MicroRNAs (miRNAs) are key regulators of gene expres-

sion that have important roles in a wide range of biologi-

cal processes, including animal and plant development,
cell proliferation and differentiation, apoptosis and
metabolism
14
. There is strong evidence to suggest that
miRNAs are also involved in the pathogenesis of human
diseases such as cancer and metabolic disorders
3,4
. The
number of miRNAs encoded by the genomes of the
organisms that have been studied so far varies con-
siderably from a handful to up to 500 in mammals
1,2
.
Computational predictions and genome-wide identifica-
tion of miRNA targets estimate that each animal miRNA
regulates hundreds of different mRNAs, suggesting that a
remarkably large proportion of the transcriptome (about
50% in humans) is subject to miRNA regulation
1,2
.
In animals, most miRNAs are processed from longer
hairpin transcripts by the consecutive action of the
RNase III-like enzymes Drosha and Dicer, whereas in
plants only Dicer is responsible for miRNA process-
ing
24
. One strand of the hairpin duplex is loaded into
an Argonaute family protein (AGO) to form the core
of miRNA-induced silencing complexes (miRISCs).
miRISCs silence the expression of target genes pre-
dominantly at the post-transcriptional level. The tar-
gets to be silenced are selected through base-pairing
interactions between the loaded miRNA and an mRNA
target that contains a partially or fully complementary
sequence
14
.
Over the past few years, remarkable progress has been
made in our understanding of miRNA biogenesis and
function
3,4
; however, the mechanisms that miRNAs use to
regulate gene expression remain unclear and several con-
troversies surround the topic
47
. In animals, the initial evi-
dence suggested that miRNAs repress their targets at the
level of translation, with little or no influence on mRNA
abundance. By contrast, plant miRNAs were thought
to act almost entirely by promoting target cleavage
and degradation. These differences in target regulation
between plants and animals stem from the fact that
the base-pairing between miRNAs and their targets is
much less extensive in animals than in plants (FIG. 1).
Nevertheless, despite differences in target recognition,
it has now become clear that miRNAs can induce mRNA
degradation in animals and, conversely, translational
repression in plants. However, the question of whether
target silencing occurs predominantly by mRNA deg-
radation or at the level of translation has been highly
controversial, with conflicting lines of evidence support-
ing both views
47
. Furthermore, in the case of animal
miRNAs, translational repression has been proposed
to occur in four distinct ways: inhibition of transla-
tion initiation; inhibition of translation elongation;
co-translational protein degradation; and premature
termination of translation
47
.
Recently, several studies have taken advantage
of advances in the fields of mass spectrometry and
Max Planck Institute for
Developmental Biology,
Spemannstrasse 35,
D72076 Tbingen, Germany.
Correspondence to E.I.
email: elisa.izaurralde@
tuebingen.mpg.de
doi:10.1038/nrg2936
Argonaute family proteins
The effectors of RNA-mediated
gene-silencing pathways.
Small RNAs (for example, small
interfering RNAs or microRNAs)
guide Argonautes to their
RNA targets; Argonautes carry
out regulation either directly
or by recruiting additional
factors. Most multicellular
eukaryotes have several
Argonaute paralogues.
Gene silencing by microRNAs:
contributions of translational
repression and mRNA decay
Eric Huntzinger and Elisa Izaurralde
Abstract | Despite their widespread roles as regulators of gene expression, important
questions remain about target regulation by microRNAs. Animal microRNAs were
originally thought to repress target translation, with little or no influence on mRNA
abundance, whereas the reverse was thought to be true in plants. Now, however, it is clear
that microRNAs can induce mRNA degradation in animals and, conversely, translational
repression in plants. Recent studies have made important advances in elucidating the
relative contributions of these two different modes of target regulation by microRNAs.
They have also shed light on the specific mechanisms of target silencing, which, although it
differs fundamentally between plants and animals, shares some common features between
the two kingdoms.
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0CVWTG4GXKGYU)GPGVKEU
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Figure 1 | RnA-target recognition in plants and animals. MicroRNAs (miRNAs)
recognize their targets by WatsonCrick base pairing. a | Plant miRNAs recognize fully
or nearly complementary binding sites, which are generally located in ORFs.
Importantly, miRNA nucleotides (N) 912 are usually engaged in WatsonCrick base
pairing, which allows target cleavage (also referred to as slicing) by Argonaute proteins
(AGOs)
2
. The PIWI domains of AGOs cleave the mRNA within the base-paired region
(between nucleotides 10 and 11, opposite the miRNA strand). The 3-most nucleotide
of plant miRNAs is modified with a 2O-methyl group that protects them from
degradation. b | Animal miRNAs recognize partially complementary binding sites,
which are generally located in 3 UTRs. Complementarity to the 5 end of the miRNA
the seed sequence, containing nucleotides 27 is a major determinant in target
recognition and is sufficient to trigger silencing. For most miRNA-binding sites the
complementarity is limited to the seed sequence (seed-matched sites) or to the seed
sequence plus miRNA nucleotide 8. However, in some rare cases complementarity to
the 3 region of the miRNA might contribute to target selection, particularly when the
mRNA has a weak seed match. Even for these sites, however, miRNA nucleotides 912
generally bulge out, preventing endonucleolytic cleavage by AGOs
1,2
. Note that in
both animals and plants the miRNA 5 terminal nucleotide (shown in grey) is buried
in the mid domain of AGOs and is not available for pairing with the target.
5-cap
Eukaryotic mRNA is modified
by the addition of an m
7
G(5)
ppp(5)N structure at the 5
terminus. Capping is essential
for several important steps
of gene expression for
example, mRNA stabilization,
splicing, mRNA export
from the nucleus and
translation initiation.
Polysome
A functional unit of protein
synthesis that consists of
several ribosomes attached
along the length of a single
molecule of RNA.
transcriptome analysis to measure, on a genome-wide
scale, changes in protein output and mRNA abundance
in response to either the removal or the ectopic expres-
sion of animal miRNAs
811
. Together, these studies
concluded that degradation of miRNA targets is a wide-
spread effect of miRNA-based regulation, which alone
accounts for most of the repression mediated by miRNAs
in mammalian cell cultures. This type of analysis has not
yet been carried out in plants, in which the contribution
of translational inhibition to miRNA regulation remains
unknown at the proteome level.
