APOBEC3G inhibits microRNA-mediated repression of translation by interfering with the interaction between Argonaute-2 and MOV10

Abstract

The apolipoprotein-B-mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) is a potent restrictive factor for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. It belongs to the cytidine deaminase family. Recent studies have shown that A3G significantly inhibits microRNA (miRNA)-mediated repression of translation. However, the mechanism underlying this action must be clarified. In this report, we have demonstrated that A3G counteracts miRNA-mediated repression of translation by inhibiting the interaction between moloney leukemia virus 10 (MOV10) protein and Argonaute-2 (AGO2), causing either abnormal assembly or abnormal maturation of miRNA-inducing silencing complex (miRISC). Through a series of MOV10 deletions, we found that A3G binds to a domain at the C terminus in MOV10, where it competitively inhibits the binding of AGO2 to that same domain. The interaction between A3G and MOV10 relies on its association with a small RNA named 7SL RNA. The A3G mutant W127L, which is unable to bind to 7SL RNA, shows significantly incapability to counteract the miRNA-mediated repression of translation. Our data demonstrate a novel mechanism involved in the regulation of miRISC activity. Although both A3G and MOV10 belong to the interferon antiviral system and inhibit HIV-1 and other retroviruses, their opposing effects on the cellular miRNA activity suggest that they play much more complicated regulatory roles in various cellular functions.

FIGURE 1.

The 7SL RNA mediates the interaction between A3G and MOV10. A, 293T cells transfected with A3G-HA- (or A3G-W127L-HA-) and MOV10-FLAG-expressing plasmids were collected and lysed. B, 293T cells transfected with A3G-HA- (or A3G-W127L-HA-) expressing plasmids were collected and lysed. A and B, after immunoprecipitation with anti-HA agarose beads, the samples were treated with or without RNase mixture and then analyzed by immunoblotting using anti-FLAG (A), anti-MOV10 (B), or anti-HA antibody (A and B). C, SRP14 mRNA in 293T cells expressing control-shRNA and SRP14-shRNA was quantified using real-time RT-PCR at 72 h post-transfection. The SRP14 mRNA in cells expressing control-shRNA was defined as 100%. D, 7SL RNA in 293T cells expressing control-shRNA and SRP14-shRNA was quantified by real-time RT-PCR at 6 days post-transfection. The 7SL RNA in cells expressing control-shRNA was defined as 100%. Data in C and D represent mean ± S.D. E, 293T cells expressing control-shRNA or SRP14-shRNA were co-transfected with MOV10-FLAG- and A3G-HA- (or A3G-W127L-HA-) expressing plasmids. F, 293T cells expressing control-shRNA or SRP14-shRNA were transfected with A3G-HA- (or A3G-W127L-HA-) expressing plasmids. E and F, after 24 h, cells were lysed and co-immunoprecipitated with anti-HA agarose beads. The precipitated samples were analyzed by immunoblotting using anti-FLAG (E), anti-MOV10 (F) or anti-HA antibody (E and F). Values in A, B, E, and F represent portions of MOV10-FLAG (or MOV10) normalized against A3G-HA (or A3G-W127L-HA) relative to control values.

FIGURE 2.

The depletion of MOV10 affects miRNA-mediated repression of protein translation in 293T cells. A, sequences of mir25 and mir16 and their target sites used for reporter gene constructs are shown. B, effects of MOV10-specific siRNA on the endogenous MOV10 expression in 293T cells. Values represent portions of MOV10 normalized against actin relative to control values. C and D, 293T cells were first transfected with MOV10-specific siRNA or siRNA for gfp as a control. After 24 h, cells were transfected with a plasmid containing the Renilla luciferase reporter gene with (C) perfect or (D) imperfect binding sites for mir25 or mir16 in the 3′-UTR (the plasmid also contained the firefly luciferase gene as an intraplasmid transfection normalization reporter). psiCHECK-2 was also transfected as a control. At 24 h post-transfection, Renilla/firefly luciferase activities were measured. E, 293T cells were first transfected with MOV10-specific siRNA (gfp-specific siRNA as a control). At 24 h post-transfection, cells were co-transfected with psiCHECK-2 and Renilla luciferase-specific siRNA (gfp-specific siRNA as a control). After 24 h, Renilla/firefly luciferase activity was measured. Data in C, D, and E represent mean ± S.D. (error bars). *, statistically significant, ≤0.05.

FIGURE 3.

