The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

Abstract

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases.

Figure 1.

Domain organization of Drosophila melanogaster GW182, Hs TNRC6C and the corresponding chimeric proteins. ABD, AGO-binding domain; ABD2, AGO-binding domain from Caenorhabditis elegans AIN-2; NED, N-terminal effector domain; UBA, ubiquitin associated-like domain; QQQ, region rich in glutamine; Mid, middle region containing the PAM2 motif (dark blue), which divides the Mid region into the M1 and M2 regions; RRM, RNA recognition motif; C-term, C-terminal region; SD, silencing domain. The position of the conserved CIM-1, CIM-2 and P-GL motifs are indicated. Amino acid positions at domain boundaries are indicated below the protein outlines. Vertical red lines indicate the positions of GW repeats. Vertical green lines indicate the positions of tryptophan residues in the M2 region that are involved in NOT1-binding (9). Sequence alignments of the PAM2, CIM-1, CIM-2 and P-GL motifs and the amino acids mutated in this study are shown in Supplementary Figure S7.

Figure 2.

Interaction of Drosophila melanogaster GW182 with NOT1, NOT2 and PAN3. (A–G) S2 cells were cotransfected with plasmids expressing GFP-tagged D. melanogaster GW182 (wild-type or mutants) and HA-tagged deadenylase subunits or V5-tagged PABP as indicated. Cell lysates were immunoprecipitated using a polyclonal anti-GFP antibody. GFP-tagged firefly luciferase served as a negative control. Inputs (1%) and immunoprecipitates (5% for GFP-tagged proteins or 40% for HA- or V5-tagged proteins) were analyzed by western blotting using the corresponding antibodies. In all panels, cell lysates were treated with micrococcal nuclease before immunoprecipitation. The presence of endogenous AGO1 in the immunoprecipitates was determined using a specific anti-AGO1 antibody (E).

Figure 3.

The Drosophila melanogaster GW182 SD is generally required for miRNA-mediated translational repression and target degradation. (A–I) Control S2 cells (treated with glutathion S-transferase (GST) dsRNA) or cells depleted of endogenous GW182 were transfected with a mixture of three plasmids: one expressing the indicated F-Luc reporters; a second expressing miRNA primary transcripts or the corresponding empty vector (−) and a third expressing Renilla luciferase (R-Luc). Plasmids encoding HA-GW182 (wild-type or deletion mutants) or HA-MBP (negative control) were included in the transfection mixtures as indicated. For each condition, firefly luciferase activities and mRNA levels were normalized to those of the Renilla luciferase transfection control and set at 100% in cells transfected with the empty vector (i.e. in the absence of the miRNAs). (A and E) Normalized firefly luciferase activities and mRNA levels in the absence or presence of miRNAs in control cells (i.e. cells treated with GFP dsRNA and transfected with a plasmid expressing MBP). (B and F) Northern blot analysis of representative RNA samples. Numbers in italics below the panels indicate the levels of the F-Luc reporters normalized to that of R-Luc mRNA and set at 100 in the absence of the miRNAs. (C and G) Relative derepression of F-Luc activity for each condition. (D and H) Relative F-Luc mRNA levels. Throughout this study, error bars represent standard deviations from at least three independent experiments. Upper and lower dashed lines indicate maximal derepression and repression, respectively, observed in depleted cells. (I) A western blot showing that GW182 mutants were expressed at levels equivalent to that of the wild-type protein.

Figure 4.

The Drosophila melanogaster GW182 SD is generally required for silencing. (A–F) Complementation assays using the indicated miRNA reporters were carried out as described in Figure 3. The graphs on the left of each panel show normalized firefly luciferase activities in the absence or presence of miRNAs in control cells (i.e. cells treated with GFP dsRNA and expressing MBP). The graphs on the right of each panel show the relative derepression of the F-Luc reporters for each condition. Mean values ± standard deviations from three independent experiments are shown. Labels are as described in Figure 3.

Figure 5.

The Drosophila melanogaster GW182 Q+SD region is sufficient for silencing. (A and B) The silencing activity of chimeric proteins containing the Caenorhabditis elegans ABD2 fused to various GW182 fragments was tested in complementation assays as described in Figure 3 except that control cells were treated with GST dsRNA and transfected with a plasmid expressing GFP. (C–E) The interactions of the chimeric ABD2–GW182 proteins with AGO1, PABP and NOT1 were analyzed as described in Figure 2.

