Dual Role for Argonautes in MicroRNA
Processing and Posttranscriptional
Regulation of MicroRNA Expression
Sven Diederichs1,* and Daniel A. Haber1,*
1Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA
*Correspondence: firstname.lastname@example.org (S.D.), email@example.com (D.A.H.)
MicroRNAs are small endogenous noncoding
RNAs involved in posttranscriptional gene
regulation. During microRNA biogenesis, Dro-
sha and Dicer process the primary transcript
(pri-miRNA) through a precursor hairpin (pre-
miRNA) to the mature miRNA. The miRNA is
incorporated into the RNA-Induced Silencing
Complex (RISC) with Argonaute proteins, the
effector molecules in RNA interference (RNAi).
Here, we show that all Argonautes elevate
mature miRNA expression posttranscription-
ally, independent of RNase activity. Also, we
identify a role for the RISC slicer Argonaute2
(Ago2) in cleaving the pre-miRNA to an addi-
tional processing intermediate, termed Ago2-
cleaved precursor miRNA or ac-pre-miRNA.
This endogenous, on-pathway intermediate re-
sults from cleavage of the pre-miRNA hairpin
12 nucleotides from its 30-end. By analogy to
removal of the nicked passenger strand from
RISC after maturation. The multiple roles of
Argonautes in the RNAi effector phase and
miRNA biogenesis and maturation suggest
coordinate regulation of microRNA expression
The microRNA (miRNA) class consists of small endoge-
nous noncoding RNAs of approximately 22 nucleotides
(nt) in length. The first isolated miRNAs were hetero-
chronic genesfrom C. elegans (Lee et al.,1993; Wightman
et al., 1993). Since then, rapidly accumulating data have
revealed a large family of evolutionary conserved miRNA
genes implicated in vertebrate development and cancer
(Bernstein et al., 2003; He et al., 2005; Hutvagner et al.,
2001; Ketting et al., 2001; Lu et al., 2005; O’Donnell
et al., 2005). miRNA genes may be single or clustered in
the genome and either comprise independent transcrip-
tion units in intergenic regions or are embedded within in-
trons of protein-encoding genes (Rodriguez et al., 2004).
miRNA biogenesis starts with transcription by human
RNA Polymerase II or III (Borchert et al., 2006; Lee et al.,
2004). Processing of the pri-miRNA by the nuclear RNase
Drosha as part of a microprocessor complex with DGCR8
(Pasha) produces the pre-miRNA hairpin precursor, which
is exported into the cytoplasm by the Exportin-5/Ran-
GTP complex (Denli et al., 2004; Gregory et al., 2004;
Han et al., 2004; Lee et al., 2003; Lund et al., 2004; Yi
et al., 2003). Cytoplasmic pre-miRNA is then bound by
the RNase Dicer as part of the RISC Loading Complex
(RLC) including TRBP and Ago2 (Chendrimada et al.,
2005; Gregory et al., 2005; Maniataki and Mourelatos,
2005). Dicer cleaves the pre-miRNA hairpin into the ma-
turemiRNA,whichenters theRISC andiscapable ofbind-
ing to partially complementary sequences within the
30UTR of the target mRNA (Hutvagner et al., 2001; Ketting
et al., 2001). Recent studies have also revealed miRNA-
specific processing steps that modulate or omit Drosha
cleavage (Guil and Caceres, 2007; Okamura et al., 2007;
Ruby et al., 2007). RISC-bound mRNA transcripts may
be suppressed in protein translation, destabilized by
deadenylation or degraded by Ago2 RNase activity
(Doench et al., 2003; Hutvagner and Zamore, 2002; Liu
et al., 2004; Meister et al., 2004; Petersen et al., 2006).
Members of the human Argonaute family are known to
be involved in the effector phase of RNA interference
(RNAi) silencing translation of mRNAs at the stage of
translational initiation or elongation (Humphreys et al.,
2005; Petersen et al., 2006; Pillai et al., 2004, 2005).
Ago2, also known as eIF2C2, is the only human Ago pro-
tein with intrinsic endonuclease activity, encoded by its
PIWI domain, which structurally resembles an RNase H
domain (Song et al., 2004). Ago2 has therefore been
described as the RISC slicer, providing the RNase activity
to cleave target mRNAs that are complementary to the
guiding siRNA or miRNA, and it is the only protein compo-
nent required for RISC activity (Liu et al., 2004; Meister
Mello and colleagues analyzed deletional mutants of all 27
members of the large Argonaute family in the nematode
C. elegans and found that functionally and structurally
Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc. 1097
distinct Ago proteins act sequentially during siRNA-medi-
ated gene silencing (Yigit et al., 2006).
Understanding the complex process by which miRNAs
are generated from their endogenous transcripts offers
important insight into potential regulatory mechanisms.
While a single miRNA can have striking effects on expres-
sion of its target proteins, global patterns of miRNA
expression have also been correlated with distinct stages
of development and cancer (Lu et al., 2005; Neilson et al.,
2007). Posttranscriptional regulation of miRNA expression
has been described previously (Obernosterer et al., 2006;
Thomson et al., 2006), but the protein factors governing
the abundance of mature miRNAs remain largely un-
known. We hypothesized that steps within the microRNA
biogenesis pathway might also have regulatory functions
and sought to identify proteins factors that control the
expression of the mature, functional microRNAs. Here,
we demonstrate the ability of all Ago proteins to enhance
the abundance of miRNAs through posttranscriptional
mechanisms. We also identify a specific role of Ago2 in
mediating cleavage of pre-miRNA to a novel intermediate
in miRNA biogenesis.
Argonaute Proteins Increase the Abundance
of Mature miRNAs
To identify protein factors that are limiting for mature
miRNA expression, we recapitulated miRNA processing
in a manipulable in vivo system. Since cell lines generally
express lowlevels of miRNAs (Lu etal.,2005),wecotrans-
fected a CMV-driven let-7a-3 primary transcript into
HEK293 (293) cells to saturate the miRNA processing
machinery. We transiently cotransfected constructs en-
coding human proteins that have been previously linked
either to RNA interference (RNAi) or miRNA biogenesis.
Ectopic expression levels are documented in Figure S1
(in the Supplemental Data available online). Northern blot
analysis revealed that expression of each member of the
Argonaute protein family (Ago1–Ago4) gave rise to
increased levels of mature let-7a expression (Figure 1A).
Other proteins implicated in miRNA biogenesis, Drosha,
DGCR8, Dicer, MOV10, TNRC6B (Figure 1A), as well as
Gemin-4, Ran and Exportin-5 (data not shown) had no
effect in this assay screening for rate-limiting factors in
miRNA processing. In addition, combinations of process-
ing factors known to act as a complex, Drosha with
DGCR8 or Dicer with TRBP, did not alter mature miRNA
expression (Figure S2A). The enhancement of mature
miRNA expression by ectopically expressed Ago proteins
was consistently observed with different transfected pri-
miRNA constructs, miR-215, miR-17-5p, miR-23b, miR-92
in two different cell lines (293: Figure 1B; U2OS: Fig-
ure S2B). Of note, for miR-215 and miR-17-5p, the mature
miRNA is derived from the 50-arm of the hairpin precursor,
30-arm. Thus, this effect is independent of the arm of the
pre-miRNA hairpin giving rise to the mature miRNA.
