Characterization of endogenous human Argonautes
and their miRNA partners in RNA silencing
Asuka Azuma-Mukai†, Hideo Oguri‡, Toutai Mituyama§, Zhi Rong Qian†, Kiyoshi Asai§¶, Haruhiko Siomi†储,and Mikiko C. Siomi†储††‡‡†Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan; ‡First Institute of New Drug Discovery, Otsuka Pharmaceutical Co.,

Ltd., Tokushima 771-0192, Japan; §Computational Biology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo
135-00664, Japan; ¶Graduate School of Frontier Science, University of Tokyo, Chiba 277-8561, Japan; ††Japan Science and Technology Agency, Core Research
for Evolutional Science and Technology, Saitama 332-0012, Japan; and 储Keio University School of Medicine, Tokyo 160-8582, Japan
Edited by Michael Rosbash, Brandeis University, Waltham, MA, and approved March 21, 2008 (received for review January 11, 2008)

Small RNAs triggering RNA silencing are loaded onto Argonautes
and then sequence-specifically guide them to target transcripts.
Epitope-tagged human Argonautes (hAgo1, hAgo2, hAgo3, andhAgo4) are associated with siRNAs and miRNAs, but only epitopetagged hAgo2 has been shown to have Slicer activity. Contrarily,
how endogenous hAgos behave with respect to small RNA association and target RNA destruction has remained unclear. Here, we
produced monoclonal antibodies for individual hAgos. Highthroughput pyrosequencing revealed that immunopurified endogenous hAgo2 and hAgo3 associated mostly with miRNAs. Endogenous hAgo3 did not show Slicer function but localized in P-bodies,suggesting that hAgo3 endogenously expressed is, like hAgo2,
involved in the miRNA pathway but antagonizes the RNAi activityof hAgo2. Sequence variations of miRNAs were found at both 5ⴕ
and 3ⴕ ends, suggesting that multiple mature miRNAs containing
different ‘‘seed’’ sequences can arise from one miRNA precursor.
The hAgo antibodies we raised are valuable tools for ascertaining
the functional behavior of endogenous Argonautes and miRNAs in
RNA silencing.
Jurkat 兩 siRNAs 兩 SlicerRNA silencing is an evolutionarily conserved physiologicalprocess regulating gene expression (1). RNA interference(RNAi), the prototype of RNA silencing, down-regulates gene
expression by cleaving target mRNAs in a sequence-dependent
manner (2). A molecule that renders sequence-specificity is theshort interfering RNA (siRNA) of 21- to 23-nt length. MicroRNAs (miRNAs) are a large set of endogenous small RNAs
(21–23 nt long) encoded in the genome of a variety of organisms(3). miRNA functions in RNA silencing by regulating theexpression of target genes by inhibiting translation or destructing
mRNAs (3, 4). The miRNA target genes identified to date havemostly been involved in vital biological processes such as development, apoptosis, neural patterning, and viral infection. The
origins of siRNAs and miRNAs differ from each other, but bothare processed from a longer double-stranded RNA precursor by
RNase III domain-containing Dicer and loaded onto a memberof the Argonaute family of proteins (2).
Members of the Argonaute family are defined by the presence of
the PAZ and PIWI domains (5). The numbers of Argonaute
members vary from species to species. In humans, the Argonautefamily has eight members, four of which (AGO subfamily members;
hAgo1–hAgo4) are expressed in numerous tissues and cell types(5). Although comprehensive analysis remains to be done, expressions of other members of the Argonaute family in humans (PIWI
subfamily members; Hiwi1, Hiwi2, Hiwi3, and Hili) appear to be
restricted to germ lines. Recent studies on rodents, fish, and flies
have revealed that Argonautes expressed only in germ lines (PIWI
subfamily members) are associated with piRNAs (Piwi-interacting
small RNAs), a subset of small RNAs exclusively expressed in germlines (6). Unlike miRNAs, piRNAs are 24 to 31 nt long and derived
from discrete piRNA loci of chromosomes in a Dicer-independent

