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MicroRNAs (miRNAs)² are 20-22-nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages, including plants, insects, vertebrate, and mammals (1–3). More than 474 miRNAs have been identified in humans so far, and ∼30% of the genes in the human genome are predicted to be subject to miRNA regulation (4). The expression of many miRNAs is usually specific to a tissue or developmental stage, and the miRNA expression pattern is altered during the development of many diseases (3). Mature miRNAs are generated from RNA polymerase II-transcribed primary miRNAs that are processed sequentially by the nucleases Drosha and Dicer. Although miRNA can guide mRNA cleavage, the basic function of miRNA is to mediate inhibition of protein translation (1, 5–8) through miRNA-induced silencing complexes (miRISCs). The guiding strand of miRNA in a miRISC interacts with a complementary sequence in the 3′-untranslated region (3′-UTR) of its target mRNA by partial sequence complementarities, resulting in translational inhibition (1). A 7-nucleotide “seed” sequence (at positions 2-8 from the 5′-end) in miRNAs seems to be essential for this action (4). The composition of the miRISC is similar to that of the RNA-induced silencing complex (RISC), which is responsible for mRNA cleavage guided by small interfering RNAs (siRNAs) (1, 3, 7). Nevertheless, some differences exist between miRISCs and siRNA RISCs. For example, the major Argonaute protein in siRNA RISC is Ago-2, whereas all four of the Ago proteins (Ago1-4) are found in miRISC (3, 8). Further, the siRNA RISC may be associated with various RNA-binding proteins such as fragile-X mental retardation protein (FMRP), TAR RNA-binding protein (TRBP), and the human homolog of the Drosophilahelicase Armitage, Mov10, possibly in a cell type-specific manner (9–13).

The miRNA-mediated translational repression consistently correlates with an accumulation of miRNA-bound mRNAs at cytoplasmic foci known as processing bodies (P-bodies) (8). Several lines of evidence have indicated that P-bodies are actively involved in miRNA-mediated mRNA repression (14). The P-body-associated protein GW182 associates directly with Ago-1 (15, 16). Depletion of P-body components such as GW182 and Rck/p54 prevents translational repression of target mRNAs (8, 14–19). Furthermore, several miRISC-related components, such as miRNAs, mRNAs repressed by miRNAs, Ago-1, Ago-2, and Mov10, are found in P-bodies (14). P-body formation is a dynamic process that requires continuous accumulation of repressed mRNAs (20). However, P-bodies serve not only as sites for RNA degradation, but also for storage of repressed mRNAs (15). These mRNAs may later return to polysomes to synthesize new proteins (14). In fact, some cellular proteins can facilitate the exit of miRNA-bound mRNAs from P-bodies. For example, a stress situation may induce the relocation of HuR, an AU-rich element-binding protein, from the nucleus to P-bodies in the cytoplasm where it binds to the 3′-UTR of its target mRNA encoding CAT-1 (21). This binding increases the stability of the miR-122-bound mRNA by assisting it to egress from the P-body and return to polysomes. However, the mechanism underlying this reverse transport of miRNA-bound mRNA out of P-bodies remains to be further clarified.

The cellular apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G protein (APOBEC3G or A3G) is a potent antiretroviral factor that belongs to the cytidine deaminase family (22, 23). A3G can be incorporated into HIV-1 particles and cause extensive C to U conversion in the viral minus-stranded DNA during reverse transcription (24–26), which can trigger its degradation by virion-associated uracil DNA glycosylase-2 (UNG2) and apurinic/apyrimidinic endonucleases (APE) or lethal hypermutation in the HIV-1 genome (26, 27). However, accumulating evidence indicates that A3G protein carrying mutations in the catalytic domain of the cytidine deaminase retains substantial anti-HIV-1 activity (24, 28–31). Interestingly, A3G is found in P-bodies and stress granules (32, 33). It is associated with a high molecular mass structure (>700 kDa) in replicating cells, and this interaction is RNase-sensitive (34, 35). Further studies indicate that A3G interacts with many RNA-binding proteins, among which are several miRNA-related proteins, such as Ago1, Ago2, Mov10, and poly(A)-binding protein 1 (PABP1). These interactions are either partially or completely resistant to RNase A digestion (32, 35, 36).³ Aside from its inhibitory function in relation to endogenous retroviruses and other retrotransposons (37–41), the major cellular function of A3G seems to be related to P-body-related RNA processing and metabolism. As recent development has indicated that the function of P-body is closely related to miRNA activity, we therefore investigated the possibility of a connection between A3G and miRNA function.

