gene silencing by micrornas- contributions of translational repression and mrna decay

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 metabolism1–4. There is strong evidence to suggest that miRNAs are also involved in the pathogenesis of human diseases such as cancer and metabolic disorders3,4. 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 mammals1,2. 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 regulation1,2.

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-ing2–4. 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 sequence1–4.

Over the past few years, remarkable progress has been made in our understanding of miRNA biogenesis and function3,4; however, the mechanisms that miRNAs use to regulate gene expression remain unclear and several con-troversies surround the topic4–7. 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 views4–7. 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 translation4–7.

Recently, several studies have taken advantage of advances in the fields of mass spectrometry and

Max Planck Institute for Developmental Biology, Spemannstrasse 35, D‑72076 Tübingen, Germany.Correspondence to E.I. e‑mail: [email protected]:10.1038/nrg2936

Argonaute family proteinsThe 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 decayEric 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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Figure 1 | RnA-target recognition in plants and animals. MicroRNAs (miRNAs) recognize their targets by Watson–Crick base pairing. a | Plant miRNAs recognize fully or nearly complementary binding sites, which are generally located in ORFs. Importantly, miRNA nucleotides (N) 9–12 are usually engaged in Watson–Crick 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 2′O-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 2–7 — 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 9–12 generally bulge out, preventing endonucleolytic cleavage by AGOs1,2. 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′-capEukaryotic mRNA is modified by the addition of an m7G(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.

PolysomeA 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 miRNAs8–11. 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 regulation3,4, 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 repressionTranslation 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 degradation12. 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 lin‑14 and lin‑28 mRNAs, with little or no influ-ence on their abundance13,14. Although protein expression was inhibited, the lin‑14 and lin‑28 mRNAs were detected in polysomes, suggesting that repression occurred after translation had been initiated13,14, an idea that was sup-ported by subsequent studies in mammalian cell cul-tures15–17. 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 subunits15–17.

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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 eIF2–GTP–MET–tRNAMeti, 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 complex84. 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 translation84.

Internal ribosome entry site(IRES). A structured RNA element, usually present in the 5′ UTR, that allows m7G-cap-independent association of a ribosome with mRNA.

Krebs-2 ascites tumour cellsTumour cells grown in vivo in the peritoneal cavity of the mouse.

5′-to-3′ mRNA decay pathwayA 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) mRNA17. 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 initiation18,19.

Subsequent studies in cell-free extracts of diverse origin have supported a role for miRNAs in inhibiting translation initiation20–24. In extracts, miRNAs silenced m7Gppp-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 IReS23,24. Consistent with these findings, in extracts from mouse Krebs-2 ascites tumour cells, silencing was suppressed with increasing concentrations of purified eIF4F23 (BOX 1). This and more recent studies25,26 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 degradationEvidence for target degradation. evidence that animal miRNAs can induce target mRNA degradation comes from studies on specific miRNA–target pairs and, more generally, from transcriptome studies showing that the abundance of miRNA targets inversely correlates with the level of miRNA8–11,27–41. For example, if specific miRNAs are introduced into cultured cells, then tran-scripts containing complementary binding sites (for example, seed matches) become less abundant8–11,27. Conversely, transcriptome profiling of cells depleted of an miRNA showed a corresponding increase in tran-scripts containing binding sites for this miRNA8,9,28. Furthermore, in cultured cells, depleting essential com-ponents of the miRNA pathway (for example, Dicer, AGOs or Gw182) increased the abundance of miRNA targets31–35,37,38. expression profiles from differentiating and developing cells also provide numerous examples showing anti-correlated expression of miRNAs and their targets40,41. 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′ uTRs35,39. 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 targets42, 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 pathway31,34–38,43, where mRNAs are first dead-enylated (FIG. 2c). mRNAs are primarily deadenylated by the CAF1–CCR4–NOT 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 profilingThis 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, 5–60% 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 miRNA18,19.

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 profilingThis determines the position of ribosomes on cellular mRNAs with high sequence resolution11,85. 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.

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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 overexpressed31,34,37,38,43,44. For example, in cells depleted of components of the CAF1–CCR4–NOT complex, most miRNA targets (both predicted and validated) are upregulated34,38. This supports the idea that deadenylation is a widespread consequence of miRNA regulation38.

A role for decapping is more difficult to demonstrate, first because decapping factors are redundant37, 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 restored37. Nevertheless, transcriptome analysis showed that in cells depleted of decapping factors, mRNA levels of predicted and validated miRNA targets increase37.

miRNA-mediated deadenylation has also been observed in cell-free extracts22,24,25,45. However, in con-trast to cultured cells, in which deadenylated mRNAs are committed to decapping and 5′-to-3′ exonucleolytic deg-radation29,34,36–38,43, 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-lation24. However, there is debate regarding the order of events, because deadenylation has been reported both to precede22,24 and to follow translational repression25,45.

Translational repression versus decayTarget 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 parallel8,9. 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 correlated8. Baek et al. analysed protein and mRNA levels in mouse neu-trophils isolated from a mir‑223-gene-knockout mice compared with wild-type mice9. 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 colleagues11 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

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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 eIF4E12. 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 domain45,66–68, 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 AGO–GW182 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.