7964 –7969 兩 PNAS 兩 June 10, 2008 兩 vol. 105 兩 no. 23

manner (7). piRNAs are necessary for gametogenesis in organisms.
In particular, it has been shown that in flies, piRNAs function insilencing retrotransposons to preserve DNA integrity in germline
cells (6).
Much of the evidence obtained to date supports the conceptthat ubiquitously expressed Argonautes (namely AGO subfamily
members) function in RNA silencing through specific association with siRNAs and miRNAs (5). It has been reported that theimmunoprecipitates of anti-hAgo2 contain miRNAs (8, 9);
however, if the antibody specifically recognized hAgo2 or crossreacted with other hAgos was, at that time, an unanswered issue.
Later, biochemical analysis of all hAgos in respect to small RNA
association was performed with epitope-tagged hAgos expressedin mammalian cells, revealing that both miRNAs and siRNAs areindiscriminately associated with hAgo1 through hAgo4 containing miRNPs (10, 11). It has also been demonstrated that Slicer
activity is exclusively associated with hAgo2 (11, 12). Disruptionof the mouse Ago2 (mAgo2) gene produced an embryonic-lethal
phenotype (10), suggesting that Argonautes are not redundantand might be biologically specialized.
Recently, biochemical approaches were established for predicting miRNA targets in human cells (12). In that study, hAgo1
and hAgo2 exogenously expressed in HEK 293 cells were
immunopurified with specific antibodies against hAgo1 andhAgo2. cDNA libraries were then generated from the mRNApools extracted from the immunoprecipitates. Identification of
cDNA clones in the libraries revealed that only a limited numberof mRNA overlap between hAgo1 and hAgo2. It was suggested
that many miRNA targets might be specific to one Ago protein.
As indicated above, functional analyses of hAgos have so far
been performed by using epitope-tagged, exogenously expressed
Argonautes. However, how endogenous hAgos behave in regard
to small RNA association and target RNA destruction has notbeen determined. To address these questions, we produced
monoclonal antibodies against each of the hAgos and identified
small RNAs associated with endogenous hAgo2 and hAgo3.
High-throughput pyrosequencing revealed that hAgo2 andhAgo3 immunopurified from Jurkat cells are mostly associatedwith miRNAs, which in the hAgo2 and hAgo3 libraries arelargely overlapped with only a few miRNAs detected discrimiAuthor contributions: A.A.-M., H.O., T.M., K.A., H.S., and M.C.S. designed research; A.A.-M.,
H.O., T.M., and Z.R.Q. performed research; A.A.-M., H.O., T.M., and Z.R.Q. contributed new
reagents/analytic tools; A.A.-M., H.O., T.M., Z.R.Q., K.A., H.S., and M.C.S. analyzed data; andH.S. and M.C.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The miRNAs found in this study [hsa-mir-1537, hsa-mir-1538, hsa-mir1539, hsa-mir-1284, and hsa-mir-103-1-as (chr5)/hsa-mir-103-2-as (chr20)] have been deposited in miRBase (http://microrna.sanger.ac.uk).
‡‡To whom correspondence should be addressed. E-mail: siomim@sc.itc.keio.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/
0800334105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800334105

nately. Sequence variations in miRNAs were observed at both 5⬘
and 3⬘ ends, raising the possibility that miRNAs containing
different ‘‘seed’’ sequences are multiply produced in vivo fromone precursor sequence. Strong accumulation of endogenous
hAgo2 and hAgo3 into P-bodies further supports hAgo3 involvement in miRNA-mediated RNA silencing pathways. However,endogenous hAgo3 associated with miRNA and siRNA showed
little or no Slicer activity in vitro, suggesting that hAgo3 antagonizes RNAi activity in cells. The antibodies we raised against
individual hAgos are valuable tools to reveal the functional
behavior of endogenous Argonautes and their miRNA partners
in RNA silencing mechanisms.

A
B

C

Results
previously reported that hAgo1, hAgo2, hAgo3, and hAgo4
associate with both siRNAs and miRNAs (10, 11). However, allsuch analyses have been performed by using exogenously expressed, epitope-tagged hAgos in mammalian cells (10, 11). By
contrast, how endogenous hAgos behave in regard to small RNA
association has remained unelucidated. This prompted us to
produce antibodies specifically recognizing individual hAgos.
We expected that these would be useful for ascertaining endogenous hAgo functions in RNA silencing. As antigens to immunize mice, we used N-terminal regions of hAgos, because theregions show relatively lower similarities compared with others,
including the highly conserved PAZ and PIWI domains [supporting information (SI) Fig. S1]. Antibody specificity wasdetermined by Western blot analysis on the peptides used for
immunizing mice. This indicated that monoclonal antibodies
specific for individual hAgos were successfully obtained (Fig.
S2). Western blot analysis data on human cell lysates with theantibodies is shown in Fig. S2F.
We assessed whether anti-hAgos work for immunoprecipitation. It has been shown that Flag-tagged-hAgo1 copurifies with
eGFP-hAgo2 from HEK 293 cells (13). In our experiments, toavoid such undesired protein–protein interactions, a strong
zwitterionic detergent, Empigen, was used. Even under harshconditions, hAgo2 and hAgo3 were efficiently immunoprecipitated from Jurkat cells (Fig. 1A). Western blot analysis determined that the protein bands indicated with arrowheads on thesilver-stained gel (Fig. 1 A) correspond to hAgo2 (green) andhAgo3 (red) (Fig. 1 B and C). No other hAgos were detected in
the hAgo2 and hAgo3 immunoprecipitates (Fig. S3). AntihAgo1 was inefficient for immunoprecipitation (data notshown). Anti-hAgo4 immunoprecipitated hAgo4 (blue arrowhead in Fig. 1 A), but it also reacted to other proteins (single anddouble asterisks in Fig. 1 A and Fig. S3C). Mass spectrometricanalysis identified the protein migrating at nearly 120 kDa(double asterisks in Fig. 1 A and Fig. S3C) as the E1B-55kDaassociated protein (accession no. CAA07548), a cellular protein
implicated in the nucleocytoplasmic transport of adenovirus andcellular mRNAs (14); however, no implication in RNA silencing
has yet been reported.
Anti-hAgo2 and -hAgo3 were also useful for immunostaining;
we found that both hAgo2 and hAgo3 localized in P-bodies(processing bodies) (Fig. 2), the cytoplasmic foci where miRNAtargets are accumulated as a consequence of translational repression (15, 16). P-bodies, where DCP1a, a factor functioning
in mRNA decay in the bodies (17), resides, were mostly
costained with anti-hAgo2 or anti-hAgo3. hAgo2 and hAgo3 areunlikely to be sorted into different sets of P-bodies.
Small RNAs Associated with Endogenous hAgo2 and hAgo3. Small

RNAs associated with endogenous hAgo2 and hAgo3 in mammalian cells are as yet undetermined. In this study, RNAs were
purified from immunopurified endogenous hAgo2 and hAgo3(Fig. 1 A) and visualized by 32P-labeling. Both proteins wereAzuma-Mukai et al.