In this Review we discuss these recent findings and
the emerging picture of the molecular mechanisms that
drive miRNA silencing, both in animals and plants.
Although recent years have provided important insights
into miRNA biogenesis and regulation
3,4
, we do not dis-
cuss them in this Review. Rather, we focus on the effec-
tor step of silencing and on what happens after a target
is recognized by the miRISC complexes that mediate
silencing. First, we briefly summarize the evidence for
miRNA-mediated translational repression, either before
or after translation initiation, in animals. we also review
the evidence for target degradation in animals and the
mechanism involved. we then describe recent studies
in which the contributions of translational repression
and target degradation to miRNA silencing have been
investigated. These studies also provide insight into the
mechanism of silencing in animals, although some key
questions remain to be answered, particularly about the
mechanistic connections between translation repression
and mRNA degradation. we discuss how understand-
ing the functions of proteins of the Gw182 family of
trinucleotide-repeat containing proteins, which are key
components of miRNA silencing complexes in animals,
is advancing our knowledge in this respect. Finally, we
discuss recent evidence that target regulation in plants
can also take place at the level of translational repression.
we outline the commonalities and differences between
silencing mechanisms in animals and plants, and
consider directions for future research.
Animal miRNAs and translational repression
Translation requires numerous factors that are involved
in the recruitment of the ribosomal subunits to the
mRNA and that ensure initiation at the correct initiation
codon, and proper elongation and termination. Although
the basic steps in translation are not reviewed here, a
fact that is relevant to our discussion is that mRNAs are
competent for translation if they posses a 5-cap struc-
ture and a 3-poly(A) tail (BOX 1). Indeed, the factors
that associate with the 5-cap and 3-poly(A) tail inter-
act: the cytoplasmic poly(A)-binding protein (PABPC,
which is associated with the poly(A) tail) interacts with
eukaryotic translation-initiation factor 4G (eIF4G,
which is associated with the cap structure through
interaction with the cap-binding protein eIF4e), giving
rise to circular mRNAs that are efficiently translated
and protected from degradation
12
. There is increasing
evidence to suggest that animal miRNAs interfere with
the function of the eIF4F complex (comprising eIF4e,
eIF4G and eIF4A) and PABPC during translation and/or
mRNA stabilization.
Evidence for repression at the post-initiation stage. The
earliest studies addressing the mechanism of miRNA
regulation were performed in Caenorhabditis elegans. It
was found that the lin-4 miRNA represses the translation
of the lin14 and lin28 mRNAs, with little or no influ-
ence on their abundance
13,14
. Although protein expression
was inhibited, the lin14 and lin28 mRNAs were detected
in polysomes, suggesting that repression occurred after
translation had been initiated
13,14
, an idea that was sup-
ported by subsequent studies in mammalian cell cul-
tures
1517
. Although the details of these studies differ, their
conclusions rest on a common observation: in sucrose
sedimentation gradients (BOX 2), miRNAs and their tar-
gets were associated with polysomes. These polysomes
were sensitive to various conditions that inhibit transla-
tion, and so were considered to be actively involved in
translation. For example, if the polysomes were briefly
incubated with translation inhibitors (for example, hip-
puristanol, puromycin or pactamycin) they dissociated
into monosomes and/or ribosomal subunits
1517
.
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0CVWTG4GXKGYU^)GPGVKEU
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Box 1 | Role of the cap structure and the poly(A) tail in translation
It is well established that both the 5-cap structure and the poly(A)
tail of mRNAs promote translation and that both cooperate to
recruit 43S pre-initiation complexes (comprising a 40S ribosomal
subunit, the eIF2GTPMETtRNA
Meti
, eukaryotic translation
initiation factor 3 (eIF3), eIF1, eIF1A and probably eIF5, not
shown)
84
. The role of the cap structure and the poly(A) tail in
translation is mediated by the proteins that recognize these specific
features. In the cytoplasm, the cap structure is recognized by eIF4F,
which consists of the cytoplasmic cap-binding protein eIF4E, the
scaffolding protein eIF4G and the RNA helicase eIF4A. The poly(A) tail is bound by the cytoplasmic poly(A)-binding
protein PABPC. PABPC interacts with eIF4G. This interaction brings the termini of an mRNA into close proximity and
increases translation efficiency by increasing eIF4E binding to the cap structure and the likelihood of recruitment of the
43S pre-initiation complex
84
. The requirement of the cap structure and of the cap-binding protein eIF4E can be bypassed
if the mRNA contains an internal ribosome entry site (IRES, not shown), which promotes 43S or 40S subunit binding
internally in an mRNA. Nevertheless, some IRESs require eIF4G, eIF4A and PABPC for optimal translation
84
.
Internal ribosome entry site
(IRES). A structured RNA
element, usually present in the
5 UTR, that allows m
7
G-cap-
independent association of a
ribosome with mRNA.
Krebs-2 ascites tumour cells
Tumour cells grown in vivo in the
peritoneal cavity of the mouse.
5-to-3 mRNA decay
pathway
A major decay pathway for
bulk mRNA that is initiated by
the removal of the poly(A)
tail by deadenylases and
is followed by decapping
and subsequent 5-to-3
exonucleolytic digestion
of the mRNA body.
In the studies mentioned above, miRNA targets
appeared in the actively translated fraction, without
the corresponding protein product being detectable.
To explain these findings, Nottrott et al.
16
proposed that
the nascent polypeptide chain might be degraded co-
translationally. By contrast, Petersen et al.
17
proposed
that miRNAs cause ribosomes to dissociate prematurely
(ribosome drop off ). This latter model had support
because, although the miRNA targets were associated
with polysomes, when treated with translational inhibi-
tors these polysomes dissociated more rapidly than
those associated with a control (unrepressed) mRNA
17
.
Further support for the idea that miRNAs act after cap
recognition (that is, after initiation) was provided by
the observation that miRNAs could silence translation
initiated independently of the cap structure through an
internal ribosome entry site (IReS)
17
(BOX 1).
Evidence for repression at initiation. Despite these data,
conflicting results indicated that miRNAs inhibit trans-
lation at initiation. For example, Pillai et al.
