miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs

Abstract

miRNA-mediated repression in animals is dependent on the GW182 protein family. GW182 proteins are recruited to the miRNA repression complex through direct interaction with Argonaute proteins, and they function downstream to repress target mRNA. Here we demonstrate that in human and Drosophila melanogaster cells, the critical repressive features of both the N-terminal and C-terminal effector domains of GW182 proteins are Gly/Ser/Thr-Trp (G/S/TW) or Trp-Gly/Ser/Thr (WG/S/T) motifs. These motifs, which are dispersed across both domains and act in an additive manner, function by recruiting components of the CCR4-NOT deadenylation complex. A heterologous yeast polypeptide with engineered WG/S/T motifs acquired the ability to repress tethered mRNA and to interact with the CCR4-NOT complex. These results identify previously unknown effector motifs functioning as important mediators of miRNA-induced silencing in both species, and they reveal that recruitment of the CCR4-NOT complex by tryptophan-containing motifs acts downstream of GW182 to repress mRNAs, including inhibiting translation independently of deadenylation.

Figure 1

The TNRC6C CED interacts with components of the CCR4–NOT complex. (a) Schematic representation of human TNRC6C and fragments analyzed in the study. Individual domains and regions of TNRC6C are indicated: N-GW, GW-repeat–rich region; UBA, ubiquitin associated–like domain; RRM, RNA-recognition motif; M2 and Cterm, regions flanking RRM, constituting—together with PAM2 and RRM—the CED region. (b) MS analysis of proteins interacting with the CED. Relevant proteins are listed along with peptide coverage and amount of assigned spectra. For full list of proteins, see Supplementary Table 1. (c) Validation of the CED interaction with selected CCR4–NOT components by GST pull-down assays and western blotting. GST-RRM was used as a control. (d) M2 and Cterm regions of the CED interact with components of the CCR4–NOT complex but not with PABP. TNRC6C CED and its subfragments were used for GST pull-down assays. Inputs (7%) and pull-down assays were analyzed by western blotting. Extracts from nontransfected cells were used as controls. (e) CNOT1 co-immunoprecipitates with endogenous TNRC6A. (f) M2 and Cterm regions of TNRC6C mediate repression of tethered mRNA. HEK293T cells were co-transfected with plasmids encoding NHA-CED or indicated fragments, and RL-5BoxB and firefly luciferase–transfection control (FL-Con) reporters. As negative controls, untethered hemagglutinin-CED (HA-CED) and tethered NHA-RRM (where ‘N’ stands for tethering λ peptide; see Supplementary Fig. 2a) were expressed. Values represent percentage of Renilla luciferase activity (normalized to firefly luciferase activity) in the presence of nontethered HA-CED or HA-CEDΔPAM2. In all luciferase assays presented in this work, values represent means ± s.e.m. from three to six experiments. Expression levels of HA- or NHA-fusion proteins were estimated by western blotting.

Figure 2

W-motifs in GW182 proteins mediate mRNA repression by recruiting CCR4–NOT and PAN2–PAN3 deadenylation complexes. (a) Mutations of tryptophan residues in W-motifs alleviate repression by the TNRC6C CED. Schematic representation of the TNRC6C CED with positions of W-motifs marked with asterisks is shown above the graph. Plasmids encoding either wild-type NHA-CED or its mutants (mutations always to alanine; when several consecutive amino acids are mutated, the number corresponds to the first residue in the mutated stretch) were co-transfected to HEK293T cells, together with RL-5BoxB and FL-Con. As negative controls, plasmids encoding untethered HA-TNRC6C or HA-CED were used. Mutants 2W through 8W contain Trp→Ala mutations in W-motifs (for details, see Online Methods). All GW, W1487 W1494 W1648 W1659; most conserved tryptophan, W1504 W1515; less conserved tryptophan, W1487 W1605 W1648 W1659. Values represent percentages of Renilla luciferase produced in the presence of untethered HA-CED control. Expression of HA- or NHA-fusion proteins was estimated by western blotting (lower panel). (b) Proteins identified as interacting with the CED in a tryptophan-dependent manner by MS (Supplementary Fig. 4a) were validated by GST pull-down assays and western blotting. Positions of protein size markers are indicated. (c) W-motifs are required for repression by the D. melanogaster GW182 CED. NHA-dGW182 CED, either wild-type or with mutations, were co-transfected with FL-5BoxB and RL-Con in S2 cells. As negative controls, plasmids encoding HA-dGW182 and HA-dGW182 CED were used. Mutants 2W through 8W contain mutations in W-motifs, with some (5Wa and 7Wa) having different combinations of mutated tryptophans (positions of W-motifs are marked with asterisks in the scheme above; for details, see Online Methods). Expression of firefly luciferase was normalized to Renilla luciferase. Values represent percentages of firefly luciferase produced in the presence of HA-CED. Expression of HA-fusions was estimated by western blotting.