A3G Counteracts miRNA-mediated Repression of Protein Translation—We first examined the effect of A3G on the expression of miRNAs. Using a miRNA microarray method, we did not find that A3G significantly changed the miRNA expression in 293T cells (supplemental Figs. S1 and S2). A3G also did not significantly change the expression of miRNA processors such as Drosha and Dicer1 or RISC components such as Ago2 and Mov10 (supplemental Fig. S3). Further, A3G also did not change the level of expression of P-body components such as GW182, Xrn1 and Lsm1 (supplemental Fig. S3). Nevertheless, the microarray data did indicate that several miRNAs, such as mir-16, mir-10b, mir-25, and let-7a, are abundant in 293T cells.

To study whether A3G affects the efficiency of miRNA-mediated translational repression, various 293T cell-enriched miRNA-binding sites with perfect or partial complementarity to their corresponding miRNAs were inserted into the 3′-UTR of luciferase (luc) or gfp (Fig. 1a). These plasmids were transfected into 293T cells, which naturally do not express A3G (22, 27), with or without an A3G-HA-expressing plasmid. Fig. 1b shows that the presence of mir-16, mir-10b, or mir-25 miRNA-binding sites in the 3′-UTR of luc gene remarkably inhibited the expression of luciferase. Interestingly, A3G significantly counteracted this inhibition. Similar phenomenon can be observed in HeLa cells (Fig. 1c). To verify this derepression, a dose dependence experiment was performed and derepression was found to correlate with the A3G expression level (Fig. 1d). Real-time PCR data showed that the expression level of luciferase mRNA also substantially increased concomitantly with the expression level of A3G (Fig. 1e). This derepression of miRNA-mediated translational inhibition still occurred when the reporter gene was changed to gfp (Fig. 1f).

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FIGURE 1.

A3G counteracts miRNA-mediated repression of protein translation in 293T and HeLa cells. a, sequences of the miRNAs mir-16, mir-25, mir-10b, and let-7a and their target sites used for reporter gene constructs are shown. b and c, 293T cells (b) or HeLa cells (c) were co-transfected with a plasmid expressing A3G (pcDNA-A3G-HA) and a plasmid containing the luciferase reporter gene with binding sites for mir-16, mir-10b, or mir-25 in the 3′-UTR. pcDNA3 and pmir-REPORT were also transfected as controls. At 48-h post-transfection, luciferase activity was measured. d and e, 293T cells were co-transfected with different amounts of A3G-expressing plasmid (ranging from 0 to 0.4 μg) and pmir16-luc. At 48-h post-transfection, luciferase activity (d) was measured, and luciferase mRNA (e) was detected by real-time RT-PCR. The means ± S.D. are shown. f, 293T cells were co-transfected with pcDNA-A3G-HA and a plasmid containing a GFP reporter gene with a binding site for let-7a in the 3′-UTR, pEGFP-c1-let-7a, or pEGFP-c1 as a control. At 48-h post-transfection, GFP expression was analyzed by FACS and the mean fluorescence intensity (MFI) of GFP was determined. The data shown are representative of at least three replicates.

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FIGURE 2.