Loss of Ago2 Leads to Decreased
Argonaute proteins have been described as effector
molecules in the RNAi pathway, executing the inhibitory
effects of mature siRNA or miRNA molecules (Pillai et al.,
2005),buttheyhavenotbeen functionally linkedto miRNA
biogenesis. Given their effect in the ectopic expression
screening assay, we tested whether expression of en-
dogenous Argonaute proteins also has an impact on the
expression levels of endogenous miRNAs. A knockout
mouse for Ago2 has recently been reported (Liu et al.,
2004), allowing us to analyze the effects of endogenous
Ago2 on miRNA expression. We used mouse embryonic
fibroblasts (MEFs) derived from Ago2 knockout (KO)
mice and measured expression of endogenous miRNAs
whose mature form is detectable in matched wild-type
(WT) MEFs. Remarkably, all five miRNAs tested, let-7a,
let-7c, miR-17-5p, miR-92, and miR-21, showed strongly
reduced expression in Ago2 null MEFs (Figure 2A). For
reconstitution and compensation experiments, we stably
expressed each Ago family member in the Ago2-deficient
MEFs (Figure S3A) and analyzed its effects on expression
of mature exogenous and endogenous miRNAs. Intro-
duction of exogenous miRNA precursors demonstrated
strongly increased expression of mature miRNAs in Ago2-
reconstituted MEFs compared to Ago2-KO cells (Fig-
ure 2B, Figure S3B). Ectopic expression of Ago1 partially
compensated for the Ago2 deficiency, but other Ago pro-
teins had no or weak effects on mature miRNA expression
in Ago2-KO cells. For members of the let-7 family, high
ectopic expression in Ago2-deficient cells also resulted
in accumulation of unprocessed precursors (Figure S4A),
which was reduced following Ago2 reconstitution (Fig-
ure S4B) suggesting a potential processing defect in
cells lacking Ago2. The effect of Ago2 reconstitution on
baseline expression of endogenous miRNAs was con-
sistent with the observations for exogenous precursors
but less pronounced. In each case, densitometric quanti-
tation of mature miRNA expression revealed increased
mature miRNA expression following Ago2 reconstitution
(Figure 2C, Figure S3C). On average, the mature miRNA
expressioninKOMEFswasreduced to40%of theirlevels
in WT MEFs and returned to 71% of wild-type levels fol-
lowing Ago2 expression. No such effect was seen with
other Ago family members, Ago1, Ago3, and Ago4. The
absence ofknockout models precludes usfromextending
our miRNA expression studies for these Ago family mem-
bers. However, the described phenotypes of Ago2 loss
and reconstitution could reflect a role for Ago2 in miRNA
maturation or indicate stabilization of the miRNA within
the Ago2-containing RLC or RISC—or a combination of
these effects. Since Ago proteins are primary effectors of
miRNA-mediated silencing, their additional role in miRNA
biogenesis raisesthepossibility ofcoordinated regulation,
providing a potential feedback mechanism to titrate the
expression of mature miRNAs with the availability of Ago
proteins. Argonaute proteins—one RISC component—
can elevate the expression of mature miRNAs—the other
1098 Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc.
RISC component—and thus coordinate expression of
both RISC components to form functional, stoichiometric
Identification of an Additional MicroRNA
The design of the initial screening experiment, using over-
expression of the let-7a miRNA and different processing
pin (pre-miRNA) (Figure 3A) as well as the mature miRNA
(Figure 1A). Ectopic expression of Argonaute proteins
Ago1, Ago2, and Ago3, as well as TRBP, gave rise to
increased levels of the known pre-miRNA precursor. Re-
markably, Ago2, but noneof the other Argonaute proteins,
gave rise to a smaller precursor band. To further charac-
terize this new precursor, we first verified its induction
following Ago2 overexpression for additional let-7 family
members in both 293 and U2OS cells by northern blotting
To uncover the identity of the additional precursor, we
undertook to isolate miRNA precursor populations from
293 cells that had been transfected with let-7a-3 pri-
miRNA and Ago2 (Figures 3C and 3D). To clone the pre-
miRNAs, we purified RNA enriched for small RNA species
(<200 nt), ligated these to an RNA-cloning linker, and used
RT-PCR with one miRNA-specific and one linker-specific
primer, followed by high-efficiency cloning and sequenc-
ing analysis. About half of the cloned miRNA precursors
corresponded to the previously described pre-miRNA
hairpin. In addition, we identified a previously unknown
otides shorter than the pre-miRNA population (Figure 3D:
ac-pre hairpin). Sequencing analysis revealed that this
smaller precursor species is derived from the pre-miRNA
hairpin by endonucleolytic cleavage within the passenger
30-arm of the hairpin. Ago2 is the only human Ago protein
with an intrinsic endonuclease domain, providing the
RNase activity to cleave mRNAs targeted by siRNA or
miRNA (Liu et al., 2004; Meister et al., 2004). The unique
endonucleolytic activity of Ago2 is of particular interest,
since it is known to cleave its mRNA substrates approxi-
mately ten nucleotides downstream of the 50-end of the
guiding siRNA (Elbashir et al., 2001). Given the 30-over-
hang of one to two nucleotides at the 30-end of the hairpin,
this size difference matches the estimated cleavage site
within the pre-miRNA to generate the hairpin identified
here. The hairpin of this endogenous precursor corre-
sponds in size to the shortened intermediate detected
by northern blotting in Ago2-overexpressing cells. Thus,
unappreciated step in miRNA biosynthesis, ‘‘Ago2-
cleaved precursor miRNA’’ or ‘‘ac-pre-miRNA.’’ For clari-
fication, in cloning and northern blotting experiments, the
ac-pre-miRNA is only detected in the form of the short
hairpin due to denaturation during RNA isolation and
PAGE. However, our further experiments (see below) indi-
cate, that the functional ac-pre-miRNA, the Dicer sub-
strate, consists of a complex of the shortened hairpin hy-
bridized to the short 30-fragment forming a nicked hairpin,
which can also be reconstituted in vitro.
of MicroRNA Processing
Having identified the ac-pre-miRNA in cells overex-
pressing Ago2 and let-7a pri-miRNAs, we wanted to
Figure 1. Induction of Different miRNA
Populations by Argonaute Proteins
(A) Identification of rate-limiting factors for pro-
cessing of pri-miRNAs. Mature miRNA expres-
sion in 293 cells cotransfected with constructs
encoding let-7a-3 pri-miRNA and protein fac-
tors involved in either miRNA biogenesis (Dro-
sha, DGCR8, Dicer, TRBP), RNA interference
(Ago1–4) or binding to Ago proteins (MOV10,
TNRC6B) wasdetermined bynorthern blotting.
