Abstract
In animals, the majority of microRNAs regulate gene expression through the RNAi machinery without inducing small RNA-directed mRNA cleavage 1. Thus, the mechanisms by which microRNAs repress their targets have remained elusive. Recently, Argonaute proteins, which are key RNAi effector components, and their target mRNAs were shown to localize to cytoplasmic foci known as P-bodies or GW-bodies 2,3. Here, we show that the Argonaute proteins physically interact with a key P-/GW-body subunit, GW182. Silencing of GW182 delocalizes resident P-/GW-body proteins and impairs the silencing of microRNA reporters. Moreover, mutations preventing Argonaute proteins from localizing in P-/GW-bodies prevent translational repression of mRNAs even when Argonaute is tethered to its target in a small RNA-independent fashion. Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA.
MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) enter the RNA-induced silencing complex, RISC, and suppress the expression of target genes, which they recognize by complementary base pairing 1,4. The precise mechanism of suppression depends upon two factors. First is the degree of complimentarity between the small RNA and its target. In cases of perfect or near-perfect complimentarity, the mRNA can be cleaved by an Argonaute protein. When complimentarity is imperfect, as normally occurs for animal microRNAs, suppression occurs without RISC-mediated cleavage 5. A second factor is the nature of the Argonaute protein that forms the core of RISC. Not all Argonaute proteins are catalytically active. In mammals, Argonaute-2 is competent for substrate cleavage while Argonautes 1,3 and 4 are inert 6,7. Thus, at least in mammals, RISC can recognize a substrate and form a complex that is incapable of cleavage even with a perfect small RNA-target interaction. The outcome of such events is presently unknown, but such interactions could potentially lead to cleavage-independent repression.
The mechanisms by which RISC can repress targets in the absence of substrate cleavage are yet to be resolved. Early studies indicated that repression by animal microRNAs occurred without changes in the overall level of the mRNA target 4,8,9. However, recent studies in mammalian cells and C. elegans have indicated that changes in mRNA abundance are observed for the proposed targets of several microRNAs 10,11. Additionally, several studies detect both bulk microRNAs and some mRNA targets on polysomes, suggesting that suppression might occur during the act of protein synthesis either by changes in initiation or elongation rates or by destabilizing nascent proteins 12–15.
Recent observations have also led to an alternative model for silencing by miRNAs wherein mRNA interactions with RISC might sequester targeted mRNAs in P-/GW-bodies 2,3,15–17. These are cytoplasmic foci that contain non-translated mRNAs and exclude the translation machinery 18. Not only are Argonaute proteins found in mammalian P-/GW-bodies 2,3, mRNA targets of microRNAs become similarly localized in a manner that depends both on the presence of the miRNA and upon miRNA binding sites in the target 2,15. Such localization could potentially embody part, or all, of the underlying cause of repression or could occur as a downstream consequence of translational repression by RISC. Thus, it is critical to examine the functional significance of the connections between P-/GW-bodies and the RNAi pathway.
To investigate mechanisms of miRNA-mediated repression, we have searched for Argonaute-interacting proteins by MudPIT (Multidimensional Protein Identification Technology) analysis of immunoaffinity purified Ago1 and Ago2 complexes. We recovered a number of previously identified Argonaute binding proteins including HSP90, Dicer, TRBP and DCP1 2,6,19,20. Additionally, we repeatedly identified a known component of P-/GW-bodies, GW182, in both Ago1 and Ago2 complexes. To verify these observations, we examined GW182 immunoprecipitates by immunoblotting for epitope tagged Argonaute (Fig. 1a). An interaction between these proteins was easily detectable and was not disrupted by treatment of extracts or immunocomplexes with RNaseA (not shown).
GW182 is present in discrete cytoplasmic foci wherein it co-localizes with the de-capping complex 21,22. To determine if these foci also contain Argonaute proteins, we used two different GW182-specific autoantisera (IC-6 and 18033) to highlight GW182-containing bodies and an anti-myc antiserum to recognize ectopically expressed Ago2. The two staining patterns show substantial overlap (Fig. 1b), suggesting that at least a portion of the total populations of each of these proteins co-localize.