A3G/F-specific siRNA restores miRNA-mediated repression of protein translation in A3G/F-rich T-lymphocytes and macrophages. PHA-activated CD4+ T cells (a) and H9 cells (b) were first transfected with A3G- and A3F-specific siRNAs via Nucleofector (AMAXA). A siRNA for luc was used as a control for transfection. After 48 h, the cells were transfected with pEGFP-c1-let-7a or pEGFP-c1. At 48-h post-transfection, GFP expression was analyzed by FACS. The MFI of GFP from pEGFP-c1 was set as 100%. The means ± S.D. are shown. c, primary monocyte-derived macrophages were first transfected with A3G- and A3F-specific siRNAs. A siRNA for luc was used as a control for transfection. After 48 h, the cells were transfected with pEGFP-c1-let-7a or pEGFP-c1. pcDNA3-A3G-HA (2 μg) was also cotransfected for overexpression experiment. At 48-h post-transfection, GFP expression was analyzed by Western blotting analysis via anti-GFP antibody. The expression of A3G and A3F were also examined by Western blotting.

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FIGURE 3.

APOBEC3 family members inhibit miRNA-mediated repression of protein translation. 293T cells were co-transfected with plasmids expressing APOBEC3 family members and pmir16-luc (a) or with plasmids expressing various A3G mutants and pmir16-luc (b). At 48-h post-transfection, luciferase activity was measured. The 4C mutant represents an A3G mutant that has four point mutations: C97A/C100A/C288A/C291A. The means ± S.D. are shown.

Furthermore, to confirm this effect, H9 T-cells, PHA-activated primary CD4+ T-lymphocytes and macrophages, which naturally harbor significant amounts of A3G and another APO-BEC3 protein, A3F, were treated with A3G- and A3F-specific siRNAs. Western blotting showed that expression of A3G and A3F could be effectively decreased by these siRNAs (Fig. 2, a-c). The depletion of A3G and A3F enhanced the efficiency of let-7a miRNA-mediated translational repression in these A3G/F-enriched cells (Fig. 2, a-c). Conversely, overexpression of A3G/F in macrophages can substantially enhance the derepression of miRNA-mediated translational inhibition (Fig. 2c, lane 1).

Other APOBEC3 Family Members Also Inhibit miRNA-mediated Repression of Protein Translation—To test whether other APOBEC3 family members also regulate miRNA repression, vectors expressing the APOBEC3 family members A3B, A3C, and A3F were transfected into 293T cells. All the tested APOBEC3 family members were able to inhibit the miRNA-mediated translational repression (Fig. 3a). Interestingly, a synergistic effect was found between various APOBEC3 family members (Fig. 3a).

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FIGURE 4.

A3G enhances the association of mir-16-targeted mRNA with polysomes. 293T cells were co-transfected with pMIR-REPORT and pcDNA3 (a), pMIR-REPORT and pcDNA3-A3G (b), pmir16-luc and pcDNA3 (c), pmir16-luc and pcDNA3-A3G (d), pmir16-luc and anti-mir16 inhibitors (e), or pmir16-luc and anti-mir28 inhibitors (f). At 48-h post-transfection, polysome profile analysis was performed and the distribution of luciferase mRNA and β-tubulin mRNA in the fractions was analyzed by RT-PCR. 293T cells were co-transfected with pmir16-luc and pcDNA3-A3G (g), or pMIR-REPORT alone (h). Prior to collection, the cells were treated with puromycin (0.3 mg/ml) for 30 min. At 48-h post-transfection, polysome profile analysis was performed, and the distribution of luciferase and β-tubulin mRNA in the fractions was analyzed by RT-PCR.

Given that A3G has cytidine deaminase activity, we examined whether this activity is responsible for the A3G inhibitory effect on miRNA translational repression. Mutation in the N-terminal zinc-binding domain of A3G important for virion incorporation and mutation in the C-terminal zinc-binding domain important for cytidine deaminase activity were examined for their possible influence on miRNA-mediated translational repression (28–31). The mutations that inactivate the N-terminal domain, C97A and C100A, had a modest effect on miRNA-mediated translational repression, whereas the C-terminal domain C288A and C291A mutations had no significant influence on the inhibitory effect of A3G (Fig. 3b), suggesting that the cytidine deaminase activity is unlikely involved in this effect.