18
showed that
in the presence of cognate miRNAs, mRNA targets do
not co-sediment with the polysomal fraction in sucrose
gradients, but shift towards lighter fractions containing
fewer ribosomes or free messenger ribonucleoproteins
(mRNPs). In this and other studies, mRNAs translated
through cap-independent mechanisms (that is, through
an IReS) were refractory to repression by miRNAs, sug-
gesting that miRNAs inhibit cap-dependent translation
initiation
18,19
.
Subsequent studies in cell-free extracts of diverse
origin have supported a role for miRNAs in inhibiting
translation initiation
2024
. In extracts, miRNAs silenced
m
7
Gppp-capped mRNAs, but not mRNAs carrying an
artificial Appp-cap structure. Moreover, in mouse and
human cell extracts, miRNAs failed to silence transcripts
if translation was driven by an IReS
23,24
. Consistent with
these findings, in extracts from mouse Krebs-2 ascites
tumour cells, silencing was suppressed with increasing
concentrations of purified eIF4F
23
(BOX 1). This and more
recent studies
25,26
argued that the silencing machinery
targets the cap structure and/or interferes with the func-
tion of the cap-binding complex eIF4F. As discussed
below, there is now increasing evidence to suggest that
if translational repression does occur, it is predominantly
at initiation.
Animal miRNAs and target degradation
Evidence for target degradation. evidence that animal
miRNAs can induce target mRNA degradation comes
from studies on specific miRNAtarget pairs and, more
generally, from transcriptome studies showing that the
abundance of miRNA targets inversely correlates with
the level of miRNA
811,2741
. For example, if specific
miRNAs are introduced into cultured cells, then tran-
scripts containing complementary binding sites (for
example, seed matches) become less abundant
811,27
.
Conversely, transcriptome profiling of cells depleted of
an miRNA showed a corresponding increase in tran-
scripts containing binding sites for this miRNA
8,9,28
.
Furthermore, in cultured cells, depleting essential com-
ponents of the miRNA pathway (for example, Dicer,
AGOs or Gw182) increased the abundance of miRNA
targets
3135,37,38
. expression profiles from differentiating
and developing cells also provide numerous examples
showing anti-correlated expression of miRNAs and their
targets
40,41
. For example, at the onset of zebrafish zygotic
transcription, a dramatic increase in miR-430 expres-
sion correlates with the degradation of a large number
of maternal mRNAs containing miR-430-binding sites
in their 3 uTRs
35,39
. Collectively, these studies provide
genome-wide evidence that mRNA destabilization is a
widespread effect of miRNA regulation.
Mechanisms of miRNA-mediated target degradation.
Although miRNAs can direct endonucleolytic cleavage
of fully complementary targets
42
, they rarely do so in ani-
mal cells, in which the vast majority of targets are par-
tially complementary (FIG. 1). In the case of such targets,
miRNAs direct their targets to the cellular 5-to-3 mRNA
decay pathway
31,3438,43
, where mRNAs are first dead-
enylated (FIG. 2c). mRNAs are primarily deadenylated
by the CAF1CCR4NOT deadenylase complex, and
then decapped by the decapping enzyme DCP2. DCP2
requires additional co-factors for full activity or stability.
In metazoa, these include DCP1, eDC4 (also known as
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Box 2 | Methods for determining the effect of microRNA-mediated regulation on translation and mRNA degradation
The most straightforward and sensitive method for
determining the contributions of translational repression
and mRNA degradation to the overall effect of
microRNAs (miRNAs) is to directly quantify protein
levels by western blot (or enzymatic activity) and
mRNA levels by northern blot. Although this approach
has been used in many studies it is not amenable to
large-scale studies. Furthermore, the analysis of protein
levels by western blot does not provide information
about the mechanism of translational repression.
Polysome profiling
This method assesses how efficiently an mRNA is
translated in vivo. Cells are normally treated with
cycloheximide to arrest translating ribosomes.
Lysates from these cells are then loaded onto a sucrose
gradient (for example, 560% sucrose) and velocity
sedimentation separates free mRNAs (the free
ribonucleoprotein (RNP) fraction) from mRNAs
associated with varying numbers of ribosomes (see
panel a in the figure). If translation is inhibited during
elongation, the sedimentation of miRNA targets should
be shifted to heavier fractions of the gradient in the
presence of the cognate miRNA. By contrast, if
translation is inhibited at initiation, miRNA targets are
expected to shift to a lighter fraction of the gradient
(with fewer ribosomes) in the presence of the miRNA
18,19
.
Hendrickson et al.
10
used polysome profiles combined
with microarray analysis to determine how the ectopic
expression of miRNAs affects the translation rates of
targets. Translation rates can be inferred by determining
the fraction of a given transcript associated with
ribosomes (ribosome occupancy) and the average number
of ribosomes per unit of coding sequence (ribosome
density) (see part b in the figure). Ribosome occupancy is
established by measuring the amount of a given mRNA
in the free RNP fraction of the polysome profile relative
to the fraction associated with one or more ribosomes.
Ribosome density for a given mRNA is estimated by
analysing each fraction of the gradient individually. This
number is normalized to the length of the ORF. For a given
mRNA, ribosome occupancy and density are normalized
to total mRNA levels (to correct for mRNA degradation),
these values are then compared in the absence and the
presence of the miRNA to determine whether the miRNA
promotes changes in translation rates.
Ribosome profiling
This determines the position of ribosomes on cellular
mRNAs with high sequence resolution
11,85
. Cells are briefly
treated with cycloheximide, then lysed and treated with
RNase I to degraded mRNA regions not protected by
ribosomes. This treatment releases monosomes that
protect RNA fragments of about 30 nucleotides in length
(ribosome protected fragments (RPFs)). After monosomes
are purified on sucrose gradients, the protected mRNA
fragments are released and sequenced, generating
millions of mRNA-sequence tags. In parallel, total mRNA
levels are also determined. For a given mRNA, changes
to RPFs in the presence of the miRNA are normalized to
changes on total mRNA levels. This normalization removes
the contribution of mRNA degradation, so that any
remaining change can be attributed to changes in
translation efficiency. 40S, small ribosomal subunit;
60S, large ribosomal subunit; 80S, complete ribosome,
or monosome; OD, optical density.
0CVWTG4GXKGYU)GPGVKEU
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Ge1), PAT and the DeAD-box protein RCK (also known
as Me31B). In vivo, decapped mRNAs are ultimately
degraded by the major cytoplasmic 5-to-3 exonuclease
XRN1 (FIG. 2f).