Considered together, our data identify GW182 as a novel Argonaute-interacting protein. We therefore examined the effects of depleting GW182 on the integrity of P-/GW-bodies and on the ability of small RNAs to silence their targets. By co-transfection with a GFP-GW182 fusion protein, we identified an siRNA that could effectively suppress GW182 expression (Fig. 2a). Transfection of HeLa cells with this siRNA caused a substantial loss of GW182, Dcp1a, and Dcp2 in P-/GW bodies 23 (Fig. 2b, 2c, 2d). In contrast, siRNAs against Dcp2p effectively reduced Dcp2p protein levels, but did not impact the number of GW182 foci observed (data not shown).
Given that GW182 suppression affected the overall integrity of mammalian P-/GW-bodies, we sought to determine whether disruption of these foci impacted small RNA-directed gene silencing. We first examined a cleavage-independent repression event in which a CXCR4 siRNA can bind to 6 imperfect sites in a Renilla luciferase mRNA 24. Co-transfection of the reporter with the CXCR4 siRNA resulted in an approximately 20-fold repression of luciferase activity under the conditions used for this assay (Fig. 3a). Repression of GW182 but not another P-body protein, XRN1, impaired the ability of the miRNA mimetic to silence its target (Fig. 3a). Suppression of DCP2 also showed a less pronounced, albeit reproducible, effect (Fig 3a). Curiously, suppression of GW182 also had an effect on the ability of the siRNA to suppress a perfectly complementary target via mRNA cleavage (Fig. 3b). Qualitatively similar results were seen with a second reporter that is targeted for repression by an endogenous microRNA, let-7 (Fig. S1). All of these outcomes correlated with inhibition of GW182 expression by the siRNA and with a reduction in the appearance of P-/GW-bodies (Fig. 2 and not shown). Notably, the pattern of Ago1 and Ago2 localization was also disrupted upon repression of GW182 (not shown). These data demonstrate that GW182 has a functional role in RISC-mediated silencing, which is correlated with maintenance of P-bodies.
Cleavage-independent suppression of an mRNA target has previously been accomplished by tethering an Argonaute protein to an mRNA 3’ UTR in a manner independent of the small-RNA-target interaction 25. This was achieved by fusing Ago1 or Ago2 to a phage RNA binding motif (λN) and placing its recognition sequence (boxB) within the reporter. It is difficult to be certain that tethered Argonaute proteins work through precisely the same mechanism as microRNA-directed RISC. However, several lines of evidence are consistent with tethered Ago proteins being able to function in the RNAi pathway similarly to their small RNA-directed counterparts. First, λN-fused Argonaute proteins can complement the silencing defect observed in Ago2 knockout MEF (Fig S2). Second, Argonaute proteins that can suppress their targets through direct protein-mRNA interactions localize to P-/GW-bodies in a manner similar to the native proteins (Fig. 4a). Our previous work indicated that a series of point mutations in the PAZ domain could prevent small RNA binding with an accompanying loss of localization to P-/GW-bodies. The same outcome was observed when this series of PAZ mutations was introduced into λN-fused Ago2 protein (Fig. 4a). Since these recognize their targets in a small-RNA independent fashion, we were afforded the opportunity to examine whether Argonaute binding, per se, or localization to P-/GW-bodies correlated with repression.
Expression of Ago2 fused at the amino terminus to λN protein reduced Renilla expression by ~2-fold, provided that the mRNA contained the λN binding site (Fig 4). Also, consistent with previous studies, a λN fusion with HIWI, an enigmatic member of a second Argonaute subfamily had no repressive effect on the reporter. λN-Ago2 (Fig. 4b) proteins containing point mutations in the PAZ domain that prevent small RNA binding neither localized to P-/GW-bodies nor repressed a boxB-containing target mRNA, despite maintaining interaction with the target (Fig. 4, not shown). Notably, Ago2-PAZ9 and -PAZ10 proteins were still present in GW182 immunoprecipitates, indicating that their potential to interact with GW182 was retained, despite the inability of these mutant proteins to localize to P-/GW-bodies or to repress their targets (Fig. 4c).
Previous studies have suggested connections between suppression by RNAi and cytoplasmic foci known as P-bodies or GW-bodies. All four of the mammalian Argonaute proteins that are known to bind to small RNAs are localized to these structures 2,3. Target mRNAs also entered P-/GW-bodies in a manner that was dependent upon their recognition by small RNAs 2. Additionally, Argonaute proteins were shown to bind to components of the de-capping complex that reside, at least in part, in P-/GW-bodies 2. Finally, exogenously added miRNAs can be seen to accumulate within P-/GW-bodies 15.