Figure 6.

PABP and deadenylase binding are required for silencing. Mutations in the PAM2 and CIM-1 motifs and the M2 region were introduced in a minimal GW182 protein consisting of Caenorhabditis elegans ABD2 fused to the GW182 Q+SD region (ABD2-Q+SD). The PAM2 mutant carries a single amino acid substitution (F961A) in the PAM2 motif. Mutations in the CIM-1 motif are shown in Supplementary Figure S7. The 6xW mutant carries alanine substitutions of all six tryptophan residues in the M2 region of the SD. (A and B) The interactions of the ABD2-Q+SD protein (wild-type or mutant) with PABP and NOT1 were analyzed as described in Figure 2. (C–F) The silencing activity of the ABD2-Q+SD protein (wild-type or mutants) was tested in complementation assays as described in Figure 5.

Figure 7.

The TNRC6 SD is sufficient for silencing. Mutations in the PAM2, CIM-1 and CIM-2 motifs were introduced in a minimal GW182 protein consisting of Caenorhabditis elegans ABD2 fused to the human TNRC6C SD region (ABD2-6C-SD). The PAM2 mutant carries a single amino acid substitution (F1389A) in the PAM2 motif. Mutations in the CIM-1 and CIM-2 motifs are shown in Supplementary Figure S7. (A and B) The silencing activity of the chimeric ABD2-6C-SD protein (wild-type or mutant) was tested in complementation assays as described in Figure 5. (C and D) The interactions of the ABD2-6C-SD protein (wild-type or mutant) with PABP and NOT1 were analyzed as described in Figure 2.

Figure 8.

Complementation assay in human cells. (A–C) Control HeLa cells (transfected with β-Gal siRNA) or cells codepleted of TNRC6A and TNRC6B were transfected with a mixture of three plasmids: the R-Luc-3xlet-7 or the corresponding reporter carrying mutations in let-7–binding sites (R-Luc-Mut), a plasmid expressing F-Luc as a transfection control, and a plasmid expressing GFP or siRNA-resistant versions of HA-TNRC6A (wild-type or mutant). For each condition, Renilla luciferase activity was measured, normalized to that of the F-Luc transfection control and set at 100% in cells expressing R-Luc-Mut. (A) Normalized Renilla luciferase activities in control cells (i.e. cells treated with β-Gal siRNA and expressing GFP). (B) Relative fold derepression for each condition. Mean values ± standard deviations are shown. (C) Western blot showing the efficiency of the TNRC6A knockdown. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. α-tubulin served as a loading control. (D) Interaction of GST-tagged TNRC6A-SD (wild-type or mutants) with CNOT1 and endogenous PABP. Inputs (1%) and bound fractions (40%) were analyzed by western blotting. (E) Western blot analysis showing that all proteins were expressed at comparable levels.

Figure 9.

Overexpression of the CNOT1-M domain inhibits silencing in a dominant negative manner. (A) Domain organization of human CNOT1. CNOT1-N, N-terminal domain; CNOT1-M, middle domain containing the MIF4G domain (31); CNOT1-C, C-terminal domain containing the NOT homology domain. Amino acid positions at domain boundaries are indicated below the protein outlines and correspond to the NCBI protein sequence NP_057368.3. (B) Interaction of GFP-tagged human CNOT1 (wild-type or fragments) and human HA-TNRC6A. Inputs (1%) and immunoprecipitates (5% for CNOT1 or 10% for TNRC6A) were analyzed by western blotting. F-Luc-GFP served as a negative control. (C and D) Human cells were transfected with the indicated Let-7 reporters or the corresponding controls (Mut) as described in Figure 8. The transfection mixtures also contained plasmids encoding GFP-CNOT1-M and GFP-CNOT7 (catalytically inactive mutant, D40A+E42A), as indicated. A plasmid encoding GFP-MBP served as a negative control. For each condition, R-Luc activities were normalized to that of a F-Luc transfection control and set at 100% for the Mut reporters (gray bars, shown only for control cells). Error bars represent standard deviations from three independent experiments.