The role of mRNA decay factors in miRNA-mediated
mRNA destabilization is evidenced by the observation
that the abundance of miRNA targets increases when
these factors are depleted or when dominant-negative
forms are overexpressed
31,34,37,38,43,44
. For example, in
cells depleted of components of the CAF1CCR4NOT
complex, most miRNA targets (both predicted and
validated) are upregulated
34,38
. This supports the idea
that deadenylation is a widespread consequence of
miRNA regulation
38
.
A role for decapping is more difficult to demonstrate,
first because decapping factors are redundant
37
, and
second because depleting decapping factors does not
restore protein levels. Although it is true that blocking
decapping causes deadenylated miRNA targets to accu-
mulate (because deadenylation preceeds decapping),
these deadenylated mRNAs are not translated efficiently,
so protein levels are not fully restored
37
. Nevertheless,
transcriptome analysis showed that in cells depleted
of decapping factors, mRNA levels of predicted and
validated miRNA targets increase
37
.
miRNA-mediated deadenylation has also been
observed in cell-free extracts
22,24,25,45
. However, in con-
trast to cultured cells, in which deadenylated mRNAs are
committed to decapping and 5-to-3 exonucleolytic deg-
radation
29,34,3638,43
, in cell extracts deadenylated mRNAs
are not further degraded and remain in a deadenylated,
translationally repressed state. These observations pro-
vided a new perspective on the question of the inhibition
of translation by miRNAs. Indeed, because deadenylation
alone could account for the reduction in protein output,
it has been proposed that miRNAs primarily cause target
mRNAs to be deadenylated, rendering them unable to
bind PABPC, unable to circularize and silenced for trans-
lation
24
. However, there is debate regarding the order of
events, because deadenylation has been reported both to
precede
22,24
and to follow translational repression
25,45
.
Translational repression versus decay
Target degradation provides a major contribution to
silencing. As discussed above, there is compelling evi-
dence both that miRNAs repress target translation and
that they trigger target degradation. until recently, it
was unclear which of these mechanisms dominates.
This question has been difficult to address because, in
contrast to mRNA levels, which can be analysed using
high-throughput methods (for example, microarrays or
deep sequencing), protein levels are difficult to quan-
tify on a proteome-wide level. However, the combina-
tion of recently developed proteomics methods with
more established methods of mRNA profiling (BOX 2)
have provided important insight and suggest that target
degradation is the predominant mode of regulation by
miRNAs in mammalian cell cultures.
Two recent studies used quantitative mass spec-
trometry approaches to measure the effect of miRNAs
on protein output at the proteome level. These studies
aimed to discern to what degree silencing was caused
by translational repression versus mRNA degradation.
To this end, miRNAs were transfected or depleted in
cultured cells and then changes in the levels of proteins
and mRNA were measured in parallel
8,9
. These studies
agree on one main conclusion: miRNAs only modestly
inhibit protein production, rarely resulting in more than
a fourfold reduction in protein levels.
However, the two studies disagree on what fraction
of targets are regulated only at the translational level.
Selbach et al. found that at an early time point (8 hours)
after transfecting an miRNA, many targets were regu-
lated only at the protein level, but at a later time point
(32 hours) protein and mRNA levels correlated
8
. Baek
et al. analysed protein and mRNA levels in mouse neu-
trophils isolated from a mir223-gene-knockout mice
compared with wild-type mice
9
. In this case, changes in
protein and mRNA levels strongly correlate. Accordingly,
changes in mRNA levels accounted for most of the regu-
lation, and only a small fraction of targets was repressed
at the translational level without detectable changes
in mRNA abundance. This rare class of targets also
displayed a modest level of regulation.
Subsequent studies have provided additional strong
evidence that target degradation provides a major con-
tribution to silencing by animal miRNAs. They have also
provided support for the findings discussed earlier that
suggest that if translational repression occurs, it is at the
level of initiation. In one of these recent studies, mRNA
levels and translational rates were measured in human
embryonic kidney (HeK)-293T cells transfected with
miR-124 (REF. 10). The authors identified 600 transcripts
that, in response to miR-124 transfection, co-immuno-
precipitated with AGOs. Comparing mRNA abundance
and translational rates revealed that mRNA degradation
accounted for about 75% of the changes observed in pro-
tein synthesis, regardless of the magnitude of the regula-
tion. Furthermore, this study did not find evidence of
targets regulated exclusively at the level of translation.
Recently, Bartel and colleagues
11
further investigated
the contribution of translational repression to silencing
of miRNA targets using ribosome profiling, which allows
the position of translating ribosomes to be mapped at
high resolution (BOX 2). In agreement with the studies
mentioned above, they found that miRNAs cause a
decrease in steady-state mRNA levels that can explain
most of the reduction (84%) in protein production. By
contrast, the mRNA fraction that was not degraded was
translated less efficiently. These results did not change
when the analysis was done at an earlier time point
(12 hours) instead of 32 hours after miRNA transfection,
suggesting that if translational repression occurs before
degradation, mRNA destabilization must immediately
follow. Therefore, regardless of whether destabilization
occurs before or immediately after a translational block,
it nevertheless provides the main contribution to the
reduction in protein output.
The common theme emerging from these studies
is that rapid mRNA degradation can explain a large
fraction of miRNA regulation in animal cell cultures.
As discussed further below, whether decay occurs as a
REVI EWS
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AGO
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Figure 2 | Mechanisms of microRnA-mediated gene silencing in animals. The
minimal requirements for microRNA (miRNA)-mediated gene silencing in animals are:
an Argonaute protein (AGO), a GW182 trinucleotide-repeat-containing protein,
cytoplasmic poly(A)-binding protein (PABPC), components of the major deadenylase
complex (CAF1, CCR4 and the NOT complex), the decapping enzyme DCP2 and
several decapping activators (for example, DCP1, EDC4 and DDX6, also known as
RCK). a | The mRNA target is represented in a closed loop conformation, which is
achieved through interactions between PABPC bound to the 3 poly(A) tail and
eukaryotic translation-initiation factor 4G (eIF4G) bound to the cap-binding protein
eIF4E
12
. b,c | Animal miRNAs bound to AGO recognize their mRNA targets by
base-pairing to partially complementary binding sites, which are predominantly
located in the mRNA 3 UTR. AGO interacts with GW182 (b), which interacts, in turn,
with PABPC bound to the mRNA poly(A) tail (c). GW182 proteins contain two
PABPC-binding sites: the PAM2 motif, which confers direct binding to the PABPC
MLLE domain
45,6668
, and a less-defined sequence comprising the M2 and
carboxy-terminal regions, which interacts indirectly with the PABPC, most likely
through additional proteins (indicated as X)
64,66
. The AGOGW182 complex directs
the mRNA to deadenylation (c). Of note, it remains unclear whether translation is
inhibited before deadenylation (this controversial step is represented in b as
establishment of silencing?). d | Depending on the cell type and/or specific target,
deadenylated mRNAs can be stored in a translationally repressed state. e,f | In animal
cell cultures, deadenylated mRNAs are decapped and rapidly degraded by the major
5-to-3 exonuclease XRN1.