Figure 3

W-motifs present in the dGW182 NED and the engineered yeast protein fragment repress tethered mRNA and recruit components of CCR4–NOT. (a) The dGW182 NED W-motifs function in mRNA repression. HEK293T cells were transfected with RL-5BoxB, FL-Con and plasmids expressing either full-length NHA-dGW182 or its NED (1–490) (WT or 6W mutant; for description of the mutant, see ref. 17). As negative controls, HA-dGW182 and HA-dGW182(1–490) were used. As positive controls, TNRC6C NHA-CED and full-length NHA-dGW182 were tethered. Values represent percentages of Renilla luciferase produced in the presence of HA-dGW182(1–490). Western blot analysis of HA- or NHA-fusion proteins is presented below. (b) GST fusions of the dGW182(1–490), WT and 6W mutant, expressed in HEK293T cells, were used for GST pull-down assays. Inputs (7% for anti-CNOT1, anti-CAF1, anti-tubulin and anti-GST; 15% for anti-PABP, anti-PAN2 and anti-PAN3) and the pulled-down material were analyzed by western blotting, using indicated antibodies. Additional western blots (on the right) for PABP, PAN2 and PAN3 represent pull-down assays done with the TNRC6C GST-CED analyzed in parallel on the same gel. Anti-PAN3 antibody cross-reacts with GST (asterisk). (c) W-motifs are sufficient to induce repression of tethered mRNA. HEK293T cells were transfected with RL-5BoxB, FL-Con and plasmids encoding engineered N-Sic-GST protein fusions having either four (N-Sic4xW-GST) or seven (N-Sic7xW-GST) W-motifs. N-Sic-GST containing no tryptophan residues, and NHA-GST, served as controls; plasmids encoding TNRC6C N-CED-GST, WT and 7W mutant were transfected for comparison. (d) GST pull-down assays with GST-Sic7xW, GST-CED (positive control), and GST-CED 7W and GST-Sic (negative controls), were done as in Figure 1d. The pulled-down material was analyzed by western blotting, using indicated antibodies.

Figure 4

W-motifs are necessary for repression by full-length GW182 and function in bona fide miRNA repression. (a) W-motifs are required for repression by tethered full-length TNRC6C. The experiment was done as in Figure 2a but included the full-length TNRC6C. The right panel shows fold derepression relative to repression induced by WT NHA-TNRC6C or NHA-CED taken to be a value of 1 (broken line). Western analysis of expression levels of relevant mutants in a and other panels, with anti-HA antibody, is shown below the graphs. (b) Mutations in W-motifs lead to partial derepression of tethered mRNAs in D. melanogaster S2 cells. The assay was done as in Figure 2c but with the full-length dGW182 and TNRC6C. 6W, 7W and EF1388 mutations were described in Figures 2 and 3a but are here introduced into the full-length proteins. 13W mutant combines 6W and 7W; PAM2mut has EF960 WK967 Thr982 mutated. NHA-Q–rich (1080–1245) and NHA-CED represent TNRC6C fragments. In the right panel, data are presented as in a. (c) W-motifs are required to rescue depletion of endogenous dGW182. Endogenous dGW182 was depleted in D. melanogaster S2 cells with dsRNA (open bars); a batch of cells was treated with GFP-specific dsRNA as a control (black bars). Cells were transfected with RL-Con, FL-nerfin, and plasmids encoding miR-9b or miR-12, or the empty vector. To rescue depletion of dGW182, increasing amounts of plasmids encoding NHA-dGW182, NHA-TNRC6C or their mutants were co-transfected. In panels c and d, extracts from cells transfected with highest plasmid concentrations were used for western blotting. (d) W-motifs are necessary to complement the knockdown of endogenous TNRC6 proteins. HeLa cells were transfected with siRNAs targeting three endogenous TNRC6 proteins (open bars) or AllStars siRNA (negative control, black bars), RL-hmga2 reporter containing let-7 sites or its mutant version (RL-hmga2 mut), and increasing amounts of plasmids expressing NHA-TNRC6A or its mutants: 8W has Trp→Ala mutations in W-motifs within the CED region (Supplementary Fig. 1 and Online Methods); EF1358 has PAM2 mutated.