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RibozymeAn 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 questionsAs 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 stem–loop structures in the 5′ uTR are nevertheless deadenylated and degraded in an miRNA-dependent manner36,38; 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 translation24,39. 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 ORF45. 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-enylation25,45, although other studies reported evidence to the contrary22,24,46. 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 stem–loop 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 silencingGw182 proteins are essential for miRNA-mediated gene silencing in animal cells31,34,47–55. 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 targets47. 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 complexes31,34,47–51 (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 orthologues34. 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 function26,51–53, although they lack some of the domains present in the vertebrate and insect proteins52,54,55.

The amino-terminal domain of Gw182 proteins con-tains multiple glycine–tryptophan repeats (Gw-repeats), and is required for silencing because it confers binding of Gw182 proteins to the AGOs34,47,56–60. The mid and carboxy-terminal regions define a bipartite silencing domain (SD), which is also required for silencing54,55,59–61. Remarkably, if the silencing domains of D. melanogaster Gw182 or human TNRC6A–C are artificially tethered to a reporter mRNA, the reporter is silenced59,61,62. As in tethering assays for the full-length Gw182 proteins, the silencing domains cause bound mRNAs to be both translationally repressed and degraded34,47,59–63. Because the silencing domains do not interact with AGOs,

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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 AGOs86. A protein fragment containing the mid and PIWI domains is sufficient for binding to the GW182 proteins34,56. 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 glycine–tryptophan repeats (GW-repeats, also known as WG-repeats)54. Most repeats are located in the N-terminal fragment, which confers binding to AGOs56–60. The mid and C-terminal regions define a bipartite silencing domain, which includes a PABPC-interacting motif 2 (PAM2 motif)45,59–61,66–68. 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 silencing54,60,61,64,66. GW182 proteins also contain a region rich in glutamine (Q-rich). c | PABPC consists of four N-terminal RRMs (1–4), a proline-rich unstructured linker, and a conserved C-terminal domain, called MLLE. The term MLLE refers to a conserved signature motif, KITGMLLE67. 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 circularization12. 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 proteins12,66. The PABPC MLLE domain interacts directly with proteins containing PAM2 motifs, including PAIP1, PAIP2 and the silencing domains of GW182 proteins45,66–68. Human PABPC1 is shown as an example.

the conclusion is that these domains have autono-mous silencing activity54,55,59–61. 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 miRNA–target pair, suggesting that this domain can induce the formation of silencing complexes in specific 3′ uTR contexts62. 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 PABPC45,64. Three lines of evidence suggest this interaction has a role in silencing. First, in both D. melanogaster and human cells, overexpression of PABPC suppresses silencing64,65. Second, depletion of PABPC from cell-free extracts abolishes miRNA-mediated deadenylation45. Third, D. melanogaster Gw182 and human TNRC6A protein mutants that no longer interact with PABPC are strongly impaired in silencing66.

PABPC consists of four N-terminal RNA-recognition motifs (RRM1–4), a proline-rich unstructured linker and a C-terminal domain termed the PABC or the

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Mlle67 (FIG. 3). The Mlle recognizes PAM2 (PABPC-interacting motif 2) motifs, which are present in several proteins involved in translation or mRNA decay12,67. 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 RRMs12.

Remarkably, like PAIP1 and PAIP2, Gw182 proteins contain two binding sites for PABPC66. 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 PABPC45,66–68 (FIG. 3); the second, less defined site is contributed by the M2 and C-terminal regions of the SDs64,66. This second binding site mediates indi-rect binding to PABPC RRMs66. 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 translation12. 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 PABPC45,64 (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 PABPC–Gw182 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 Gw182–PABPC complex might provide a platform for additional interactions required in silencing; these could include interactions with the CAF1–CCR4–NOT1 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 extracts22,24,25,45,69. 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 34,36–38,43) (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 plantsAs 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 cleavage2,70–72 (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, respectively73 (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-phosphate70. 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 miRNA–target paired region, as expected70,74,75 (FIG. 1). Together with earlier transcriptome analysis that showed mRNA target degradation in response to miRNA expression76, these studies indicated that endonucleolytic cleavage of highly complementary targets is a prominent mechanism of miRNA-based regulation in plants.

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′ ′ ′ ′

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 domain86. 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, respectively73. 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 77–79). However, recent studies suggest that translation inhibition may be more common in plants than previously anticipated80–83. 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. thaliana81. 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 repression37. 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 metazoans37,80. Previous studies identified eDC4 as a suppressor of miRNA-mediated gene silencing in D. melanogaster cells37: 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 levels80.

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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.

ConclusionmiRNAs 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 cells8–11 have not been performed in plants. Moreover, although plant miRNAs can repress targets with partially complementary binding sites82 (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 degraded8,13–19,26,34. 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 embryos69. 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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AcknowledgementsThe research in this laboratory is supported by the Max P lanck Soc i e t y, by g ran t s f rom the Deu t s che 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 statementThe authors declare no competing financial interests.

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