All Argonaute proteins gave rise to increased
expression of mature miRNAs. Reprobing of
the blot for U6 snRNA served as loading con-
trol. As additional loading control, Ethidium-
bromide staining was used to visualize tRNAs
and 5S and 5.8S ribosomal RNAs under UV
light. This control was obtained for all northern
blots throughout the manuscript and will be
provided upon request.
(B) Effects of Ago proteins on diverse pri-
miRNAs. Northern blot analysis of 293 cells,
cotransfected with either EGFP or one of
the Ago proteins, together with different pri-
miRNAs indicated induction of mature exoge-
nous miRNAs by all Ago proteins. U6 snRNA
served as loading control.
Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc. 1099
analyze whether this new precursor was also evident in
cells only expressing endogenous Ago2 and endoge-
nous miRNAs. The currently known miRNA biosynthesis
pathway was initially defined in vitro, using synthetic
precursors and isolating precursor miRNAs that had
been processed in vitro (Han et al., 2004; Lee et al.,
2003). To determine the physiological prevalence of pre-
cursor miRNAs in vivo, we used untransfected, cultured
human cell lines to directly clone the rare population of
endogenous miRNA precursor species using the linker-
mediated cloning method described above for ectopic
Precursors of three members of the let-7 miRNA family
(let-7a-1, let-7a-3, let-7f-1) and the miR-20a fromthe miR-
17/-92 cluster were cloned (Figures 4A–4E). These miRNA
species were selected for initial analysis based on their
relative abundance incultured cellsanddefined functional
properties (He et al., 2005; O’Donnell et al., 2005). The
most frequently cloned miRNA precursor population
corresponded to the previously described pre-miRNA
hairpin, with an overhang of non-base-paired nucleotides
at the 30-end. Hairpins lacking one or two nucleotides at
the 30-end were also cloned, suggestive of either exonu-
cleolytic trimming or a lack of accuracy of Drosha cleav-
age, as previously described for in vitro derived cleavage
products (Han et al., 2004).
Endogenous ac-pre-miRNA species were identified,
comprising 27% of all endogenous precursors isolated
from these cells. As described for the ectopic miRNA pre-
cursors, endogenous ac-pre-miRNA hairpins were also
eleven to twelve nucleotides smaller than the pre-miRNA
population (Figure 4A: ac-pre hairpin). Of note, the abun-
dance of the ac-pre-miRNA derived from nontransfected
cells expressing endogenous miRNA and endogenous
Ago2 was lower than that observed in cells expressing
ectopic miRNA and ectopic Ago2 (Figure 3C) explaining
by northern blotting. Endonucleolytic cleavage of the pre-
miRNA hairpin within the passenger 30-arm hairpin arm
presumably generates both the shortened hairpin and
a small 30-fragment forming a nicked hairpin (Figures
4B–4E). Individual cloning frequencies and the structures
of the cloned hairpin sequences are detailed in Figure S5.
For all four miRNAs studied, the new precursors com-
prised a distinct RNA population, clustered around a de-
fined number of deleted nucleotides at the 30-end, and
consistent with a new endogenous intermediate in the
biogenesis pathway of these miRNAs.
Figure 2. miRNA Expression in Ago2-
Deficient and -Reconstituted Cells
(A) Reduced expression of endogenous ma-
ture miRNAs in Ago2 null MEFs. Northern blots
of wild-type (WT) and Ago2-knockout (KO)
MEFs showed lower expression of different
mature endogenous miRNAs in KO cells. 5S
rRNA served as loading control. RT-PCR for
Ago2 mRNA demonstrates loss of expression
in KO MEFs with Actin as loading control.
(B) Increased expression of mature exogenous
miRNAs after reconstitution of KO MEFs with
Ago2. Ago2 null MEFs stably expressing ec-
topic Ago family members were transiently
transfected with pri-miRNAs. Northern blot
analysis revealed elevated levels of mature
miRNA expression following reconstitution
with Ago2. Expression levels of stably ex-
pressed Ago proteins were determined by
qRT-PCR (Figure S3A). Loading controls are
shown in Figure S3B.
miRNAs in Ago2-KO cells and in cells stably
reconstituted with different Ago proteins.
Northern blot analyses revealed partial rescue
of endogenous miRNA expression only in
Ago2-KO MEFs reconstituted with Ago2, not
with other Ago family members. Densitometric
analysis is shown for multiple northern blots
(+SEM) which are depicted in Figure S3C.
1100 Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc.
The RISC Slicer Argonaute-2 Generates the
Additional miRNA Precursor
using transfected Ago2 and primary miRNA expression
constructs allowed us to test required features of both
the enzyme and the miRNA substrate. To test whether
the RNase activity of Ago2 was necessary for the genera-
tion of the ac-pre-miRNA, we created a set of Ago2 con-
structs harboring mutations in residues implicated in its
RNase activity and tested their effects on ac-pre-miRNA
generation using northern blotting (Figures 5A, S6A, and
S6B).Theaspartate residuesD597 andD669areessential
for the catalytic endonuclease activity of the Ago2 PIWI
domain (Liu et al., 2004; Song et al., 2004) and were
indeed required for formation of the ac-pre-miRNA as
indicated by the absence of the band from the shorter
ac-pre-miRNA hairpin. In contrast, mutations of nones-
sential residue Q633 did not affect generation of the pre-
of the pre-miRNA. The absent contribution of Q633 and
H634 differs from their previously reported roles in
siRNA-guided mRNA cleavage (Liu et al., 2004), possibly
reflecting differences in the relevant substrates and guid-
ing RNA structures. Notably, the increased expression of
the mature miRNA was not affected by the abrogation
of RNase activity. This indicates that the two functions of
Ago proteins in miRNA biogenesis reported here are truly
independent: an Ago2 RNase-dependent generation of
the ac-pre-miRNA and an RNase-independent enhanced
production of mature miRNAs by all Ago proteins. While
Figure 3. Additional miRNA Precursor Associated with Ago2 Expression
(A) Detection of an additional precursor band in the presence of Ago2. Northern blot analysis of 293 cells cotransfected with constructs encoding let-
7a-3pri-miRNA andproteinfactors revealedthat Ago2 wastheonlyprotein togive risetoanadditional,smaller precursorband (‘‘ac-pre’’).Reprobing
of the blot for U6 snRNA served as loading control (complete blot from Figure 1A).
(B) Multiple miRNAs display an additional precursor band. Northern blot analysis of U2OS and 293 cells cotransfected with different let-7 constructs,
together with EGFP or Ago2, demonstrated a previously unknown, shorter miRNA precursor hairpin associated with Ago2 expression.
(C and D) Cloning of a precursor hairpin with a shorter 30-end. Precursor miRNAs were cloned from 293 cells cotransfected with let-7a-3 and Ago2.