A3G counteracts the interaction between MOV10 and AGO2. A and B, 293T cells were co-transfected with a plasmid expressing A3G and a plasmid containing the Renilla luciferase reporter gene with perfect/non-perfect binding sites for mir25 or mir16 in the 3′-UTR. This plasmid also contains the firefly luciferase gene as an intraplasmid transfection normalization reporter. pcDNA3.1 and psiCHECK-2 were also transfected as controls. At 48 h post-transfection, Renilla/firefly luciferase activity was measured. C, 293T cells were co-transfected with pcDNA3.1-A3G-HA, psiCHECK-2, and Renilla luciferase-specific siRNA. pcDNA3.1 and gfp-specific siRNA were also transfected as controls. At 48 h post-transfection, Renilla/firefly luciferase activity was measured. Data in A, B, and C represent mean ± S.D. (error bars). **, statistically significant, ≤0.01. D, 293T cells were co-transfected with HA-AGO1- (or HA-AGO2-), A3G-FLAG-, and MOV10-FLAG-expressing plasmids. E, 293T cells were co-transfected with HA-AGO2-, MOV10-FLAG- and different amounts of A3G-expressing plasmids. D and E, transfected-cells were lysed and immunoprecipitated with anti-HA antibody at 24 h post-transfection. The precipitated samples were then analyzed by immunoblotting using anti-FLAG and anti-HA antibodies. Values in D and E represent percentage of MOV10-FLAG normalized against HA-AGO1 (or HA-AGO2) and compared with control.

FIGURE 4.

A3G and AGO2 both bind to the C terminus of MOV10. A, C, and E, scheme of MOV10 series deletion. A series of MOV10 deletions were generated via PCR-based mutagenesis and all of them were tagged with HA-tag. B, D, and F, 293T cells were co-transfected with A3G-FLAG- (or FLAG-AGO2-) expressing plasmid and a series of MOV10-HA deletion mutants. At 24 h post-transfection, cells were lysed and co-immunoprecipitated with anti-HA-agarose beads. The precipitated samples were then analyzed by Western blotting using anti-FLAG or anti-HA antibody.

FIGURE 5.

The 7SL RNA affects the activity of A3G to counteract miRNA-mediated gene silencing. A, 293T cells were co-transfected with A3G-FLAG- (or A3G-W127L-FLAG-), MOV10-FLAG- and HA-AGO2-expressing plasmids. After 24 h, cells were lysed and co-immunoprecipitated with anti-HA agarose beads. The immunoprecipitated samples were then detected by Western blotting using anti-FLAG and anti-HA antibody. B, 293T cells were co-transfected with pcDNA3.1-A3G-HA (or pcDNA3.1-A3G-W127L-HA) and a plasmid containing the Renilla luciferase reporter gene with binding sites for mir25 in the 3′-UTR (the plasmid also contains the firefly luciferase gene as an intraplasmid control). pcDNA3.1 and psiCHECK-2 were also transfected as controls. At 48 h post-transfection, Renilla/firefly luciferase activity was measured. C, 293T cells expressing control-shRNA or SRP14-shRNA were first transfected with SRP14 expressing plasmid (pcDNA3.1 as a control). After 48 h post-transfection, 7SL RNA in transfected-cells was quantified by real-time RT-PCR. The level of 7SL RNA in cells expressing control-shRNA and pcDNA3.1 was defined as 100%. D, 293T cells expressing control-shRNA and those expressing SRP14-shRNA were first transfected with SRP14 expressing plasmid. At 24 h post-transfection, the transfected cells were then co-transfected with MOV10-FLAG- and A3G-HA- (or A3G-W127L-HA-) expressing plasmids. After 24 h, cells were lysed and co-immunoprecipitated with anti-HA-agarose beads. The precipitated samples were analyzed by immunoblotting using anti-FLAG or anti-HA antibody. E, 293T cells expressing control-shRNA or SRP14-shRNA were first transfected with SRP14 expressing plasmid. After 12 h, the transfected cells were then further transfected with A3G-expressing plasmid and a plasmid containing the Renilla reporter gene with binding sites for mir25 in the 3′-UTR (the plasmid also contains the firefly luciferase gene as an intraplasmid control). At 36 h post-transfection, the cells were harvested and disrupted, and Renilla/firefly luciferase activity was measured. Values in A and D represent portion of MOV10-FLAG normalized against HA-AGO2 (or A3G-HA) and compared with control. Data in B, C, and E represent mean ± S.D. (error bars). **, statistically significant, ≤0.01.

FIGURE 6.

A3G and MOV10 do not affect tethered AGO1- or AGO2-mediated down-regulation of protein synthesis. A, scheme of the tethering assay system. psi-5BoxB Renilla mRNA, containing five 19-nt BoxB hairpins in the 3′-UTR of psiCHECK-2, interacting with λN-AGO1 or λN-AGO2. B, 293T cells were co-transfected with A3G-expressing plasmid, pλN-AGO1 (or pλN-AGO2) and psi-5BoxB. pAGO1 (or pAGO2) and pcDNA3.1 were transfected as controls. At 48 h post-transfection, Renilla/firefly luciferase activity was measured. C, 293T cells were first transfected with MOV10-specific siRNA (gfp-specific siRNA as a control). After 24 h, cells were co-transfected with psi-5BoxB and pλN-AGO1 (or pλN-AGO2). pAGO1 (or pAGO2) was transfected as a control. At 24 h post-transfection, Renilla/firefly luciferase activity was measured. Data in B and C represent mean ± S.D. (error bars). D, competitive inhibition of interaction between AGO2 and MOV10 by A3G.