Fig. 1. Characterization of immunoprecipitates with anti-hAgo2, -hAgo3,
and -hAgo4 antibodies from Jurkat cells. (A) Jurkat cell lysates prepared witha cell lysis buffer containing Empigen, and immunoprecipitation performedby using anti-hAgo2, -hAgo3, and -hAgo4 antibodies. Protein components in
the immunoprecipitates were stained with silver. Protein bands indicated witharrowheads in green, red, and blue are hAgo2, hAgo3, and hAgo4, respectively. Immunoprecipitates of anti-hAgo4 contain proteins other than hAgo4(shown with single and double asterisks), demonstrating undesired crossreactivity of the antibody. The protein indicated with double asterisks was
identified by mass spectrometric analysis as E1B-55kDa-associated protein.
h.c., heavy chains of the antibodies used for immunoprecipitation. (B) Immunoprecipitates of anti-hAgo2 analyzed by Western blot analysis with antihAgo2. (C) Immunoprecipitates of anti-hAgo3 analyzed by Western blot
analysis with anti-hAgo3.

revealed to associate with small RNAs ranging from 21 to 23 nt(Fig. 3A). It was noted that the exposure time for hAgo3 small
RNAs was approximately three times longer than that of hAgo2
small RNAs, indicating that the amount of hAgo3 small RNAsA

B

Fig. 2. Immunofluorescence on HeLa cells with anti-hAgo2 and anti-hAgo3.
(A) HeLa cells immunostained with anti-hAgo2 and anti-DCP1a antibodies. It
is shown that hAgo2 localizes in P-bodies (green), the cytoplasmic foci where
miRNA targets are accumulated as a consequence of translational repression
mediated by Argonaute–miRNA complexes (15, 16). The staining patters ofhAgo2 were quite similar to those of epitope-tagged hAgo2 exogenously
expressed in HEK 293 or U2-OS cells (10, 11, 13). Almost all of the P-bodies,
where DCP1a, a factor functioning in mRNA decay in the bodies (17), resides(red), are costained with anti-hAgo2 (yellow arrowheads in merged image).
Part of the merged image is enlarged (enlarged), where a P-body costainedwith anti-hAgo2 and anti-DCP1a is indicated by a yellow arrowhead. We
occasionally found foci only stained with anti-hAgo2 and anti-DCP1a, which
are indicated by green and red arrowheads, respectively. (B) HeLa cells stainedwith anti-hAgo3 (green) and anti-DCP1a (red). Almost all of the P-bodies arestained with both of the antibodies (yellow arrowheads). The cytoplasmic focicontaining only hAgo3 or DCP1a are indicated by green and red arrowheads,
respectively (in the enlarged image).
PNAS 兩 June 10, 2008 兩 vol. 105 兩 no. 23 兩 7965

BIOCHEMISTRY

Monoclonal Antibodies for Individual Endogenous hAgos. It has been

B

A

C

Fig. 3. Identification of small RNAs associated with hAgo2 and hAgo3 in
Jurkat cells. (A) Small RNAs associated with hAgo2 and hAgo3 in Jurkat cells
visualized by 32P-labeling. Both hAgos are associated with small RNAs ranging
from 21 to 23 nt in length. (B) Compositions of small RNAs in the hAgo2 andhAgo3 libraries. The most abundant class of small RNAs in both libraries was
miRNAs. Small RNAs corresponding to sequences of piRNA (piwi-interactingRNA) (0.03%), rRNA (ribosomal RNA) (0.08%), tRNA (transfer RNA) (0.004%),
snRNA (small nuclear RNA) (0.002%), snoRNA (small nucleolar RNA) (0.01%),
and scRNA (small cytoplasmic RNA) (0.004%) appear in the hAgo2 library. The
hAgo3 library had piRNA (0.13%), tRNA (0.001%), snRNA (0.01%), snoRNA(0.005%), and scRNA (0.007%). Their appearance was negligible; thus, they
apparently did not show up in the graphs. (C) Top 30 miRNAs appearing ineach library.

obtained in the experiments was lower than that of hAgo2 small
RNAs. This would have been due to the fact that hAgo3
expression levels are lower than those of hAgo2 in Jurkat cells(data not shown). Other factors, such as immunoprecipitation
efficiency, could also be at play. To identify the small RNAs withhAgo2 and hAgo3, cDNA libraries were constructed and highthroughput pyrosequencing was performed. Total reads fromhAgo2 and hAgo3 libraries were 68,976 and 85,690, respectively.
Compositions of small RNAs identified are shown in Fig. 3B. It
was clearly indicated that the majority in both libraries were
miRNAs [⬇75% (51,780) and ⬇88% (76,084) in hAgo2 andhAgo3, respectively] representing 203 (hAgo2) and 181 (hAgo3)
distinct known miRNAs (Dataset S1). Five novel miRNA candidates were also found in the hAgo2 library (Fig. S4).
miRNA profiles in the hAgo2 and hAgo3 libraries overlapped
to a large extent. The Top 30 miRNAs in both libraries are
7966 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800334105