Ribozyme
An RNA molecule with
a catalytic activity.
consequence of an initial block in translation remains
an open question. However, if this is the case, the block
is most likely to occur at initiation, rather than at a
subsequent stage of translation. Indeed, although the
number of ribosomes present on target mRNAs that
were not degraded decreased, this decrease was constant
throughout the length of the message, which is incom-
patible with the proposal that miRNAs cause ribosomes
to drop off
10,11
. These results also conflict with the idea
that miRNAs inhibit translation elongation, because
in this case ribosome density should have increased.
Moreover, because changes in protein abundance closely
matched changes in mRNA levels, these studies exclude
the possibility that the nascent polypeptide is degraded
co-translationally, as in this case mRNA levels should
have remained unchanged.
Silencing mechanisms: new views, new questions
As mentioned above, an important question that remains
unresolved is whether target degradation occurs as a
consequence of an initial block in translation. Much evi-
dence suggests that deadenylation and subsequent deg-
radation are not coupled to active translation. Indeed,
miRNA-dependent target degradation is seen even when
translation of miRNA targets is precluded. For example,
miRNA target reporters that are poorly translated
because of strong stemloop structures in the 5 uTR are
nevertheless deadenylated and degraded in an miRNA-
dependent manner
36,38
; this indicates that degradation
occurs even when the target is not translated. likewise,
in zebrafish embryos and human cell extracts, miRNA
targets are deadenylated despite having a defective
cap structure (Appp-cap) that impairs translation
24,39
.
Accordingly, miRNA-mediated deadenylation can be
observed in the absence of translation (for example, in
the presence of cycloheximide or hippuristanol)
24,37,45
.
Finally, in cell-free extracts, miRNAs triggered dead-
enylation of a short, Appp-capped RNA containing
miRNA-binding sites and a poly(A) tail but lacking an
ORF
45
. Collectively, these results indicate that miRNAs
trigger deadenylation and decay independently of the
translation status of the mRNA target.
Nevertheless, it is not yet clear whether silencing of
polyadenylated miRNA targets can be entirely attributed
to deadenylation or whether additional mechanisms
repress protein production. For example, in some cell-
free extracts, translational repression preceded dead-
enylation
25,45
, although other studies reported evidence
to the contrary
22,24,46
. Another finding suggesting that an
additional mechanism of translational repression might
operate is that miRNA reporters in which the poly(A)
tail is replaced by a histone mRNA stemloop struc-
ture or by a self-cleavable ribozyme are still repressed by
miRNAs (albeit less efficiently than their polyadenylated
counterparts)
36,38,47
. This indicates that although dead-
enylation contributes to and consolidates silencing of
polyadenylated mRNAs, it is not absolutely required for
the establishment of silencing.
Role of GW182 proteins in silencing
Gw182 proteins are essential for miRNA-mediated
gene silencing in animal cells
31,34,4755
. In cells depleted
of Gw182 proteins, the expression levels of miRNAs or
AGO proteins are unaffected, indicating that AGOs alone
cannot trigger silencing of partially complementary
targets
47
. This and additional observations, summarized
below, indicate that Gw182 proteins act at the effec-
tor step of silencing, downstream of AGOs. Therefore
the study of the Gw182 proteins is of crucial impor-
tance to understanding the mechanisms of silencing
in animals.
Essential silencing roles in animals. The Gw182 pro-
teins which interact with AGOs and are required
for miRNA-mediated silencing were identified by
genetic screens in C. elegans, RNAi screens in Drosophila
melanogaster and biochemical purification of AGO
complexes
31,34,4751
(FIG. 3). vertebrates contain three
Gw182 paralogues (TNRC6A (also known as Gw182),
TNRC6B and TNRC6C), D. melanogaster has one
(Gw182), but fungi and plants lack orthologues
34
. The
C. elegans genome encodes two divergent members of the
Gw182 protein family, AIN-1 and AIN-2. Both interact
with AGOs and are required for miRNA function
26,5153
,
although they lack some of the domains present in the
vertebrate and insect proteins
52,54,55
.
The amino-terminal domain of Gw182 proteins con-
tains multiple glycinetryptophan repeats (Gw-repeats),
and is required for silencing because it confers binding
of Gw182 proteins to the AGOs
34,47,5660
. The mid and
carboxy-terminal regions define a bipartite silencing
domain (SD), which is also required for silencing
54,55,5961
.
