Multiple independent domains of dGW182 function in miRNA-mediated repression in Drosophila

Abstract

miRNA-mediated repression affects a wide range of biological processes including development and human pathologies. The GW182 protein is a key component of miRNA repression complex, recruited by Argonaute and functioning downstream to repress translation and accelerate mRNA degradation, but little is known about how GW182 proteins act. Using both tethered function and complementation assays, we identify three independent domains of the Drosophila GW182 protein (also termed Gawky) that are sufficient to repress mRNA. Each of these domains also functions independently of poly(A) tails. These results indicate that miRNA-mediated repression is facilitated by multiple domains of GW182.

FIGURE 1.

Three separate domains of Drosophila GW182 are sufficient to repress tethered mRNA. (A) Schematic representation of Drosophila GW182 protein and generated dGW182 deletion mutants. The numbers correspond to the amino acid positions. (B) Schematic representation of reporter constructs: FLuc-boxB contains firefly luciferase coding sequence and 3′-UTR with five boxB sites specifically binding to λN peptide; RLuc contains Renilla luciferase coding sequence and no boxB sites (Rehwinkel et al. 2005). (C) Repression of FLuc-boxB mRNA by NHA-dGW182 and its deletion mutants. Drosophila S2 cells were co-transfected with plasmids encoding for FLuc-boxB, RLuc, and full-length NHA-dGW182 or one of the NHA-dGW182 deletion mutants depicted in A. As negative controls, either NHA alone, NHA fused to β-galactosidase (NHA-lacZ), or mutant NHA-Ago1 F594VF629V (designated as 2F2V) not able to recruit dGW182, or HA-dGW182 without λN peptide was transfected instead of NHA-dGW182 or NHA-dGW182 deletions. Cells were lysed 2 or 3 d post-transfection, and protein levels were measured with dual luciferase assay. Expression of firefly luciferase was normalized to that of the Renilla luciferase. Values are presented as a percentage of firefly luciferase produced in the presence of NHA-lacZ. Values represent the average of at least four experiments. The error bar shows the standard deviation. (D) Expression of different HA-fusion proteins was estimated by Western blotting with antibodies directed against HA-peptide.

FIGURE 2.

Drosophila GW182 and its effector domains decrease stability of tethered mRNA. Northern blotting was used to estimate the levels of firefly and Renilla luciferase mRNAs from the experiment described in Figure 1. mRNA levels were quantified by PhosphorImaging, and amounts of FLuc-boxB were normalized according to Renilla control and expressed as a percentage of FLuc-boxB level in the presence of NHA-lacZ protein (numbers below the figure represent an average of two experiments). (Lanes 1–6) FLuc-boxB and RLuc RNA levels in the presence of tethered NHA-lacZ, NHA-dGW182, and dGW182 fragments: NHA-1-605, NHA-605-830, NHA-940-1215, and NHA-605-1215, accordingly. (Lanes 7,8) To demonstrate that degradation is the effect of tethering, lanes 7 and 8 show untethered FLuc and RLuc in the presence of either (lane 7) NHA-lacZ or (lane 8) NHA-dGW182. The last lane contains total RNA from untransfected S2 cells (no target control).

FIGURE 3.

Repression mediated by dGW182 and its sufficiency domains is independent of poly(A) tail. Drosophila S2 cells were transfected with a boxB-containing firefly luciferase reporter, either (open bars) FLuc-boxB or (black bars) FLuc-boxB-HSL. Unlike FLuc-boxB, FLuc-boxB-HSL lacks polyadenylation signal and bears histone H1 stem–loop structure in its 3′-UTR. Together with a firefly luciferase reporter, RLuc as a normalization control and one of the NHA-fusion proteins (NHA-dGW182, or NHA-dGW182 deletion mutant, or NHA-lacZ as a negative control) were co-transfected. For the rest, the assay was performed as described in Figure 1.

FIGURE 4.