summarized in Fig. 3C and Table S1. miRNAs such as miR-150
and miR-142-3p appeared frequently in both [13.0% and 16.7%(miR-150) and 8.4% and 24.2% (miR-142-3p) of total miRNAs
in the hAgo2 and hAgo3 libraries, respectively] (Fig. 3c). We also
observed that some miRNAs were discriminated by hAgo2 andhAgo3. The Top 20 miRNAs observed differentially in thehAgo2 and hAgo3 libraries are summarized in Dataset S2.
Although it remains unknown how such miRNA loading discrimination occurs in vivo, it is suggested that hAgo2 and hAgo3
are occasionally directed to specific target mRNAs.
miRNA profiling studies have been performed in 26 different
organ systems and cell types of human and rodents (18), where
sequence variations in mature miRNA are reported. As well,Morin et al. (19) have recently reported from massively parallel
sequencing that miRNAs expressed in human embryonic stem
cells (hESCs) and embryoid bodies (EBs) exhibited sequencevariation at their 5⬘ and 3⬘ ends. In our study, we also foundend-sequence variations for nearly all of the miRNAs we identified in the hAgo2 and hAgo3 libraries (Fig. S5). 3⬘-Terminal
additions were more apparent than 5⬘-terminal additions (Fig.
4). Some of these nucleotide additions likely occurred after
miRNA maturation by Dicer and Drosha processing, because
those nucleotides were not identical to the precursor sequences(nontemplated extensions) (Fig. 4A). We further examined thevariations and found that the addition of A, U, and C, but little
or no G addition, occurred at both ends (Fig. S6). Other variants
had additional nucleotides that were identical to the precursor
sequences (alternative ends) (Fig. 4B), indicating that they might
be produced directly through Dicer and Drosha miRNA processing activities. miRNAs with alterative ends appeared more
frequently than those with nontemplated extensions (Fig. 4).
Although the prevalence was lower compared with that of 3⬘-end
variations, 5⬘-end variations were also clearly observed. This
raised the possibility that various miRNAs containing different‘‘seed’’ sequences, the core region of miRNAs functioning in
identifying miRNA target transcripts (20), are produced fromone precursor sequence in vivo.
Endogenous hAgo2 Slicer Activity. Transfection experiments haverevealed that in humans only hAgo2 has Slicer activity (10, 11).
However, we wondered whether endogenous hAgo2 could indeed exhibit Slicer activity. In the transfection experiments, it
was unclear whether hAgo2 was purified alone or copurified withother hAgos. In our case, immunopurified hAgo2 in an Empigen
buffer did not copurify with other hAgos (Fig. S3) but still
associated with miRNAs (Fig. 3). hAgo2 immunopurified fromHeLa cells under presumably milder Nonidet P-40 conditions
was able to cleave a target RNA containing a sequence completely matching to miR-21 (miR-21 target) (Fig. 5A). We also
observed that hAgo2 immunopurified from HeLa cells in an
Empigen buffer was able to exhibit Slicer activity (Fig. 5A).
When isolated in Empigen, hAgo2 showed less activity to bindwith miR-21 and to cleave the target than itself isolated in
Nonidet P-40 (Fig. 5 A and B). This was most likely due toEmpigen’s affecting the strength of the antibody to bind theantigen (data not shown).
Endogenous hAgo3 Does Not Function as Slicer. We assessed

whether endogenous hAgo3 shows Slicer activity like endogenous hAgo2. The appearance of miR-150 in the hAgo2 andhAgo3 libraries was 13.0% and 16.7%, respectively (Fig. 3C).
Thus, in vitro target RNA cleavage assays were set up to target
RNA (miR-150 target) harboring a sequence fully complementary to miR-150. hAgo3 was immunopurified from three volumesof the Jurkat cell lysate that was used for hAgo2 immunopurification; the amount of hAgo3 immunopurified was ⬇78% ofthat of hAgo2 (Fig. S7). Northern blot analysis confirmed thatthe amounts of miR-150 associated with hAgo3 under such
Azuma-Mukai et al.