The cloned precursors correspond to pre-miRNA (?69 nt) and shorter ac-pre-miRNA hairpins (?58 nt) lacking parts of the 30-end. Depicted are the
relative cloning frequencies of the different precursors (C). The structures of the most abundant cloned pre-miRNA and ac-pre-miRNA hairpins are
Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc. 1101
independent of Ago2 RNase activity, the increased
expression of mature miRNAs does depend on the direct
interaction between the miRNA and Ago2, as demon-
strated by using an Ago2 mutant deficient in binding small
RNAs (PAZ9; (Liu etal., 2005) (Figures 5Band S6C). All the
effects of Ago2 were confirmed in the presence and
absence of an N-terminal tag (Figures S6D and S6E).
Recognition elements within the miRNA that determine
processing efficiency in vivo have not been identified in
detail (Diederichs and Haber, 2006; Zeng and Cullen,
2005). To characterize such elements within the pre-
miRNA that are necessary for Ago2 cleavage, we gener-
ated a set of mutations in the let-7a-3 precursor, altering
the initial nucleotides of the double-stranded stem from
a non-base-paired U U to either a weakly base-paired
U-A or U-G, or to the strongly base-paired C = G (Figures
5C and S7A–S7C). Optimal Ago2 cleavage was achieved
with the pre-miRNA containing the non-base-paired
wild-type ends, giving rise to a prominent ac-pre-miRNA
band. Ago2 cleavage was reduced by introduction of
Figure 4. Identification of Endogenous miRNA Precursors
(A) Cloning of endogenous, in vivo processed miRNA precursors from nontransfected human cell lines reveals two distinct precursor hairpin popu-
lations. The cloning frequency in correlation to the size of the cloned precursor for let-7a-1, let-7a-3, let-7f-1 and miR-20a is illustrated. The largest
cloned pre-miRNA for each miRNA was set as ‘‘n,’’ and shorter precursors are listed relative to this size. The larger precursors represent the known
pre-miRNA hairpins, whereas the approximate eleven to twelve nt smaller forms constitute the hairpins of the additional ac-pre-miRNA class.
(B–E) Nucleotide sequencing analysis of cloned endogenous miRNA precursors. Depicted are the structures of the most abundantly cloned ac-pre-
miRNAscomprisedof the shortened hairpin (black) hybridized to the30-fragment (green) for let-7a-1 (B), let-7a-3 (C), let-7f-1 (D),and miR-20a (E) with
the cleavage site indicated by a red arrow. For these examples, the mature miRNA is derived from the 50-arm of the hairpin, with the passenger strand
of the hairpin targeted by cleavage.
1102 Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc.
a weak base pair at this position, independent of the re-
spective sequence change to A or G, and it was almost
absent with a strong initial base pair. Therefore, the first
base pair of the hairpin contributes a potential recognition
element for Ago2-mediated pre-miRNA cleavage, either
by its base-pairing properties or its sequence. This asym-
metry resembles that of siRNA incorporation into Ago2-
containing RISC, which is also largely governed by the
thermodynamic stability of the first base pair (Khvorova
et al., 2003; Schwarz et al., 2003).
The Ago2-Cleaved Precursor Serves
as Dicer Substrate
The product of Ago2-mediated pre-miRNA cleavage, the
ac-pre-miRNA, may be an intrinsic on-pathway intermedi-
ate during miRNA biogenesis and hence a substrate to
Dicer, or it could be a byproduct which cannot be further
processed toward the mature miRNA. To test whether
the ac-pre-miRNA precursor was indeed a substrate for
Dicer, we reconstituted the Ago2 cleavage product by
synthesizing the short hairpin, hybridized at its 30-end to
the elevento twelve nucleotide fragment, i.e., reproducing
the ac-pre-miRNA with a single-strand cleavage in the
30-arm (Figure 6A). In vitro processing by Dicer with
50-labeled radioactive substrates was as efficient for the
Ago2-nicked ac-pre-miRNA as it was for the pre-miRNA
itself, as demonstrated by the generation of the expected
for the precursors of let-7a as well as miR-20a. Site-
specific photo-activated crosslinking with 4-Thio-Uracil
theac-pre-miRNA (Figure S8).Presence of the hybridized,
but not covalently linked 30-fragment was required for effi-
cient Dicer cleavage of the precursor, since the short hair-
and ac-pre-miRNA was comparable, indicating similar
processing kinetics for the two precursor substrates
(Figure 6C). These data identify the ac-pre-miRNA as
a pathway intermediate in microRNA biogenesis that is
generated from the pre-miRNA by Ago2 and serves as
a substrate for Dicer to mature into the active miRNA.
Two Functions of Argonaute Proteins in miRNA
Processing and Posttranscriptional Regulation
We have shown that Ago proteins have two independent
functions in miRNA biogenesis: first, all Agoproteins post-
transcriptionally enhance production or stability of mature
miRNAs giving rise to elevated levels of mature miRNAs.
Loss of Ago2 reduces expression of mature endogenous
miRNAs. This effect depends on direct binding of the
Ago protein to the miRNA, but not on the RNase activity
of Ago2, and is mediated by all Ago proteins. Therefore,
it is likely that Ago proteins can bind and stabilize mature
miRNAs and thereby increase their abundance at a post-
transcriptional level. Second, Ago2, the only Ago protein
with endonucleolytic activity, specifically generates an
additional miRNA precursor, the Ago2-cleaved precursor
miRNA or ac-pre-miRNA. The ac-pre-miRNA harbors
a single strand cleavage in the 30-arm of the pre-miRNA
and thus consists of a shortened hairpin bound to a frag-
ment of about eleven to twelve nucleotides.
Figure 5. Ago2 Is a Candidate RNase Generating the
(A) Requirement of Ago2 RNase activity for ac-pre-miRNA generation.
293 cells were cotransfected with let-7a-3 and Ago2 constructs in
which RNase activity of Ago2 was abrogated by mutations in residues
D597 or D669. Northern blotting revealed that the endonucleolytic
activity of Ago2 was necessary to generate the ac-pre-miRNA band.
Mutation of Q633 did not interfere with pre-miRNA cleavage, whereas
H634 mutations increased ac-pre-miRNA abundance.
(B) Requirement for miRNA binding, but not RNase activity, for in-
creased mature miRNA expression. 293 cells were cotransfected
with let-7a-3 and Ago2 constructs, in which either miRNA binding
(PAZ9) or RNase activity of Ago2 (D597A, D669A) were abrogated.
Enhanced abundance of mature miRNAs in northern blots following
Ago2 overexpression was dependent on binding of the miRNA to
Ago2 (PAZ9), but independent of its endonucleolytic activity (D597A,
(C) Importance of base pairing by the first residues in the hairpin (red
bar in schematic) for Ago2-mediated pre-miRNA processing. 293 cells
were cotransfected with different let-7a-3 constructs and with either
EGFP or Ago2. Alterations in the thermodynamic stability or sequence
dependent cleavage and formation of the ac-pre-miRNA. U6 snRNA
served as loading control.
Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc. 1103
Functional Implications of Ago2-Mediated
The identification of an additional step in miRNA biogene-
sis raises the question of its biological function. As our in
vitro data suggest, the ac-pre-miRNA is probably not
a more efficient substrate for Dicer compared with the
pre-miRNA, since both appear to be processed efficiently
into the miRNA duplex. However, the cleavage in the
30-arm of the hairpin could serve two functions: first,
the 22 nt miRNA. Previous work has shown that strand se-
of the hairpin (Khvorova et al., 2003; Schwarz et al., 2003),
which also seems to determine Ago2-mediated cleavage
of the pre-miRNA. Second, cleavage in the 30-arm could
facilitate removal of the passenger strand and unwinding
of the miRNA duplex after Dicer cleavage. Cleavage in
the middle of the passenger strand would be expected
to reduce the annealing temperature and the free energy
of duplex formation that has to be invested by the heli-
case to separate the mature miRNA strands, as docu-
mented for siRNAs (Leuschner et al., 2006; Matranga
et al., 2005). Consistent with this hypothesis, blocking
siRNA passenger strand cleavage during in vitro RISC
formation has been shown to inhibit strand dissociation
and suppress RISC formation (Leuschner et al., 2006;
Matranga et al., 2005). Thus, the new processing
Figure 6. The Ac-pre-miRNA Is a Substrate for Dicer Cleavage Generating a Mature miRNA
(A) RNA substrates for in vitro Dicer cleavage of miRNA precursors. RNA oligonucleotides are schematically illustrated for the pre-miRNA hairpin, the
shortened miRNA hairpin alone, truncated by Ago2, and the reconstituted ac-pre-miRNA, a nicked hairpin including the short 30-fragment hybridized
to the Ago2-truncated, short hairpin.
(B)InvitroDicercleavageofmiRNAprecursors.RNAoligonucleotidesoflet-7a-3 andmiR-20awere[32P]-labeled atthe50-endandsubjected toDicer
cleavage in vitro. Both ac-pre-miRNAs were processed by Dicer as efficiently as the respective pre-miRNAs, as indicated by the abundance of the
mature miRNA detected by autoradiography. The short hairpin alone was not processed. The undigested ac-pre-miRNA was detected as short
hairpin since the 11/12 nt fragment was released from the complex with the short hairpin under denaturing PAGE conditions.
(C) Similar kinetics for pre-miRNA and ac-pre-miRNA cleavage by Dicer. RNA oligonucleotide hairpins of let-7a-3 and miR-20a were [32P]-labeled at
were processed equally well as indicated by the timing of the appearance of mature miRNA. From both precursors, the maximum signal of mature
miRNA for let-7a was reached approximately after 2–3 hr, whereas mature miR-20a reached a plateau after 8 hr.
1104 Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc.
intermediate may facilitate strand dissociation of the ma-
MicroRNA Biogenesis Model Including
In light of the data presented here, we propose a modified
model for microRNA biogenesis from the pre-miRNA hair-
pin to the mature functional miRNA (Figure 7). After the
pre-miRNA is exported from the nucleus, it is known to
bind to the preformed complex of Ago2, Dicer and TRBP
(Chendrimada et al., 2005; Gregory et al., 2005; Maniataki
and Mourelatos, 2005). In this complex, Ago2 cleaves the
30-arm of the hairpin which comprises the passenger
strand destined not to give rise to the mature miRNA (right
pathway in model). This step is followed by Dicer-medi-
ated cleavage of the nicked hairpin, generating the miRNA
As currently understood, Dicer and TRBP then dissociate
from Ago2, leaving the mature miRNA strand to form an
active RISC, together with Ago2 or other Ago proteins,
inhibiting target gene expression. Since miRNA matura-
tion is not completely absent in Ago2-KO MEFs, it is likely
that a salvage processing pathway exists, as it does for
siRNA passenger strand cleavage (Leuschner et al.,
2006; Matranga et al., 2005; Rand et al., 2005), either by
omitting the Ago2 cleavage (left pathway in model) or
through an alternative endonuclease activity. We found
the Ago2-mediated pre-miRNA cleavage applicable to
all four miRNAs analyzed, but it could also apply to the
many other miRNAs derived from the 50-arm of the pre-
miRNA hairpin that do not have mismatches at the imme-
diate cleavage site (Han et al., 2006), since mismatches
outside of the cleavage site do not significantly interfere
with Ago2-mediated cleavage (Leuschner et al., 2006).
Mature miRNAs derived from the 30-arm of the hairpin
could be spared from cleavage by their prevalent mis-
match at the cleavage position (Han et al., 2006). Given
the proposed function of the Ago2-mediated cleavage
after Dicer cleavage), we hypothesize that miRNAs with
mismatches in the middle of the duplex would dissociate
due to their inherent lower thermodynamical stability,
whereas miRNAs with high complementarity in their
duplex depend on Ago2-mediated cleavage to facilitate
Mammalian Argonautes have not been previously func-
tionally implicated in miRNA biogenesis, although they are
known to form a complex with Dicer and TRBP before the
pre-miRNA is incorporated into the RLC (Chendrimada
et al., 2005; Gregory et al., 2005; Maniataki and Mourela-
O’Carroll and colleagues described an impact of Ago2 on
mature miRNA expression independent of its Slicer activ-
ing mature miRNA expression. In C. elegans, the greatly
expanded family of Argonaute proteins has recently
been described to function at multiple stages in the
RNAi pathway, exerting multiple functions in siRNA bio-
genesis and the effector phase of RNAi (Yigit et al.,
2006). While these studies focused on siRNA pathways,
the C. elegans Argonaute protein alg-1 has also been de-
scribed to facilitate mature miRNA expression (Grishok
et al., 2001). Thus, while the C. elegans and vertebrate
Argonaute proteins are divergent in structure and gene
number, this gene family may display a broad functional
conservation in both siRNA and miRNA biogenesis and
Role of Ago2 in miRNA and siRNA Processing
Together with the studies described above, our data sug-
gest a consistent role for Ago2 in both siRNA and miRNA
pathways. However, significant differences are also evi-
dent in the structural requirements for Ago2 cleavage in
these two processing pathways. Cleavage of the siRNA
passenger strand by Ago2 prior to incorporation into
RISC has been described in Drosophila and human cells
(Leuschner et al., 2006; Matranga et al., 2005; Miyoshi
et al., 2005; Rand et al., 2005). The contribution of human
Ago2 to miRNA biogenesis differs from this reported role
in siRNA passenger strand cleavage in two major re-
spects: first, in miRNA processing, Ago2 cleaves the pre-
cursor before Dicer-mediated cleavage of the hairpin,
whereas in siRNA processing, Ago2 nicks the mature 22
nt double-stranded RNA. Second, while the siRNA duplex
contains the passenger strand perfectly base-paired with
the guide siRNA, the pre-miRNA hairpin does not provide
a guide siRNA, but instead presents a bulky loop structure
atone end andmismatches in the imperfectlybase-paired
stem of most pre-miRNA hairpins. Thus, the presumed
structural requirements for Ago2 cleavage of the pre-
miRNA differ considerably fromthose described forcleav-
age of either mature double-stranded siRNA orsiRNA-tar-
get mRNA hybrids. Along with the heterogeneity of the
in vivo Drosha cleavage products that we document
here, the abovementioned differences couldalso account
for the apparent heterogeneity of Ago2-mediated pre-
miRNA cleavage sites pointing to a less precise mecha-
nism than siRNA-mediated target cleavage. Alternatively,
in aso faruncharacterized mechanism, themature miRNA
could serve in trans as guide in pre-miRNA cleavage. De-
spite these important differences, our findings unite the
general biogenesis mechanisms described for siRNA
and miRNAprecursors, sharingtheAgo2-mediated cleav-
age of the passenger strand.