Remarkably, if the silencing domains of D. melanogaster
Gw182 or human TNRC6AC are artificially tethered
to a reporter mRNA, the reporter is silenced
59,61,62
. As
in tethering assays for the full-length Gw182 proteins,
the silencing domains cause bound mRNAs to be both
translationally repressed and degraded
34,47,5963
. Because
the silencing domains do not interact with AGOs,
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2011 Macmillan Publishers Limited. All rights reserved
0CVWTG4GXKGYU)GPGVKEU
C
8S0 221
Humun AGO2
430
PlWl mid N
3S0
PAZ
8inds 3-OH
ol miPNAs
8inds S-P
ol miPNAs
Cuulyic
domuin
GW182-bindinq
1
D
Humun TNPC6A isolorm 2
mid
Siloncinq domuin (SD)
PPM C O-ricb U8A
031 888 1011 1208 1000 1 1100 1388 1S2S
AGO-bindinq domuin
N-orm (GW-roous)
PAM2
Humun PA8PC1
Linlor MLLL PPM3
Diroc bindinq o
oll4G und PAM1 moils
ol PAlP1 und PAlP2
lndiroc bindinq o
GW182 SDs
Diroc bindinq o
PAM2 moils in PAlP1,
PAlP2 und GW182 SDs
E
636 1 00 200 360 S44
Diroc bindinq
o PA8PC1 MLLL
lndiroc bindinq o
PA8PC1 PPMs
M2
1600
101
PPM1 PPM2 PPM4
C-orm
13S1
M1
S18
Figure 3 | Proteins involved in the effector step of microRnA-mediated gene silencing. Although in plants only
Argonaute proteins (AGOs) are known to mediate silencing, in animals AGOs associate with proteins of the GW182
family of trinucleotide-repeat-containing proteins, which, in turn, interact with cytoplasmic poly(A)-binding protein
(PABPC). a | AGOs consist of four domains: the amino-terminal domain; the PAZ domain, which binds the 3-end of
microRNAs (miRNAs); the mid domain, which provides a binding pocket for the 5-phosphate of miRNAs; and the PIWI
domain, which adopts an RNase H-like fold and has endonucleolytic activity in some AGOs
86
. A protein fragment
containing the mid and PIWI domains is sufficient for binding to the GW182 proteins
34,56
. Human AGO2 is shown as a
representative example of this family. b | Human TNRC6A (isoform 2) is shown as a representative family member of the
GW182 proteins. GW182 proteins contain two globular domains: an ubiquitin-associated-like domain (UBA) and an
RNA-recognition motif (RRM). These domains are embedded in N-terminal (N-term), middle (mid) and carboxy-terminal
(C-term) unstructured regions containing a variable number of glycinetryptophan repeats (GW-repeats, also known as
WG-repeats)
54
. Most repeats are located in the N-terminal fragment, which confers binding to AGOs
5660
. The mid and
C-terminal regions define a bipartite silencing domain, which includes a PABPC-interacting motif 2 (PAM2 motif)
45,5961,6668
.
The mid domain is further divided into the M1 and M2 regions (upstream and downstream of the PAM2 motif,
respectively). The RRM is not required for PABPC binding and is not essential for silencing
54,60,61,64,66
. GW182 proteins also
contain a region rich in glutamine (Q-rich). c | PABPC consists of four N-terminal RRMs (14), a proline-rich unstructured
linker, and a conserved C-terminal domain, called MLLE. The term MLLE refers to a conserved signature motif,
KITGMLLE
67
. PABPC interacts with various proteins involved in translational regulation and mRNA decay. Through the
N-terminal RRM domains, PABPC interacts with eukaryotic translation-initiation factor 4G (eIF4G), which binds to
the mRNA 5-cap structure; this interaction triggers mRNA circularization
12
. The RRMs also interact directly with the
PAM1 motifs of PABP-interacting protein 1 (PAIP1) and PAIP2 and indirectly with the silencing domains (SDs) of GW182
proteins
12,66
. The PABPC MLLE domain interacts directly with proteins containing PAM2 motifs, including PAIP1, PAIP2
and the silencing domains of GW182 proteins
45,6668
. Human PABPC1 is shown as an example.
the conclusion is that these domains have autono-
mous silencing activity
54,55,5961
. In this context, it is
important to mention that the N-terminal domain of
D. melanogaster Gw182 exhibits silencing activity when
tethered to an mRNA reporter and can complement
silencing of a specific miRNAtarget pair, suggesting
that this domain can induce the formation of silencing
complexes in specific 3 uTR contexts
62
. However, the
Gw182 N-terminal domain is not sufficient to silence
the majority of miRNA targets tested, indicating that
the silencing activity of D. melanogaster Gw182 resides
primarily in the C-terminal silencing domain (e.H. and
e.I., unpublished observations).
Important clues to how the bipartite SDs elicit silenc-
ing have come from recent studies revealing that these
domains interact with PABPC
45,64
. Three lines of evidence
suggest this interaction has a role in silencing. First, in
both D. melanogaster and human cells, overexpression
of PABPC suppresses silencing
64,65
. Second, depletion of
PABPC from cell-free extracts abolishes miRNA-mediated
deadenylation
45
. Third, D. melanogaster Gw182 and
human TNRC6A protein mutants that no longer interact
with PABPC are strongly impaired in silencing
66
.
PABPC consists of four N-terminal RNA-recognition
motifs (RRM14), a proline-rich unstructured linker
and a C-terminal domain termed the PABC or the
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2011 Macmillan Publishers Limited. All rights reserved
Mlle
67
(FIG. 3). The Mlle recognizes PAM2 (PABPC-
interacting motif 2) motifs, which are present in several
proteins involved in translation or mRNA decay
12,67
. The
PAM2 motif was first identified in the translational regu-
lators PABP-interacting protein 1 (PAIP1) and PAIP2
(REFS 12,67). These proteins contain, in addition, a PAM1
motif that interacts with the PABPC RRMs
12
.
Remarkably, like PAIP1 and PAIP2, Gw182 proteins
contain two binding sites for PABPC
66
. These binding
sites are located in the SDs. One binding site is a PAM2
motif that interacts directly with the C-terminal Mlle
domain of PABPC
45,6668
(FIG. 3); the second, less defined
site is contributed by the M2 and C-terminal regions
of the SDs
64,66
. This second binding site mediates indi-
rect binding to PABPC RRMs
66
. It is worth noting that,
although both PAIP1 and PAIP2 contain PAM1 and
PAM2 motifs and interact with PABPC in a similar man-
ner, they affect translation in opposite ways: PAIP1 stim-
ulates translation, whereas PAIP2 inhibits translation
12
.
It now seems clear that Gw182 proteins are most likely
to function similarly to PAIP2 and interfere with PABPC
function during translation and/or mRNA stabilization.
How does GW182 interfere with PABPC function? By
analogy with PAIP2, Gw182 proteins may compete with
eIF4G for binding to PABPC
45,64
(FIG. 2), thereby preventing
mRNA circularization, and consequently inhibiting trans-
lation. Moreover, an mRNA in the open conformation
may be more exposed to mRNA decay enzymes.
Alternatively, the PABPCGw182 interaction could
contribute to silencing by reducing the affinity of PABPC
for the poly(A) tail, as described for PAIP2 (REF. 12).
This could expose the poly(A) tail to deadenylases and
thus indirectly interfere with mRNA circularization.
However, it is not yet known whether Gw182 can reduce
PABPC affinity for the poly(A) tail.