AB

circumstances were approximately half of that bound to hAgo2(Fig. S7). Nonetheless, we found that hAgo3 immunopurified
from Jurkat cells showed little or no Slicer activity (Fig. 5C). We
then examined whether hAgo3 cleaves target RNA when associated with siRNA. hAgo3 bound with luc guide siRNA was
subjected to target RNA cleavage assays for a luc RNA target.
Cleavage activity was not observed for hAgo3 (Fig. 5D), although single-stranded luc guide siRNA was loaded onto hAgo3
to the same extent as on hAgo2 (Fig. 5E).
Functional Significance of 5ⴕ-End Variations. Ago2 cleaves its cog-nate RNA target across from nucleotides 10 and 11, measured
from the 5⬘ end of the guide small RNA (21). Thus, if a miRNA
had variants with an extension of one or two nucleotides at the5⬘ end, the hAgo2-miRNA-mediated cleavage should give rise to
two or more cleaved products that differed in size. We found thatmiR-142-5p has one very abundant variant with a CC additionat the 5⬘ end (Fig. S5). Thus, in vitro target RNA cleavage assays
with a target harboring a sequence completely complementary
to miR-142-5p (miR-142-5p target) was performed. The target
was predominantly cleaved at two major sites; one corresponding
to the site of the cleavage directed by registered miR-142-5p, andthe other corresponding to the site of the cleavage directed by
one very abundant miR-142-5p variant that had two additional
nucleotides (mostly CC) at the 5⬘ end (miR-142-5p⫹CC; Fig.
S8). miR-142-5p target modified with a methyl group at the
expected cleavage site by miR-142-5p⫹CC showed strong resistance to miR-142-5p⫹CC but not to registered miR-142-5p
cleavage (Fig. S8). Our data clearly indicated that miRNAvariants with a 5⬘ extension existed in vivo and that miRNAs thathave variants can at least exhibit slightly different target
specificities.
Discussion
From our experience, especially from studies on Drosophila
RNA silencing mechanisms, we were aware that producing andusing specific antibodies against individual Argonautes was aneffective means to understand their functional contributions in
RNA silencing (22–24). In this study, to gain a better insight into
the functions of human endogenous Argonautes in RNA silencing, we produced monoclonal antibodies against each member
and carried out various experiments. Anti-hAgo1 and -hAgo4
worked for Western blot analysis but showed inefficiencies in
immunoprecipitation. Anti-hAgo2 and anti-hAgo3 worked quite
efficiently for immunoprecipitation, even under the harsh conditions that were intentionally selected to avoid undesired protein–
Azuma-Mukai et al.

protein interactions that are presumed to occur in vivo. Through theexperiments, we found that (i) endogenous hAgo2 and hAgo3 in
Jurkat cells associate with various kinds of miRNAs, and that (ii)endogenous hAgo3 most likely does not function as Slicer, as
opposed to hAgo2. The anti-hAgo2 and -hAgo3 antibodies weraised were very versatile and revealed that hAgo2 and hAgo3
localize in P-bodies. miRNA targets are accumulated to P-bodies asa consequence of translational repression for being finally degradedor stored until the mRNAs are once again pulled back to thecytoplasm to be translated (16). Our findings indicated that endogenous hAgo3 functions in a translational repression pathway mediated by miRNAs. The hAgo3–siRNA complex showed little or no
Slicer activity. This new finding regarding endogenous hAgo3
raised two possibilities in human RNAi: (i) hAgo3 antagonizes
RNAi activity, whereas hAgo2 functions as Slicer in RNAi in vivo;and (ii) siRNA loading onto hAgo3 apparently requires otherprotein(s) that need to unwind siRNA duplex before loading
single-stranded siRNA. It has previously been shown that hAgo2
Slicer activity is closely involved in the siRNA duplex unwinding
process in mammalian RNAi (25, 26).
miRNAs associated with hAgo2 and hAgo3 overlapped to a large
extent. However, some seemed to be discriminately loaded onto
either hAgo2 or hAgo3. It is reported that in Caenorhabditis elegans,structural features of small RNA precursors determine Argonauteloading (27). In this study, such structural discrimination in regard
to miRNA loading was not observed, although comprehensive
analysis remains to be performed. It has been shown that the
reduced levels of miR-24 are observed in mAgo2⫺/⫺ MEF (mouseembryo fibroblast) cells but not in mAgo3⫺/⫺ MEF cells, suggestingthat mAgo1, mAgo3, and mAgo4 do not compensate for the loss ofmAgo2 activity (28). Here, we found that in Jurkat cells, albeit ata very low rate (0.21%), miR-24 was observed only with hAgo2 but
did not appear at all with hAgo3. Thus, it may be possible thatmiR-24 is unloaded onto mAgo3 even in mice, and that it must be
the reason that loss of mAgo3 did not affect the miR-24 levels in
MEFs. mAgo2 has been shown as essential for RNAi in MEFs. It
has also been shown that MEFs lacking mAgo2 were devoid of
RNAi activity although mAgo3 was likely expressed normally in theparticular cells (10). It is possible that Ago3 also has no Slicer
activity in mice, as in humans. How some miRNAs are loaded onto
specific hAgos remains an open question. However, a regulatory
mechanism may be involved in regulating the expression levels of
individual miRNAs in different cell types and tissues, where thelevels of hAgos themselves may likewise be specified by unknown
mechanisms.
PNAS 兩 June 10, 2008 兩 vol. 105 兩 no. 23 兩 7967

BIOCHEMISTRY

Fig. 4. Occurrence of variations at the 5⬘ and 3⬘ ends of miRNA with hAgo2 and hAgo3. (A) Occurrence of nontemplated extensions at the 5⬘ and 3⬘ ends ofmiRNA compared for hAgo2 and hAgo3. The Wilcoxon matched-pair signed-rank test was used to test for any significant difference between nontemplated
extensions occurring at 5⬘ and 3⬘ ends. There is a significant difference between the occurrence of nontemplated extensions at 5⬘ and 3⬘ in both hAgo2 (P ⱕ 2.9 ⫻10⫺5) and hAgo3 (P ⱕ 1.6 ⫻ 10⫺6). The blank square represents the interquartile range (IQR) of the first quartile (Q1) and third quartile (Q3) where 50% of theobserved points are included. The thick horizontal line drawn in the square represents the median of the distribution. The lower whisker represents Q1 ⫺ 1.5 ⫻IQR, whereas the upper whisker represents Q3 ⫹ 1.5 ⫻ IQR. Observations outside of the whiskers are ‘‘outliers,’’ represented as white circles. (B) Occurrencesof alternative ends at 5⬘ and 3⬘ ends compared for hAgo2 and hAgo3. There are significant differences between the occurrence of alternative ends at 5⬘ and3⬘ in both hAgo2 (P ⱕ 3.9 ⫻ 10⫺2) and hAgo3 (P ⱕ 5.5 ⫻ 10⫺3).