Repression mediated by dGW182 and its sufficiency domains appears to be independent of endogenous dGW182 and Argonaute 1. (A) S2 cells were treated with dsRNAs indicated above the lanes (dGW182, Ago1, or GFP as a negative control), and efficiencies of endogenous dGW182 and Ago1 depletions were analyzed by Western blotting with antibodies shown on the left. Expression of tubulin was estimated as a loading control. (B) dGW182 and its effector domains were transfected in S2 cells depleted of endogenous dGW182 or Ago1 and, as negative controls, in GFP-dsRNA-treated or untreated cells. Transfections were largely performed and analyzed as in Figure 1. In tethering experiments, firefly luciferase activity was expressed as a percentage of that in the presence of NHA-lacZ, for each RNAi depletion. To additionally control for efficient depletion of endogenous Ago1 and dGW182, firefly luciferase reporter containing miRNA-9b target sites (FLuc-nerfin) (Behm-Ansmant et al. 2006) was co-transfected with either miRNA-9b-encoding plasmid or the empty vector. As in all transfection experiments, RLuc was coexpressed as a transfection control. Firefly luciferase activity was normalized to that of Renilla luciferase and presented as a percentage of FLuc-nerfin expression in the presence of the empty vector.

FIGURE 5.

Overexpression of dGW182 and its N-terminal fragments rescues RNAi knockdown of endogenous dGW182. (Black bars) Endogenous dGW182 was depleted in Drosophila S2 cells with dsRNA as described in Figure 4; (open bars) a batch of cells was treated with GFP-specific dsRNA, as a negative control. Cells were transfected with firefly luciferase reporter containing miRNA-9b target sites (FLuc-nerfin) and with either miRNA-9b-encoding plasmid or the empty vector. As in all transfection experiments, RLuc was coexpressed as a transfection control; expression levels of firefly luciferase were normalized to Renilla luciferase activity and expressed as a percentage of the firefly luciferase activity in the presence of the empty vector. To rescue the knockdown of endogenous dGW182, increasing amounts (from 0 to 100 ng per well, in a 96-well plate) of plasmids encoding NHA-dGW182 or its deletion mutants were co-transfected: 1–605 construct, encoding N-terminal GW-repeats, and 1–830 construct, encoding GW-repeats together with the Q-rich region. Expression of NHA-dGW182 and its deletions was estimated by Western blotting with anti-HA antibody; the squared bands correspond to the minimal expression levels sufficient to rescue miRNA-9b-mediated repression to the level observed in GFP dsRNA-treated cells. Increasing amounts of NHA-lacZ were co-transfected for a negative control. To test the dependence of rescue on the N-terminal region of dGW182 required for dGW182–Ago1 interaction, we also co-transfected increasing amounts of NHA-605-830 and NHA-940-1215 plasmids encoding for Q-rich and DUF-RRM regions, accordingly (30 and 100 ng/well).

FIGURE 6.

Human homolog of dGW182, TNRC6C, and its Q-rich and DUF-RRM regions are able to repress tethered mRNA in Drosophila S2 cells. (A) The tethering assay was performed essentially as in Figure 1C, with human protein TNRC6C and its deletion mutants tethered instead of dGW182. The N-terminal tethered fragment of TNRC6C (amino acids 1–1035) includes GW-rich repeats; the middle portion (amino acids 1080–1245) covers the Q-rich region; the C-terminal part (amino acids 1370–1690) comprises conserved DUF and RRM. Full-length NHA-dGW182 was tethered as a positive control, NHA and NHA-lacZ as negative controls. (B) Expression levels of NHA-fusion proteins were estimated by Western blotting with anti-HA antibody. (C) Summary of the repressive effects by TNRC6C, dGW182, and their deletions. The numbers show the percent translation in the presence of tethered full-length proteins, N-terminal, Q-rich, and C-terminal (DUF-RRM) fragments, accordingly.

FIGURE 7.

Scheme illustrating possible modes of action of dGW182 protein in translational repression by miRNAs. See the text for details.