Figure 5

The CED W-motifs and CCR4–NOT complex contribute to repression of poly(A)⁻ mRNAs in fly cells. (a) The CED W-motifs contribute to repression of poly(A)⁻ mRNA in fly cells. S2 cells were co-transfected with plasmids encoding NHA fusions of the WT dGW182 CEDs (NHA-CED or NHA-CED*) or its indicated mutants, together with plasmids encoding the indicated reporters (FL-5BoxB, FL-5BoxB-HSL or FL-5BoxB-HhR) and RL-Con. Normalized firefly luciferase activity is indicated as the percentage of activity in cells expressing NHA-lacZ set as 100%. Expression of relevant HA- and NHA-fusion proteins was estimated by western blotting and is shown in the panel on the right. (b) Tethering dCAF1 or human CNOT1 represses poly(A)⁺ and poly(A)⁻ mRNAs in fly cells. Cells were co-transfected with plasmids expressing HA or NHA fusions of dCAF1 or human CNOT1 and plasmids encoding indicated reporters. Normalized firefly luciferase activity is indicated as the percentage of activity in cells expressing HA fusions of dCAF1 or human CNOT1 set as 100%. Expression levels of HA- and NHA-fusion proteins were estimated by western blotting (shown above the graph). HA- and NHA-CNOT1 were only detectable after enrichment by anti-HA antibody immunoprecipitation. Lower signal of the NHA-tagged, compared to HA-tagged protein, may be partially due to the lower reactivity of anti-HA antibody with the internally located epitope. Analysis of mRNA levels by northern blotting is shown below the graph. Identity of analyzed reporters (including Renilla luciferase mRNA as a reference) is shown on the left, and the co-transfected CCR4–NOT complex components are indicated at the bottom.

Figure 6

Repression of poly(A)⁻ RNA by tethering dGW182 or its fragments depends on NOT1, but repression by tethered CCR4–NOT components is dGW182-independent. (a) Repression of FL-5BoxB-HSL reporter by tethering dGW182 or its fragments is alleviated in S2 cells depleted of NOT1. S2 cells treated with dsRNA targeting GFP or NOT1 were co-transfected with plasmids expressing either NHA fusions of dGW182 and its fragments or the PIN domain (either WT or a catalytic mutant thereof) of the endonuclease SMG6, and also reporter plasmids FL-5BoxB-HSL and RL-Con. Normalized firefly luciferase activity is indicated as percentage of the activity in cells expressing NHA-lacZ or SMG6-PINmut, set as 100%. The NOT1 depletion affected the repression by dGW182 and its fragments but had no effect on repression by SMG6-PIN that targets mRNA for endonucleolytic degradation, supporting the specificity of the effect. (b) Repression of FL-5BoxB and FL-5BoxB-HSL reporters by tethered dCAF1 and human CNOT1 is unaffected in S2 cells depleted of dGW182. Normalized firefly luciferase activity is indicated as the percentage of activity in cells expressing HA-dCAF1 or HA-CNOT1, or cells transfected with pAC5.1 (empty vector), each set as 100%. The efficiency of GW182 depletion was analyzed by western blotting (lower panel). Lanes 1–5, dilutions of the extract from S2 cells treated with GFP-specific (control) dsRNA. (c) Scheme illustrating a possible mode of action of GW182 proteins in miRNA-mediated repression. GW182 proteins are recruited to mRNA through direct interaction with the miRNA–AGO complex. The GW182 NED and CED regions both recruit, through the W-motifs, the CCR4–NOT complex that represses translation and leads to mRNA deadenylation. Interaction of the GW182 PAM2 motif with PABP may interfere with the PABP-eIF4G association, thus contributing to translational inhibition and mRNA deadenylation. The PABP interaction with the CED M2/C-term regions (broken line) may be mediated by the CCR4–NOT complex (see text).