Finally, it should be noted that some PABPC-binding
proteins do not affect PABPC function, but rather use
PABPC as a binding platform for hooking onto mRNAs.
Analogously, a Gw182PABPC complex might provide a
platform for additional interactions required in silencing;
these could include interactions with the CAF1
CCR4NOT1 deadenylase complex. Indeed, Fabian
et al.
45
showed that PABPC is required for deadenyla-
tion of miRNA targets in vitro. Therefore, the Gw182
PABPC interaction may contribute to silencing through
multiple mechanisms.
Emerging model of silencing in animals. To bring
together the accumulated data in the field, we propose
a stepwise model for silencing that begins with the rec-
ognition of the target by an miRNA in complex with an
AGO protein (FIG. 2). The AGO interacts with Gw182,
which, in turn, interacts with PABPC bound to the
mRNA poly(A) tail. The assembly of this complex on
the mRNA ultimately triggers deadenylation (FIG. 2),
although the precise mechanism remains to be deter-
mined. Depending on the cell type and/or specific target,
deadenylated mRNAs can be stored in a translationally
repressed state, as observed in cell-free extracts or in
embryonic extracts
22,24,25,45,69
. However, as mentioned
before, in animal cell cultures deadenylated mRNAs are
generally decapped and rapidly degraded by the major
5-to-3 exonuclease XRN1 (REFS 34,3638,43) (FIG. 2).
One important question that remains open is
whether translation is inhibited before deadenylation
or whether there is an initial triggering event that renders
the target more accessible to the decay enzymes and
simultaneously interferes with translation. For example,
although the idea is completely speculative, miRNAs
could promote the dissociation of PABPC from the
poly(A) tail or eIF4F components from the cap struc-
ture. In such cases, both translational repression and
mRNA decay would be a consequence of this primary
effect, and whether or not deadenylation occurs first
would depend on deadenylation rates. This hypothesis,
if confirmed, resolves the apparent dichotomy between
translational repression and target degradation because
both modes of regulation would be a consequence of a
common initial triggering event.
miRNA-based regulation in plants
As in animals, miRNAs in plants can induce translational
repression and mRNA degradation. In this section we
summarize similarities and differences on the mechanisms
of silencing between these two kingdoms.
miRNA-directed target mRNA degradation. As men-
tioned earlier, it is well established that plant miRNAs
recognize fully or nearly complementary mRNA targets
and direct endonucleolytic mRNA cleavage
2,7072
(FIG. 1).
Cleavage occurs between nucleotides 10 and 11, opposite
the miRNA strand, and is catalysed by the AGOs. The
resulting 5 and 3 mRNA fragments are degraded from
the newly generated 3 and 5 ends, respectively
73
(FIG. 4).
Degradation of the 5 fragment is thought to be catalysed
by the exosome, a multiprotein complex with 3-to-5
exonuclease activity, whereas the 3 fragment is degraded
by the 5-to-3 exonuclease XRN4 (a plant paralogue of
metazoan XRN1 (REF. 73)). Consistent with this model,
3 fragments of miRNA targets accumulate in plants lack-
ing XRN4 (REFS 73,74). As in animals, it is not yet clear
whether the decay enzymes are recruited to the mRNA
decay intermediates through specific interactions with
miRISC. Also, how miRISC complexes dissociate from
the target after endonucleolytic cleavage, and give access
to the decay enzymes, has not been determined.
In contrast to full-length mRNAs, which contain a
5-cap structure, the 3 fragments resulting from AGO-
mediated endonucleolytic cleavage contain a 5-mono-
phosphate
70
. This feature was used to specifically sequence
these 3 fragments in wild-type plants or in plants lack-
ing XRN4 (REFS 74,75). This approach validated predicted
miRNA targets and identified new endogenous targets on
a genome-wide scale. Furthermore, the site of cleavage
was mapped precisely and shown to lie at the centre
of the miRNAtarget paired region, as expected
70,74,75

(FIG. 1). Together with earlier transcriptome analysis that
showed mRNA target degradation in response to miRNA
expression
76
, these studies indicated that endonucleolytic
cleavage of highly complementary targets is a prominent
mechanism of miRNA-based regulation in plants.
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2011 Macmillan Publishers Limited. All rights reserved
0CVWTG4GXKGYU)GPGVKEU
AGO
AGO
AGO
mPNA
oll4G AAAAAAAAAA
oll4L
oll4G
mPNA
AAAAAAAAAA
oll4L
XPN4
mPNA
AAAAAAAAAA
oll4L
oll4G
3-o-S docuy S-o-3 docuy
P- -OH
'ZQUQOG
G+()
O40#
##########
G+(' 2#$2% 2#$2%
2#$2% 2#$2%
2#$2% 2#$2%
%CR
%CR
%CR %CR
C6CTIGVTGEQIPKVKQP
D6CTIGVENGCXCIG E6TCPUNCVKQPCNTGRTGUUKQP
2#$2% 2#$2%
OK40#IWKFG
UVTCPF
Figure 4 | Mechanisms of microRnA-mediated gene silencing in plants. a | Plant microRNAs (miRNAs) that are
bound to Argonaute (AGO) recognize mRNA targets containing fully or nearly complementary binding sites, which
are predominantly located in the ORF. b | AGOs cleave the mRNA in the base-paired region (between nucleotides 10 and
11, opposite the miRNA strand, at the red arrowhead). The endonucleolytic activity of AGOs resides in the PIWI domain
86
.
The mRNA fragments resulting from the endonucleolytic cleavage are degraded from the newly generated 3 and 5 ends
by the exosome and the exonuclease XRN4, respectively
73
. XRN4 is related to the major 5-to-3 exonuclease XRN1 in
animals. c | Alternatively, endonucleolytic cleavage by AGOs is prevented and the mRNA target is translationally repressed
by an unknown mechanism. eIF, eukaryotic translation-initiation factor; PABPC, cytoplasmic poly(A)-binding protein.
In summary, miRNAs have evolved two divergent
ways of promoting target degradation: endonucleo-
lytic cleavage (prominent in plants) or exonucleolytic
degradation (prominent in animals). In both cases, the
general mRNA decay enzymes are involved, but only
the XRN paralogues are shared (FIGS 2,4).