MicroRNA Biogenesis and the Dual Functions
We have identified two mechanisms by which Argonaute
proteins impact miRNA biogenesis: increased miRNA
abundance mediated by all Ago proteins and Ago2-spe-
cific cleavage of pre-miRNAs. While we have documented
these effects for multiple independent miRNAs, it remains
to be determined whether this activity is relevant for all
sion, suchaglobalmechanism hasbeensought to explain
Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc. 1105
the coordinated differences in miRNA levels characteristic
of different tissues, e.g., with high global miRNA expres-
sion in mature, differentiated organs versus lower levels
worthy that broadly reduced miRNA levels in tumors
compared to matched normal tissues were correlated
with lower expression of Ago2 in cancer (Lu et al., 2005).
In conclusion, we define two additional roles for Ago
proteins upstream in the pathway of miRNA maturation
and function. As primary effectors of miRNA-mediated
silencing and modulators of miRNA processing and abun-
dance, Argonaute proteins are candidate master regula-
tors of the miRNA biogenesis and effector pathways.
MicroRNA Northern Blotting
Total RNA was isolated from adherent cells using TRIzol (Invitrogen,
Carlsbad, CA) and resuspended in 50 ml preheated nuclease-free wa-
ter. After 45 min of gel prerun, 10–25 mg of total, denatured RNA were
loaded onto 15% polyacrylamide TBE-Urea gels (Bio-Rad, Hercules,
CA) and run in 0.53 TBE at 125 V for 90 min. RNA markers from the
miRVana Probe and Marker kit (Ambion, Austin, TX) were used. After
Ethidium Bromide staining as loading control, RNA was transferred
by electroblotting to Hybond-N+ nylon membranes (Amersham/GE
Healthcare, Piscataway, NJ) at 400 mA for one hour. The membranes
were crosslinked and hybridized with ExpressHyb hybridization buffer
(Clontech, Mountain View, CA) for 30 min at 37?C. 20 pmol DNA oligo-
nucleotide probe were labeled with 20 mCi [g-32P]ATP at the 50-end
Figure 7. Schematic Model of MicroRNA Processing Including the Ac-pre-miRNA
After nuclear export,thepre-miRNA bindstoapreformedcomplex ofDicer,TRBPand Ago2tobuildtheRISC-LoadingComplex(RLC)(Chendrimada
et al., 2005; Gregory et al., 2005; Maniataki and Mourelatos, 2005). Our modified model of miRNA processing includes an additional endonuclease
step in which Ago2 cleaves the pre-miRNA within the RLC generating the nicked ac-pre-miRNA hairpin (shown in gray box). Since the impact of Dicer
and TRBP on the Ago2 cleavage step, as well as the influence of Ago2 to Dicer cleavage, have not been determined, these proteins are depicted with
adashed outline.Since miRNA maturation is diminished butnotcompletelyabrogatedinAgo2-KOMEF cells,aless efficientsalvagepathway islikely
also likely used for miRNAs derived from the 30-arm of the pre-miRNA and miRNAs with mismatches at the cleavage site. After cleavage by Dicer
(Hutvagner et al., 2001; Ketting et al., 2001), the resulting miRNA duplex is unwound, Dicer and TRBP dissociate, the passenger strand of the miRNA
duplex is degraded, and the mature miRNA forms the RISC together with Ago2 (Doench et al., 2003; Hutvagner and Zamore, 2002; Liu et al., 2004;
Meister et al., 2004; Petersen et al., 2006). The stabilization of mature miRNAs by Ago proteins is independent of this Ago2-specific processing step
and not depicted in this model.
1106 Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc.
using T4 Polynucleotide-Kinase (New England Biolabs, Ipswich, MA)
for one hour at 37?C followed by purification on a MicroSpin G-25 col-
umn (Amersham/GE Healthcare, Piscataway, NJ). Probe sequences
are listed in Table S1. The purified probe was hybridized to the mem-
brane in ExpressHyb buffer for one hour at 37?C. After hybridization,
the membranes were washed three times at 37?C for 15 min in 23
SSC/0.05% SDS and twice at room temperature for 20 min in 0.13
SSC/0.1% SDS and exposed at ?80?C to Kodak BioMAX XAR films.
Densitometric quantitation of northern blots was performed using
MicroRNA Precursor Cloning
RNA was isolated from nontransfected human cell lines 293, U2OS,
SAOS-2 and HeLa to clone endogenous, in vivo processed miRNA
precursors using the miRVana isolation kit (Ambion, Austin, TX) with
the protocol for specific enrichment of small RNA species (<200 nt).
The microRNA Cloning Linker-2 (IDT, Coralville, IA) was ligated to
2.5 mg RNA using T4 ssRNA Ligase I (New England Biolabs, Ipswich,
MA). After ethanol precipitation, the linker-ligated RNA was reverse
transcribed with Thermoscript (Invitrogen, Carlsbad, CA) using
a primer complementary to Linker-2. After dilution, the cDNA was
amplified by polymerase chain reaction (PCR) using Pfu Turbo poly-
merase (Stratagene, La Jolla, CA) with a miRNA-specific forward
primer and a reverse primer complementary to the cloning linker.
The PCR products were analyzed by 4% Agarose gel electrophoresis
and positive PCR products were diluted and cloned into pcDNA3.1D
using the pcDNA3.1D Directional TOPO cloning kit (Invitrogen, Carls-
bad, CA). Individual clones were picked, cultured and DNA was iso-
lated using a miniprep kit (QIAgen, Hilden, Germany). The DNA was
digested using Hind III and Xho I, analyzed on a 4% Agarose gel and
all clones with digested products between 50 and 200 bp were
sequenced.All primer sequencesare listed in Table S1.The same pro-
tocol was used to clone miRNA precursors from 293 cells transfected
with let-7a-3 and Ago2.
Dicer Cleavage Assays
RNA oligonucleotides of the pre-miRNA, the ac-pre-miRNA, the frag-
ment cleaved off the pre-miRNA generating the ac-pre-miRNA and
this fragment containing the photo-activatable 4-Thio-Uracil (4-S-U)
for let-7a-3 and miR-20a were synthesized (Dharmacon, Lafayette,
CO). 5 pmol of the pre-miRNA and ac-pre-miRNA were labeled at
the 50-end with 20 mCi [g-32P]ATP using T4 Polynucleotide Kinase
(New England Biolabs, Ipswich, MA) for one hour at 37?C followed
by purification on a MicroSpin G-25 column (Amersham/GE Health-
care, Piscataway, NJ). For hybridization to the fragment, 1 pmol la-
beled ac-pre-miRNA was combined with 100 pmol of fragment in a hy-
bridization buffer containing 10 mM Tris-Cl, pH8.0, and 20 mM NaCl.
