCACAUAUCGAGGUGAACAUCACGUACGUCAACAUCAGUC
UGAUAAGCUAUCGGUUGGCAGAAGCUAU-3′ and 5′-CGGAAAGAUCGCC
GTGTAAUUCUAGACUCGAGCCGGAAGUUUUCACUCCAGCUAACACCG
GAUCGCGGGCCCGUUUAAACCCGCUGAUC-3′. We incubated radiolabeled
miR-21 or CXCR4 target RNAs with or without ssRNAs or dsRNAs and with or
without purified AGOs in 50-µl reactions in cleavage buffer (3 µg of yeast tRNAs,
25 mM HEPES-KOH, pH 7.5, 50 mM potassium acetate, 5 mM magnesium
acetate and 5 mM DTT) at 37 °C for 90 min. We terminated these reactions by
adding equal volumes of phenol:chloroform:isoamy-alcohol, precipitating in
ethanol and separating the resuspension by 20% PAGE with 7 M urea for small
RNA cleavage or by 8% PAGE for target mRNA cleavage.
AGO1 and AGO2 precipitation in cells. For immunoprecipitation of AGO1 and
AGO2 from cells, we cotransfected HEK 293T cells with hemagglutinin (HA)tagged AGO1 or AGO2 plasmid and miR-21P or miR-21−miR-21*, lysed the
transfected cells with lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES,
pH 7.0, 0.5% (w/v) NP-40, 100 U ml−1 of RNase Out (Invitrogen) and complete protease inhibitor cocktail (Roche)), added the lysate to HA-probe (F-7)
AC (SC Biotechnology) and incubated the samples for 4 h at 4 °C. We treated
the beads with DNase I in NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl,
1 mM MgCl2 and 0.05% (w/v) NP-40) at 37 °C for 10 min, and then added
protease K and SDS in NT2 buffer. We extracted the RNAs and assayed target
mRNA expression by quantitative PCR using the miScript PCR kit (Qiagen).
In vitro translational repression assay. We performed translational repression assay as described27,28,47. We incubated 0.025 pmol of firefly luciferase
reporter (FL6X) and 0.025 pmol of control Renilla luciferase RNA (RL0X) with
0.15 pmol of small RNAs and 7 µl of RRL in 10-µl reactions with or without
AGO2 at 30 °C for 15 min and performed dual luciferase assays.
46.Izzo, A., Regnard, C., Morales, V., Kremmer, E. & Becker, P.B. Structure-function
analysis of the RNA helicase maleless. Nucleic Acids Res. 36, 950–962 (2008).
47.Wang, B., Doench, J.G. & Novina, C.D. Analysis of microRNA effector functions
in vitro. Methods 43, 91–104 (2007).
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