A

B

D

C

E

Fig. 5. Endogenous hAgo2 and hAgo3 Slicer activities. (A) Immunopurified
hAgo2 from HeLa cells, which contained miR-21 (shown in B), subjected to in
vitro target RNA cleavage assays. Even hAgo2 immunopurified from HeLa cells
in an Empigen buffer shows Slicer activity. (B) miR-21 associated with hAgo2
in HeLa cell lysates prepared in a buffer containing Nonidet P-40 or Empigen
visualized by Northern blot analysis. Total RNAs from HeLa cells and DNA
oligos with sequences of miR-21 (sense) and miR-21* (antisense) were used ascontrols. (C) Immunopurified hAgo2 and hAgo3 from Jurkat cells in an Empigen buffer were subjected to in vitro target RNA cleavage assays. hAgo3 does
not cleave a target RNA (miR-150 target), whereas hAgo2 does. Association ofhAgo3 with miR-150 is confirmed by Northern blot analysis (Fig. S7B). (D)hAgo2 and hAgo3 associated with luc guide siRNA subjected to in vitro target
RNA cleavage assays. hAgo2 but not hAgo3 cleaves the luc target RNA. (E)Northern blot analysis showing that hAgo2 and hAgo3 are able to be loadedwith luc guide siRNA under the same circumstances.

miRNA sequence variations have been observed in humans
and rodents (18). In the study, the A and U additions at the 3⬘
ends were discovered. Morin et al. (19) have recently revealed
that there are variations at the 5⬘ and 3⬘ ends of miRNAs
expressed in hESCs and EBs. In this study, we also found thatmiRNAs associated with hAgo2 and hAgo3 showed various
variations at the 5⬘ and 3⬘ ends. We found the 5⬘-end variationsquite interesting, because they presumably alter miRNA abilities
with respect to recognizing targets in vivo. In miRNA sequences,
the second to eighth nucleotides from the 5⬘ ends are called‘‘seed’’ sequences, and the regions function in defining miRNAtargets in the miRNA pathway (20). Sequence variations at the5⬘ ends mean that these miRNA variants, although produced
from the same precursor sequence, might recognize a different
set of target sequences from those of the miRNAs first registeredin the databases. Indeed, in this study we showed by in vitro target
RNA cleavage assays that miRNA variants with a 5⬘ extension
existed in vivo, and that miRNAs that have variants can at least
exhibit slightly different target specificities.
The accumulation of data on endogenous Argonautes andother crucial factors in RNA silencing, including triggering small
7968 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800334105