Results presented here strengthen the correlation and begin to build a functional link between P-/GW-bodies and small RNA-dependent silencing. An analysis of Argonaute complexes revealed a physical interaction with GW182, a core component of P-/GW-bodies. Importantly, silencing of GW182 both disrupts these foci and attenuates suppression of microRNA reporters. Although de-repression was not complete, neither was silencing of GW182 or loss of foci (see Fig. 2b). Thus, it remains possible that a complete ablation of GW182 and P-/GW-bodies might abrogate miRNA function. A similar correlation between P-/GW-body localization and suppression emerged from an analysis of Argonaute proteins that recognize their target by a direct RNA-protein interaction and without the need for a small RNA. Introduction of mutations into the PAZ domains of these λN-fusion proteins both alters their subcellular localization and prevents them from repressing their targets. These results are consistent with observations, reported while this manuscript was under consideration, from C. elegans wherein mutations in a homolog of GW182 gave phenotypes very similar to those resulting from defects in core miRNA pathway components and in Drosophila, where silencing of a GW182 homolog had effects on both miRNA and siRNA function 26,27.
Recent studies suggest P-bodies represent a pool of translationally repressed mRNPs, which is in equilbrium with the translating pool 18,28. Thus, the translation status of an mRNA could reflect the competition between interactions favoring assembly of a translation complex and interactions favoring assembly of a translationally repressed mRNP that can aggregate into P-bodies. Given this, an integrated hypothesis it that miRNAs and associated proteins, minimally Argonaute and GW182, alter this equilibrium, either by directly promoting assembly of the repressed mRNP, and/or by directly inhibiting the function of specific translation initiation factors. This model envisions situations in which provision of strong translation promoting signals could override the function of microRNAs, perhaps in a regulated fashion, to retain mRNAs in an actively translating pool.
Interestingly, other mRNA specific translation repression mechanisms that have been correlated with P-bodies require a combined series of events to achieve repression 28. For example, in Drosophila the Oskar mRNA assembles a tripartite complex wherein eIF-4E is bound to the cap, but prevented from interaction with eIF-4G by the eIF-4E binding protein Cup. Cup is delivered to the mRNA by Bruno binding the 3’ UTR 29. Despite the presence of this complex, efficient repression of the Oskar mRNA during early development requires the Drosophila Me31b protein, whose homolog in yeast and mammals contributes to targeting bulk mRNAs to translational repression and P-bodies 28,30.
Any of the aforementioned models is hard pressed to explain the effects that are seen on silencing by siRNAs that direct mRNA cleavage. One possibility is that product release and turnover of RISC occurs only once the complexes have translocated to P-bodies. Irrespective of what model is considered, emerging links between the RNAi machinery and specific cellular locales suggest that the process can no longer be viewed solely from a biochemical perspective without consideration of the impact that subcellular compartmentalization may have on the assembly and activity of RISC.
Methods
DNA constructs
HA- and Myc-tagged Ago expression plasmids were as described in 2,6. Ago2 PAZ mutants were subcloned into the LamdaN-HA-fusion vector as described in 25. GFP-tagged GW182 plasmid was as described in 23. The miRNA, siRNA and tethering mRNA reporters were as described in 24 and 25.
Cell culture and transfection
Human U2-OS, HeLa and 293 cells were cultured in DMEM (10% FBS) at 37°C with 5% CO2. Cell transfections were carried out using Mirus TransIT reagent for DNA plasmids and Invitrogen Oligofectamine reagent for siRNAs. Control siRNA, CXCR4 siRNA and siRNAs targeting Dcp2, Xrn1 and GW182 were purchased from Dharmacon. Procedures for immunoprecipitation, immunoblotting and immunoflurescence were described previously 2,6. Dual luciferase assays were performed as directed by the manufacturer (Promega).
Acknowledgments
We thank Stephen Hearn from the CSHL microscopy shared resource for assistance, and Niels Gehring (EMBL, Heidelberg), Witold Filipowicz (Friedrich Miescher Institute), Megerditch Kiledjian (Rutgers University), Jens Lykke-Andersen (U of Colorado), Marvin Fritzler (U of Calgary) and Edward Chan (U of Florida) for reagents. F.V.R. is a fellow of the Jane Coffin Childs Foundation, and J. L. is supported as a Special Fellow of the Leukemia and Lymphoma Society. This work was supported by grants from the NIH (G.J.H and R.P). G.J.H. and R. P. are Investigators of the Howard Hughes Medical Institute.
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