A3G Enhances the Association of miRNA-targeted mRNA with Polysomes—To examine whether the A3G inhibitory effect on mir-16-mediated repression was at the level of translation, a polysome profile analysis was performed (Fig. 4). As shown in Fig. 4c, mir-16 decreased the association of its target mRNA with polysomes, which is consistent with previous reports (45, 46). However, A3G, as well as an antisense anti-mir-16 inhibitor, significantly enhanced the association of the target mRNA with polysomes (Fig. 4, d and e). Puromycin treatment can disrupt this association, further confirming the complex that luciferase mRNA bound with is polysome (Fig. 4g).

A3G Facilitates the Dissociation of miRNA-targeted mRNA from P-bodies—As A3G can be found in P-bodies (32, 33), and can increase the amount of miRNA-targeted mRNA (Fig. 1e), we then investigated whether A3G could be directly associated with GW182, a key component for P-body. We found that A3G can interact with GW182. This interaction is partially resistant to RNase digestion. Mutation at C-terminal catalytic domain of A3G (C288A/C291A) cannot eliminate this interaction (Fig. 5a). Further, we also confirmed that A3G co-localized with GW182 (Fig. 5b) (32, 33). Moreover, we have found that the depletion of GW182 with GW182-specific siRNA had a synergistic effect with A3G in counteracting miRNA-mediated translational repression (Fig. 5c), which is consistent with previous reports regarding the role of GW182 in miRNA function (15, 16).

We then examined whether A3G had any effect on the interaction between miRNA-targeted mRNA and P-bodies by performing in situ hybridization with confocal microscopy, as described (21). The location of luciferase mRNA was detected with a Cy3-conjugated oligonucleotide probe, and the location of P-bodies was visualized with GFP-GW182 (19). The mRNA without miRNA-binding sites did not associate with GW182 (Fig. 6, a and b). In the absence of A3G, mir-16-targeted luciferase mRNA was found associated with GW182 and in P-bodies (Fig. 6c), indicating that miRNAs such as mir-16 mediate the association of mRNA with P-bodies. However, in the presence of A3G, mir-16-targeted luciferase mRNA was not found in the P-body (Fig. 6d), suggesting that A3G either facilitates the exit of miRNA-bound mRNA from P-bodies or prevents miRNA-bound mRNA from entering P-bodies. As a control, an anti-mir-16 antisense inhibitor, which can specifically block the function of mir-16, but not an anti-mir28 inhibitor, also prevented the miRNA-targeted luciferase mRNA from associating with GW182 and P-bodies (Fig. 6, e and f).

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FIGURE 5.

Interaction between A3G and GW182. a, 293T cells were transfected with pcDNA3-A3G-HA or pcDNA3-A3G-C97A/C100A-HA. At 48-h post-transfection, cells were collected and lysed. Lysates were treated with and without RNase A, followed by immunoprecipitation with mouse anti-GW182 antibody. The precipitated samples were then subjected to SDS-PAGE electrophoresis. After transferring, A3G was detected with rabbit anti-A3G antibody. b, HeLa cells were co-transfected with pcDNA-A3G-HA and pGFP-GW182delta1 (19). At 48-h post-transfection, the localization of GW182 was visualized with GFP-GW182 fluorescence (green) and A3G was detected with mouse anti-A3G and visualized with Texas Red-conjugated goat anti-mouse antibody (red). c, 293T cells were transfected with GW182-specific siRNA. At 48-h post-transfection, the cells were co-transfected with pcDNA-A3G-HA and pmir16-luc. After another 48 h, a luciferase assay was performed. The means ± S.D. are shown.

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FIGURE 6.