RNA molecules, will contribute to a comprehensive understanding of RNAi and other RNA silencing mechanisms. Our study
clearly indicated that the hAgo antibodies we raised are toolsthat can accurately reveal the functional behavior of Argonautes
and their miRNA partners in RNA silencing. Thus, they canpotentially provide us with valuable information about how
physiological and biological aspects of our lives are regulated
through such mechanisms.
Methods
Production of Monoclonal Antibodies. The N termini of hAgo1, hAgo2, hAgo3,
and hAgo4 (accession nos. NM㛭012199, NM㛭012154, NM㛭024852, andNM㛭017629) (171, 148, 175, and 164 aa from the N-terminal ends of hAgo1,
hAgo2, hAgo3, and hAgo4, respectively) were fused either with GST (hAgo1
and hAgo2) or 6⫻ His-tag (hAgo3 and hAgo4). Each of the fusion proteins wasproduced in and purified from Escherichia coli and used as an antigen to
immunize mice. Anti-hAgo monoclonal antibodies were produced essentiallyas described in ref. 24. Western blot analysis was performed essentially as
described in ref. 24.
Immunofluorescence. Immunofluorescence was performed essentially as described in ref. 29. After fixation, cell permeabilization was carried out with
0.1% Triton X-100 containing PBS, instead of cold acetone. Anti-DCP1a wasused at 1:400 dilution. Anti-hAgo2 and -hAgo3 used were culture supernatants of the hybridomas. Alexa Fluor 488-conjugated anti-mouse IgG and
Alexa Fluor 546-conjugated anti-rabbit IgG (Molecular Probes) were used as
secondary antibodies to detect anti-hAgos and anti-DCP1a, respectively. All
images were obtained by using a Zeiss LSM510 laser scanning microscope.
Immunoprecipitation. Immunoprecipitation was performed from HeLa andJurkat cells. Cell lysis buffer with Nonidet P-40 contained 30 mM Hepes-KOH(pH 7.3), 150 mM KOAc, 2 mM MgOAc, 5 mM DTT, 0.1% Nonidet P-40, 2 ␮g/ml
pepstatin, 2 ␮g/ml leupeptin, and 0.5% aprotinin. Empigen-containing cell
lysis buffer was 1⫻ PBS containing 1 mM EDTA, 0.1 mM DTT, 1% Empigen(Calbiochem), 2 ␮g/ml pepstatin, 2 ␮g/ml leupeptin, and 0.5% aprotinin. After
immunoprecipitation, total RNAs were isolated from the immunoprecipitateswith phenol:chloroform and precipitated with ethanol. RNAs were dephosphorylated with CIP (NEB) and labeled with [␥-32P]ATP with T4 polynucleotide
kinase (Takara) for visualization. Silver staining was performed by using a
SilverQuest kit (Invitrogen).
Small RNA Library Construction. Cloning of small RNAs associated with hAgo2
and hAgo3 was carried out essentially as described in ref. 24, using a DynaExpress miRNA cloning kit (BioDynamics Laboratory). First-strand cDNA synthesis
was performed with Reverse Transcriptase (BioDynamics Laboratory). PCR was
performed with Ex Taq polymerase (Takara) first and then KOD plus
(TOYOBO). High-throughput pyrosequencing was performed on a GS20 system (Roche). RNA sequences between 5⬘ and 3⬘ adapters were analyzed by
Takara. Briefly, each of the small RNAs was located in the human genome byusing the BLAT program in the UCSC Genome Browser (http://genome.
ucsc.edu/cgi-bin/hgBlat) and then searched by using the National Center for
Biotechnology Information BLAST (www.ncbi.nlm.nih.gov/blast) and Ensemble BlastView (www.ensembl.org/Multi/blastview) to determine matching torRNA, tRNA, snRNA, snoRNA, and scRNA. The miRBase (http://microrna.
sanger.ac.uk/sequences) was used to identify miRNA clones in humans andother species.
Northern Blot Analysis. Northern blot analysis was performed essentially asreported in ref. 22. Total RNAs were isolated from immunoprecipitates withphenol:chloroform and precipitated with ethanol. The probe used for miR-21
was 5⬘-TCAACATCAGTCTGATAAGCTA-3⬘. The sequence of a probe used for
detecting luc guide siRNA is described in ref. 30. The DNA oligo was labeledwith T4 polynucleotide kinase in the presence of [␥-32P]ATP. Control oligos
used were as follows: sense strand (miR-21), 5⬘-CTCAACATCAGTCTGATAAGCTAG-3⬘; antisense strand (miR-21), 5⬘-AATTCTAGCTTATCAGACTGATGTTGAGGTAC-3⬘; luc guide oligo, 5⬘-GAATTCTCGAAGTATTCCGCGTACGTGGGTACC-3⬘. First, Jurkat cell lysate was prepared as described in ref. 30, andluc siRNA duplex was then added to the lysate. After incubation at 37°C for 1 h,hAgo2 and hAgo3 were immunopurified by using the specific antibodies in an
Empigen buffer, and guide siRNA bound to the proteins was detected by
Northern blot analysis.

Azuma-Mukai et al.

with alternative ends. Sequences that had more than one base mismatches totheir precursors were discarded, as were sequences with ⬍10 reads. We used
the top 30 abundant sequences for our sequence data analysis. The mostabundant sequence with perfect base matches to its precursor sequence wasconsidered to be the canonical mature form. Other sequences with perfectbase matches were considered as mature miRNA sequences with alternativeends. A mature miRNA sequence with nontemplated bases was a sequence
having at its 5⬘- or 3⬘-end one mismatch with the corresponding precursor
sequences.

Bioinformatics. Small RNA sequences were aligned to miRNA precursor sequences obtained from miRBase (http://microrna.sanger.ac.uk) by our custom
perl script to identify mature miRNA sequences with nontemplated bases or

ACKNOWLEDGMENTS. We thank J. Lykke-Andersen for providing the antiDCP1a antibody; K. Saito for technical advice; T. Mori, T. N. Okada, and Y.
Kawamura for technical assistance; and other members of the Siomi laboratory for discussions and comments on the manuscript. For help in MS analysis,
we acknowledge I. Sagawa (University of Tokushima Faculty of Medicine
Support Center for Advanced Medical Sciences). This work was supported inpart by grants to M.C.S. and H.S. from the Ministry of Education, Culture,
Sports, Science and Technology (Japan) and the New Energy and Industrial
Technology Development Organization. M.C.S. is supported by Core Research
for Evolutional Science and Technology of the Japan Science and Technology
Agency. M.C.S. is Associate Professor of Global COE for Human Metabolomics
Systems Biology of the Ministry of Education, Culture, Sports, Science, andTechnology. H.S. is a member of the Genome Network Project.