The hybridization mixture was heated to 90?C for 3 min and then grad-
ually cooled down to 4?C over 90 min. For in vitro Dicer cleavage as-
says, recombinant Dicer enzyme was used (Genlantis/Gene Therapy
Systems, San Diego, CA). In the cleavage reaction, 0.5 pmol labeled
or labeled and hybridized pre-miRNA or ac-pre-miRNA was incubated
with 0.5 units Dicer, 10 units RNaseOut (Invitrogen, Carlsbad, CA) in
13 Dicer Reaction Buffer, 2.5 mM MgCl2and 1 mM ATP at 37?C for
15 hr. After 45 min of gel prerun, the entire Dicer cleavage reaction
was denatured with RNA loading buffer at 90?C for 3 min and loaded
onto 15% TBE-Urea gels (Bio-Rad, Hercules, CA) along with 0.5
pmol of labeled and undigested pre-miRNA and ac-pre-miRNA and
run in 0.53 TBE at 125 V for 90 min. RNA markers from the miRVana
Probe and Marker kit (Ambion,Austin, TX) were used.After gel electro-
phoresis, the gel was transferred onto filter paper and exposed to Ko-
dak BioMAX XAR films.
Supplemental Data include Supplemental Experimental Procedures,
eight figures, and one table and can be found with this article online
We thank Drs. Thomas Tuschl (Rockefeller University), Ramin Shie-
khattar (Wistar Institute), Gideon Dreyfuss (University of Pennsylvania),
Mien-Chie Hung (University of Texas), and Ian G. Macara (University of
mell and Gregory J. Hannon (Cold Spring Harbor Laboratory) for pro-
viding the WT and Ago2-KO MEF cells. We also thank Dr. Gromoslaw
Smolen forcriticalreading ofthe manuscript. Thiswork wassupported
by grants from the German Research Foundation DFG (Di 1421/1-1, to
S.D.), the National Institutes of Health (NIH RO1 95281, to D.A.H.), and
a grant from the National Foundation for Cancer Research (to D.A.H.).
Received: April 18, 2007
Revised: July 6, 2007
Accepted: October 2, 2007
Published: December 13, 2007
Bernstein, E., Kim, S.Y., Carmell, M.A., Murchison, E.P., Alcorn, H.,
Li, M.Z., Mills, A.A., Elledge, S.J., Anderson, K.V., and Hannon, G.J.
(2003). Dicer is essential for mouse development. Nat. Genet. 35,
Borchert, G.M., Lanier, W., and Davidson, B.L. (2006). RNA polymer-
ase III transcribes human microRNAs. Nat. Struct. Mol. Biol. 13,
Chendrimada, T.P., Gregory, R.I., Kumaraswamy, E., Norman, J.,
Cooch, N., Nishikura, K., and Shiekhattar, R. (2005). TRBP recruits
the Dicer complex to Ago2 for microRNA processing and gene silenc-
ing. Nature 436, 740–744.
Denli, A.M., Tops, B.B., Plasterk, R.H., Ketting, R.F., and Hannon, G.J.
(2004). Processing of primary microRNAs bythe Microprocessor com-
plex. Nature 432, 231–235.
Diederichs, S., and Haber, D.A. (2006). Sequence variations of micro-
RNAs in human cancer: alterations in predicted secondary structure
do not affect processing. Cancer Res. 66, 6097–6104.
Doench, J.G., Petersen, C.P., and Sharp, P.A. (2003). siRNAs can
function as miRNAs. Genes Dev. 17, 438–442.
Elbashir, S.M., Lendeckel, W., and Tuschl, T. (2001). RNA interference
is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200.
Gregory, R.I., Chendrimada, T.P., Cooch, N., and Shiekhattar, R.
(2005). Human RISC couples microRNA biogenesis and posttranscrip-
tional gene silencing. Cell 123, 631–640.
Gregory, R.I., Yan, K.P., Amuthan, G., Chendrimada, T., Doratotaj, B.,
Cooch, N., and Shiekhattar, R. (2004). The Microprocessor complex
mediates the genesis of microRNAs. Nature 432, 235–240.
D.L., Fire, A., Ruvkun, G., and Mello, C.C. (2001). Genes and mecha-
nisms related to RNA interference regulate expression of the small
temporal RNAs that control C. elegans developmental timing. Cell
Guil, S., and Caceres, J.F. (2007). The multifunctional RNA-binding
protein hnRNP A1 is required for processing of miR-18a. Nat. Struct.
Mol. Biol. 14, 591–596.
Han, J., Lee, Y., Yeom, K.H., Kim, Y.K., Jin, H., and Kim, V.N. (2004).
The Drosha-DGCR8 complex in primary microRNA processing. Genes
Dev. 18, 3016–3027.
Cho, Y., Zhang, B.T., and Kim, V.N. (2006). Molecular basis for the rec-
ognition of primary microRNAs by the Drosha-DGCR8 complex. Cell
He, L., Thomson, J.M., Hemann, M.T., Hernando-Monge, E., Mu, D.,
Goodson, S., Powers, S., Cordon-Cardo, C., Lowe, S.W., Hannon,
Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc. 1107
G.J., et al. (2005). A microRNA polycistron as a potential human onco- Download full-text
gene. Nature 435, 828–833.
Humphreys, D.T., Westman, B.J., Martin, D.I., and Preiss, T. (2005).
MicroRNAs control translation initiation by inhibiting eukaryotic initia-
tion factor 4E/cap and poly(A) tail function. Proc. Natl. Acad. Sci.
USA 102, 16961–16966.
Hutvagner, G., McLachlan, J., Pasquinelli, A.E., Balint, E., Tuschl, T.,
and Zamore, P.D. (2001). A cellular function for the RNA-interference
enzyme Dicer in the maturation of the let-7 small temporal RNA. Sci-
ence 293, 834–838.
Hutvagner, G., and Zamore, P.D. (2002). A microRNA in a multiple-
turnover RNAi enzyme complex. Science 297, 2056–2060.
Ketting, R.F., Fischer, S.E., Bernstein, E., Sijen, T., Hannon, G.J., and
Plasterk, R.H. (2001). Dicer functions in RNA interference and in syn-
thesis of small RNA involved in developmental timing in C. elegans.
Genes Dev. 15, 2654–2659.
Khvorova, A., Reynolds, A., and Jayasena, S.D. (2003). Functional siR-
NAs and miRNAs exhibit strand bias. Cell 115, 209–216.