A3G facilitates the dissociation of mir-16-targeted mRNA from P-bodies. HeLa cells were co-transfected with pcDNA-A3G-HA, pmir16-luc, pGFP-GW182delta1, or various antisense miRNA inhibitors, as indicated. At 48-h post-transfection, P-bodies were visualized with GFP-GW182 fluorescence (green), and luciferase mRNA was visualized by in situhybridization with Cy3-conjugated oligonucleotide probes (red). DAPI staining of the nuclei is shown inblue. A magnification of the regions enclosed by the boxes is shown in the insets at the upper left corners.

Discussion

Endogenous A3G can be found in various cells such as H9 T-cells, primary CD4 T-cells, macrophages, and many other normal tissues/organs such as spleen, thymus, testis, ovary, small intestine, mucosal lining of colon (22, 47). They can effectively inhibit the replication of vif-defective HIV-1 (22, 48, 49). Although miRNAs are still able to mediate translational inhibition in H9 T-cells, primary CD4 T-cells at a moderate level and in macrophage at a significant level, we believe that their activity has been restricted by endogenous A3G/A3F. As shown in Fig. 2, a-c, A3G/F-specific siRNAs, which effectively deplete A3G/F in these cells, can significantly further enhance the miRNA-mediated translational inhibition, indicating endogenous A3G or A3F are functional to prevent the activity of miRNA. Furthermore, overexpression of A3G/F can effectively counteract the miRNA-mediated inhibitory effect on translation, supporting this argument (Fig. 2c). Nevertheless, the result from overexpression of exogenous A3G/F also suggests that the either quantity or quality of endogenous A3G/F could need to be improved for an efficient counteraction to miRNA activity. Recently, we and others have found that interferon-(IFN)-α/β can significantly enhance the expression of A3G/F in various primary cells such as resting CD4 T-lymphocytes, macrophages, endothelial cells, hepatocytes, myeloid dendritic cells, and plasmacytoid dendritic cells (42, 50–54).³ Therefore, it is interesting to further investigate the correlation of IFN regulatory system and the miRNA activity in these primary cells.

Our data demonstrate that A3G facilitates recruitment of miRNA-targeted mRNA to polysomes to synthesize more proteins and drives dissociation of miRNA-targeted mRNA from P-bodies. Given that A3G is associated with mRNA, localizes to P-bodies and stress granules (32, 33, 36), and can substantially enhance the expression of miRNA-targeted mRNA (Fig. 1e), it is unlikely that A3G directly improves the interaction between mRNA and polysomes or inhibits the interaction between miRNA and its target mRNA in miRISC. Instead, A3G may block miRNA-targeted mRNA from entering P-bodies or stress granules, may prevent the miRNA-targeted mRNA from engaging the RNA degradation machinery in P-bodies, or may directly facilitate the egress of miRNA-targeted mRNA from P-bodies and stress granules. By one or more of these approaches, A3G may inhibit the degradation or storage of miRNA-targeted miRNA in P-bodies and stress granules. Subsequently, more of the mRNA could associate with polysomes, and the translation efficiency would therefore be enhanced. However, as the mechanism of the regulation of mRNA degradation and storage in P-bodies or stress granules remains to be clarified and the relationship between miRNA-mediated translational repression and P-bodies is still under intensive investigation, further experiments are required to demonstrate the exact mechanism underlying this cellular function of A3G.

Interestingly, the mutations C228A and C291A inactivated the cytidine deaminase activity of A3G, but A3G was still able to enhance the expression of luciferase when luc was controlled by miRNA (Fig. 3b). Therefore, the derepression of miRNA-mediated inhibition of protein translation by A3G is separable from its cytidine deaminase activity. As described in many reports, the cytidine deaminase activity of A3G is only partially responsible for viral infectivity (24, 28–31). It remains to be determined whether this cellular function of A3G in protein translation regulation is related to its cytidine deaminase-independent antiviral activity.