1. Zaratiegui M, Irvine DV, Martienssen RA (2007) Noncoding RNAs and gene silencing.
Cell 128:763–776.
2. Tomari Y, Zamore PD (2005) Perspective: Machines for RNAi. Genes Dev 19:517–529.
3. Kloosterman WP, Plasterk RH (2006) The diverse functions of microRNAs in animal
development and disease. Dev Cell 11:441– 450.
4. Bushati N, Cohen SM (2007) MicroRNA function. Annu Rev Cell Dev Biol 23:175–205.
5. Peters L, Meister G (2007) Argonaute proteins: Mediators of RNA silencing. Mol Cell
26:611– 623.
6. O’Donnell KA, Boeke JD (2007) Mighty Piwis defend the germline against genome
intruders. Cell 129:37– 44.
7. Aravin AA, Hannon GJ, Brennecke J (2007) The Piwi-piRNA pathway provides anadaptive defense in the transposon arms race. Science 318:761–764.
8. Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme
complex. Science 297:2056 –2060.
9. Mourelatos Z, et al. (2002) miRNPs: A novel class of ribonucleoproteins containing
numerous microRNAs. Genes Dev 16:720 –728.
10. Liu J, et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science
305:1437–1441.
11. Meister G, et al. (2004) Human Argonaute2 mediates RNA cleavage targeted bymiRNAs and siRNAs. Mol Cell 15:185–197.
12. Beitzinger M, Peters L, Zhu JY, Kremmer E, Meister G (2007) Identification of humanmicroRNA targets from isolated Argonaute protein complexes. RNA Biol 4:e1– e9.
13. Sen GL, Blau HM (2005) Argonaute 2/RISC resides in sites of mammalian mRNA decay
known as cytoplasmic bodies. Nat Cell Biol 7:633– 636.
14. Gabler S, et al. (1998) E1B 55-kilodalton-associated protein: A cellular protein with
RNA-binding activity implicated in nucleocytoplasmic transport of adenovirus andcellular mRNAs. J Virol 72:7960 –7971.
15. Liu J, Valencia-Sanchez MA, Hannon GJ, Parker R (2005) MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat Cell Biol 7:719 –723.
16. Pillai RS, et al. (2005) Inhibition of translational initiation by let-7 microRNA in humancells. Science 309:1573–1576.

17. Fenger-Gron M, Fillman C, Norrild B, Lykke-Andersen J (2005) Multiple processing bodyfactors and the ARE binding protein TTP activate mRNA decapping. Mol Cell 20:905–
915.
18. Landgraf P, et al. (2007) A mammalian microRNA expression atlas based on small RNA
library sequence. Cell 129:1401–1414.
19. Morin RD, et al. (2007) Application of massively parallel sequencing to microRNA
profiling and discovery in human embryonic stem cells. Genome Res 18:610 – 621.
20. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB (2003) Prediction of
mammalian microRNA targets. Cell 115:787–798.
21. Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and22-mucleotide RNAs. Genes Dev 15:188 –200.
22. Saito K, et al. (2006) Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev 20:2214 –2222.
23. Gunawardane LS, et al. (2007) A Slicer-mediated mechanism for repeat-associatedsiRNA 5⬘ end formation in Drosophila. Science 315:1587–1590.
24. Nishida KM, et al. (2007) Gene silencing mechanisms mediated by Aubergine-piRNAcomplexes in Drosophila male gonad. RNA 13:1911–1922.
25. Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD (2005) Passenger-strand cleavage
facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell
123:607– 620.
26. Leuschner PJ, Ameres SL, Kueng S, Martinez J (2006) Cleavage of the siRNA passenger
strand during RISC assembly in human cells. EMBO Rep 7:314 –320.
27. Steiner FA, et al. (2007) Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans. Nat Struct Mol Biol 14:927–933.
28. O’Carroll D, et al. (2007) A Slicer-independent role for Argonaute 2 in hematopoiesis
and the microRNA pathway. Genes Dev 21:1999 –2004.
29. Siomi H, Dreyfuss G (1995) A nuclear localization domain in the hnRNP A1 protein. J Cell
Biol 129:551–560.
30. Miyoshi K, Tsukumo H, Nagami T, Siomi H, Siomi MC (2005) Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev 19:2837–2848.

Azuma-Mukai et al.

PNAS 兩 June 10, 2008 兩 vol. 105 兩 no. 23 兩 7969

BIOCHEMISTRYIn Vitro Target RNA Cleavage Assay. Target RNA cleavage assays using immunoprecipitates were performed in a reaction buffer containing 25 mM HepesKOH (pH 7.4), 50 mM KOAc, 5 mM MgOAc, 5 mM DTT, 0.1 units/ml RNasin(Promega), and 1 ␮g of yeast RNA (Ambion). After reaction for 3 h at 37°C,
RNAs were isolated from the whole reaction mixtures, run on gels, andvisualized on a BAS 2500. To make miR-21 target RNA, a set of DNA oligos(5⬘-CTCAACATCAGTCTGATAAGCTAG-3⬘ and 5⬘-AATTCTAGCTTATCAGACTGATGTTGAGGTAC-3⬘) was annealed and inserted into a pBS SKII⫹ vector
digested with KpnI and EcoRI. To make a target for miR-150, a short DNA
fragment produced by annealing a set of oligo DNAs (5⬘- TCGAGCACTGGTACAAGGGTTGGGAGAG-3⬘ and 5⬘-AATTCTCTCCCAACCCTTGTACCAGTGC-3⬘)
was inserted in a pBS SKII⫹ vector digested with XhoI and EcoRI. PCR was
performed by using primers for the T7 and T3 promoter sequences, and the
PCR products were then used as templates for in vitro transcription with a
MEGAscript T7 kit (Ambion). To make a luc target, pBS-luc-target plasmid (30)
was digested with BamHI, and transcription was performed with a MEGAscript
T7 kit. The resultant RNAs were radiolabeled with [␥-32P]ATP by using T4
polynucleotide kinase and were gel-purified.