Lee, R.C., Feinbaum, R.L., and Ambros, V. (1993). The C. elegans het-
erochronic gene lin-4 encodes small RNAs with antisense comple-
mentarity to lin-14. Cell 75, 843–854.
Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P.,
Radmark, O., Kim, S., et al. (2003). The nuclear RNase III Drosha initi-
ates microRNA processing. Nature 425, 415–419.
Lee, Y., Kim, M., Han, J., Yeom, K.H., Lee, S., Baek, S.H., and Kim,
V.N. (2004). MicroRNA genes are transcribed by RNA polymerase II.
EMBO J. 23, 4051–4060.
Leuschner, P.J., Ameres, S.L., Kueng, S., and Martinez, J. (2006).
Cleavage of the siRNA passenger strand during RISC assembly in
human cells. EMBO Rep. 7, 314–320.
Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M.,
Song, J.J., Hammond, S.M., Joshua-Tor, L., and Hannon, G.J.
(2004). Argonaute2 is the catalytic engine of mammalian RNAi.
Science 305, 1437–1441.
Liu, J., Valencia-Sanchez, M.A., Hannon, G.J., and Parker, R. (2005).
MicroRNA-dependent localization of targeted mRNAs to mammalian
P-bodies. Nat. Cell Biol. 7, 719–723.
Lu, J., Getz, G., Miska, E.A., Alvarez-Saavedra, E., Lamb, J., Peck, D.,
Sweet-Cordero, A.,Ebert,B.L.,Mak,R.H.,Ferrando,A.A., etal.(2005).
MicroRNA expression profiles classify human cancers. Nature 435,
Lund, E., Guttinger, S., Calado, A., Dahlberg, J.E., and Kutay, U.
(2004). Nuclear export of microRNA precursors. Science 303, 95–98.
Maniataki, E., and Mourelatos, Z. (2005). A human, ATP-independent,
RISC assembly machine fueled by pre-miRNA. Genes Dev. 19, 2979–
Matranga, C., Tomari, Y., Shin, C., Bartel, D.P., and Zamore, P.D.
(2005). Passenger-strand cleavage facilitates assembly of siRNA into
Ago2-containing RNAi enzyme complexes. Cell 123, 607–620.
Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G.,
and Tuschl, T. (2004). Human Argonaute2 mediates RNA cleavage
targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197.
Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H., and Siomi, M.C.
(2005). Slicer function of Drosophila Argonautes and its involvement
in RISC formation. Genes Dev. 19, 2837–2848.
Neilson, J.R., Zheng, G.X., Burge, C.B., and Sharp, P.A. (2007).
Dynamic regulation of miRNA expression in ordered stages of cellular
development. Genes Dev. 21, 578–589.
O’Carroll, D., Mecklenbrauker, I., Das, P.P., Santana, A., Koenig, U.,
Enright, A.J., Miska, E.A., and Tarakhovsky, A. (2007). A Slicer-inde-
pendent role for Argonaute 2 in hematopoiesis and the microRNA
pathway. Genes Dev. 21, 1999–2004.
O’Donnell, K.A., Wentzel, E.A., Zeller, K.I., Dang, C.V., and Mendell,
J.T. (2005). c-Myc-regulated microRNAs modulate E2F1 expression.
Nature 435, 839–843.
Obernosterer,G.,Leuschner,P.J.,Alenius,M.,and Martinez, J.(2006).
Post-transcriptional regulation of microRNA expression. RNA 12,
Okamura, K., Hagen, J.W., Duan, H., Tyler, D.M., and Lai, E.C. (2007).
The Mirtron Pathway Generates microRNA-Class Regulatory RNAs in
Drosophila. Cell 130, 89–100.
Petersen, C.P., Bordeleau, M.E., Pelletier, J., and Sharp, P.A. (2006).
Short RNAs repress translation after initiation in mammalian cells.
Mol. Cell 21, 533–542.
Pillai, R.S., Artus, C.G., and Filipowicz, W. (2004). Tethering of human
Ago proteins to mRNA mimics the miRNA-mediated repression of pro-
tein synthesis. RNA 10, 1518–1525.
Pillai, R.S., Bhattacharyya, S.N., Artus, C.G., Zoller, T., Cougot, N.,
Basyuk, E., Bertrand, E., and Filipowicz, W. (2005). Inhibition of trans-
lational initiation by Let-7 MicroRNA in human cells. Science 309,
Rand, T.A., Ginalski, K., Grishin, N.V., and Wang, X. (2004). Biochem-
ical identification of Argonaute 2 as the sole protein required for RNA-
induced silencing complex activity. Proc. Natl. Acad. Sci. USA 101,
Rand, T.A., Petersen, S., Du, F., and Wang, X. (2005). Argonaute2
cleaves the anti-guide strand of siRNA during RISC activation. Cell
Rivas, F.V., Tolia, N.H., Song, J.J., Aragon, J.P., Liu, J., Hannon, G.J.,
and Joshua-Tor, L. (2005). Purified Argonaute2 and an siRNA form re-
combinant human RISC. Nat. Struct. Mol. Biol. 12, 340–349.
Rodriguez,A., Griffiths-Jones, S., Ashurst, J.L., and Bradley, A. (2004).
Identification of mammalian microRNA host genes and transcription
units. Genome Res. 14, 1902–1910.
Ruby, J.G., Jan, C.H., and Bartel, D.P. (2007). Intronic microRNA pre-
cursors that bypass Drosha processing. Nature 448, 83–86.
Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore,
P.D. (2003). Asymmetry in the assembly of the RNAi enzyme complex.
Cell 115, 199–208.
Song, J.J., Smith, S.K., Hannon, G.J., and Joshua-Tor, L. (2004). Crys-
tal structure of Argonaute and its implications for RISC slicer activity.
Science 305, 1434–1437.
Thomson, J.M., Newman, M., Parker, J.S., Morin-Kensicki, E.M.,
Wright, T., and Hammond, S.M. (2006). Extensive post-transcriptional
regulation of microRNAs and its implications for cancer. Genes Dev.
Wightman, B., Ha, I., and Ruvkun, G. (1993). Posttranscriptional regu-
lation of the heterochronic gene lin-14 by lin-4 mediates temporal pat-
tern formation in C. elegans. Cell 75, 855–862.
Yi, R., Qin, Y., Macara, I.G., and Cullen, B.R. (2003). Exportin-5 medi-
ates the nuclear export of pre-microRNAs and short hairpin RNAs.
Genes Dev. 17, 3011–3016.
Yigit, E., Batista, P.J., Bei, Y., Pang, K.M., Chen, C.C., Tolia, N.H.,
Joshua-Tor, L., Mitani, S., Simard, M.J., and Mello, C.C. (2006). Anal-
act sequentially during RNAi. Cell 127, 747–757.
Zeng, Y., and Cullen, B.R. (2005). Efficient processing of primary
microRNA hairpins by Drosha requires flanking nonstructured RNA
sequences. J. Biol. Chem. 280, 27595–27603.
1108 Cell 131, 1097–1108, December 14, 2007 ª2007 Elsevier Inc.