Translational repression in plants. until recently,
miRNA-mediated translational repression in plants had
been reported only for a few targets, such as APETALA2,
a target of miR172, and the SBP-box gene SPL3, which is
silenced by miR-156/157 (REFS 7779). However, recent
studies suggest that translation inhibition may be more
common in plants than previously anticipated
8083
.
Brodersen et al.
80
isolated Arabidopsis thaliana mutants
defective in silencing of a GFP reporter containing an
miR171 binding site in the 3 uTR. In two of these
mutants, reporter GFP protein expression was upreg-
ulated; however, mRNA levels were not restored, sug-
gesting that the mutations suppressed miRNA-mediated
translational repression but not mRNA endonucleolytic
cleavage. That these mutants can uncouple the effects on
translation and mRNA levels was confirmed for a hand-
ful of endogenous targets containing miRNA-binding
sites at different locations, including the ORF. However,
it is important to note that these mutants lack overt
phenotypes and the mechanism by which the iso-
lated genes participate in silencing remains unknown.
Nevertheless, the observations of Brodersen et al. suggest
that plant miRNAs can repress translation in the absence
of target degradation. Consistent with this possibility,
both AGO1 and a subset of miRNAs were shown to asso-
ciate with polysomes in an mRNA-dependent manner in
A. thaliana
81
. Although studies in animals have shown
that, on its own, this observation is not sufficient evi-
dence for translational repression, it indicates that
miRISC complexes associate with actively translated
mRNAs in plants.
To investigate whether translational repression by
plant miRNAs involves a set of factors similar to those
in animals, the authors analysed the effects of mutat-
ing enhancer of decapping 4 (eDC4; also known as
Ge1 and HeDlS). This is surprising, in part because
eDC4 (which is known as vARICOSe in A. thaliana)
is required in animals for miRNA-mediated mRNA
decay but not translational repression
37
. Indeed, eDC4
interacts with the decapping enzyme DCP2 and the
decapping factor DCP1 in both plants and animals,
and is required for mRNA decapping in metazoans
37,80
.
Previous studies identified eDC4 as a suppressor of
miRNA-mediated gene silencing in D. melanogaster
cells
37
: depleting eDC4 suppressed mRNA degrada-
tion mediated by miRNAs, restoring transcript levels.
Because the transcripts accumulated in the deade-
nylated form, protein levels were only partially restored.
By contrast, in plants, vARICOSe mutants showed
increased levels of protein expression but no increase in
mRNA levels
80
.
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2011 Macmillan Publishers Limited. All rights reserved
The reason for the different effects of eDC4 depletion
in animal cells compared with the effects of vARICOSe
mutations in plants remains unclear. Nevertheless, the
identification of eDC4 (or vARICOSe) as a suppressor of
miRNA-mediated gene silencing in such distant species
as D. melanogaster and A. thaliana points to similarities
in the mechanism of silencing. Because eDC4 acts
as a decapping activator, and so downstream of
deadenylation, the results by Brodersen et al.
80
also suggest
the intriguing possibility that, as in animals, miRNA
targets may be subject to deadenylation and decapping
in plants.
Conclusion
miRNAs were initially thought to inhibit translation in
animals and to predominantly promote target endonu-
cleolytic cleavage in plants. However, recent evidence
has changed this view by showing that miRNAs can
trigger translational repression and mRNA destabili-
zation in both kingdoms. However, in both plants and
animals, the current evidence suggests that target mRNA
degradation provides a major contribution to silencing
by miRNAs.
It is important to note, however, that this current view
of miRNA regulation in animals came mainly from stud-
ies in a limited set of cell types, such as cultured mam-
malian cells (for example, Hela and HeK293 cells),
which divide rapidly. It will be interesting to investigate
whether the number of miRNA targets that are repressed
at the translational level alone remains small if natural
targets are analysed in differentiated cell types and in
their physiological context.
Although miRNA targets can be degraded in the
absence of translation, an important question for future
studies will be whether in vivo degradation occurs after
an initial block on translation initiation and, if so, what
the mechanism involved is. we expect that answers
to these questions will emerge as more studies exam-
ine the molecular structure and function of silencing
factors, and how they interact to assemble into active
effector complexes.
The study of the mechanism of translational repres-
sion in plants is likely to provide important insight
into silencing mechanisms because plants seem to lack
Gw182 orthologues, although it is possible that analo-
gous proteins with a similar function exist. In addition,
at present it is not clear how much translational control
is exerted by miRNAs on the plant proteome because
experiments similar to those that have been done in
animal cells
811
have not been performed in plants.
Moreover, although plant miRNAs can repress targets
with partially complementary binding sites
82
(that is, in
the absence of target cleavage), it remains unclear how
widespread this type of regulation is.
Finally, despite the fact that target degradation
is a widespread effect of miRNA regulation, a sub-
set of targets and reporters seems to avoid being fully
degraded
8,1319,26,34
. The most likely explanation is that pro-
teins associated with these targets influence the outcome
of miRNA regulation. This opens up the possibility that
target degradation could be subject to regulation in many
ways, for example in a tissue- or developmental-stage-
specific manner. Indeed, one can imagine that, in some
cell types (for example, oocytes, embryonic or neuronal
cells), in which deadenylated mRNAs are often stable,
miRNA targets may accumulate in a deadenylated,
silenced form. Accordingly, miRNA targets were found
to be stored in such a form in C. elegans embryos
69
.
These silenced mRNAs might eventually be derepressed
and polyadenylated, and return to the pool of actively
translating mRNAs. A related question is how endonu-
cleolytic cleavage is prevented in plants so that targets
are stored in a translationally repressed state, without
undergoing degradation.
In summary, the past few years have seen significant
advances in our understanding of the mechanism of
silencing, both in animals and plants. Although the molec-
ular details remain to be elucidated, new findings have
revealed unanticipated mechanistic similarities. Future
studies in either of these two kingdoms promise to benefit
from one another and to further our understanding
of the mechanisms of silencing by miRNAs.
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Acknowledgements
The research in this laboratory is supported by the Max
Pl anc k Soc i et y, by gr ant s f r om t he Deut s c he
Forschungsgemeinschaft (DFG, FOR855 and the Gottfried
Wilhelm Leibniz Program awarded to E.I.), and by the Sixth
Framework Programme of the European Commission through
the SIROCCO Integrated Project LSHG-CT-2006-037900.
Competing interests statement
The authors declare no competing financial interests.
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