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. 2014 Apr 15;28(8):888-901.
doi: 10.1101/gad.237289.113.

Structural Basis for the Nanos-mediated Recruitment of the CCR4-NOT Complex and Translational Repression

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Free PMC article

Structural Basis for the Nanos-mediated Recruitment of the CCR4-NOT Complex and Translational Repression

Dipankar Bhandari et al. Genes Dev. .
Free PMC article

Abstract

The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4-NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1-3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1-3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1-3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4-NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4-NOT complex as the main effector complex for Nanos function.

Keywords: SLiM; deadenylation; decapping; mRNA decay; translational repression.

Figures

Figure 1.
Figure 1.
Human Nanos1–3 interact with the CNOT1 SHD directly through conserved NIMs. (A) The domain organization of human Nanos1–3. Nanos proteins consist of a conserved C-terminal CCHC-type zinc finger domain (ZnF; orange) and variable N-terminal and C-terminal extensions (gray). The NIMs are shown in red. The numbers below the protein outlines indicate the residues at the domain/motif boundaries. (B) Sequence alignment of vertebrate NIMs. The residues conserved in all of the aligned vertebrate sequences are shown with a salmon background. The asterisks indicate the residues that were mutated in this study. The species abbreviations are as follows: Hs (Homo sapiens), Xt (Xenopus tropicalis), and Dr (Danio rerio). (C–F) Interaction of V5-SBP-tagged Nanos1–3 (full length or the indicated mutants) with endogenous CNOT1 and CNOT3 in HEK293T cells. A V5-SBP-tagged GFP-MBP fusion served as a negative control. The inputs (0.5%) and bound fractions (3% V5 proteins and 35% CNOT1 and CNOT3) were analyzed by Western blotting. (G) A GST pull-down assay showing the interaction of the GST-Nanos1–3 NIMs with the recombinant NOT module, the CNOT1 SHD, and CNOT2–CNOT3 heterodimers. GST served as a negative control.
Figure 2.
Figure 2.
Nanos1–3 NIMs cause degradation of bound mRNAs. (A–L) Tethering assays using the β-globin-6xMS2bs reporter and the indicated MS2-HA-tagged proteins. A plasmid expressing an mRNA lacking MS2-binding sites (control) served as a transfection control. The β-globin-6xMS2bs mRNA levels were normalized to those of the control mRNA and set to 100 in the presence of MS2-HA-GFP. The mean values ± standard deviations from three independent experiments are shown in B, E, H, and K. (A,D,G,J) Northern blots of representative RNA samples. (C,F,I,L) Western blot analysis showing the equivalent expression of the MS2-HA-tagged proteins used in the corresponding tethering assays.
Figure 3.
Figure 3.
Nanos1–3 promote deadenylation-dependent decapping. (A–D) mRNA degradation assay using the R-Luc-Asb9 reporter (lacking MS2-binding sites) in cells coexpressing the indicated MS2-HA-tagged proteins. A plasmid expressing F-Luc mRNA served as a transfection control. The R-Luc-Asb9 mRNA levels were normalized to the control mRNA and set to 100 in the presence of MS2-HA-GFP. The mean values ± standard deviations from three independent experiments are shown in B and D. (A,C) Northern blots of representative RNA samples. (E–J) Tethering assays using the β-globin-6xMS2bs reporter were performed as described in Figure 2 with the exception that plasmids expressing the DCP2* or POP2* catalytically inactive mutants were included in the transfection mixtures as indicated. (E,H) Northern blots of representative RNA samples. (F,G,I,J) Normalized levels of the β-globin-6xMS2bs mRNA. The expression of the DCP2* and POP2* proteins is shown in Supplemental Figure 2, G and H. (K–M) The effect of Nanos1 and Nanos2 on the expression of the R-Luc-Asb9 mRNA reporter was tested as described in A–D with the exception that a plasmid expressing the POP2* catalytically inactive mutant was included in the transfection mixtures as indicated.
Figure 4.
Figure 4.
Nanos1–3 NIMs repress translation in the absence of mRNA degradation. (A–H) Tethering assay using the R-Luc-6xMS2bs-MALAT1 reporter and the indicated MS2-HA-tagged proteins. A plasmid expressing F-Luc served as a transfection control. The R-Luc activities and mRNA levels were normalized to those of the F-Luc transfection control and set to 100 in the presence of MS2-HA-GFP. (A,EH) Normalized R-Luc activities obtained in three independent experiments. (B) Northern blot of representative RNA samples. The Northern blots corresponding to the samples shown in G and H are shown in Supplemental Figure 2K. (C) Normalized R-Luc mRNA levels. The mean values ± standard deviations from three independent experiments are shown. (D) Western blot analysis showing the expression of the MS2-HA-tagged proteins.
Figure 5.
Figure 5.
Structure of CNOT1–Nanos1 NIM complex. (A) Cartoon representation of the Nanos1 peptide (orange) bound to the CNOT1 SHD. The HEAT-like repeats that form the CNOT1 SHD are numbered. Both N-SD and C-SD (N-terminal and C-terminal subdomains, respectively) are colored in a gradient from gray to blue from their N termini to the C termini, respectively. (B) Surface representation of the Nanos-binding pocket of CNOT1 colored in a gradient from white to yellow with increasing hydrophobicity. The electron difference density for the Nanos1 NIM peptide (chain B) is shown as a black mesh (difference density [F0 − FC] contoured at 2.0 σ using the refined CNOT1 model before the NIM peptide was built), and the corresponding structural model is displayed as orange sticks. (C,D) Close-up views of the binding interface. The residues of CNOT1 and the Nanos1 peptide are shown as gray and orange sticks, respectively. The residues of CNOT1 and Nanos1 mutated in this study are highlighted in dark gray and red, respectively. (E) Structural model showing the Nanos1 NIM peptide bound to the human NOT module. The model was created by the superposition of the human NOT module (Boland et al. 2013) onto the CNOT1–Nanos1 structure. CNOT1 from the CNOT1–Nanos1 structure and CNOT2–CNOT3 from the NOT module are shown in surface representation. Nanos1 is represented as a cartoon (orange). The conservation of surface residues in the NOT module is indicated by color gradients from light (no conservation) to dark (100% conservation) for CNOT1 (blue), CNOT2 (green), and CNOT3 (cyan). The conservation scores were calculated based on well-balanced multiple alignments covering all eukaryotic strata. (G–I) Conformational change of the CNOT1 α23 helix in the complex with the Nanos1 NIM peptide. The CNOT1 SHD structures from the human and yeast NOT modules and the C. thermophilum (Ct) NOT1 SHD are shown in salmon (G), yellow (H), and green (I), respectively. The CNOT1 SHD bound to the Nanos1 NIM peptide is shown in gray, and the Nanos1 NIM peptide is shown in orange. The black lines illustrate the orientation of the α23 helix in the two structures. The angle between the two conformations is indicated. PDB codes are as follows: 4C0D (human NOT module), 4BY6 (Saccharomyces cerevisiae NOT module), and 4C0E (C. thermophilum NOT1).
Figure 6.
Figure 6.
Validation of the interaction interface. (A) A GST pull-down assay showing the interaction of the GST-Nanos1–3 NIMs with the recombinant NOT module containing the wild-type CNOT1 SHD or the indicated CNOT1 mutants. GST served as a negative control. (B) A GST pull-down assay showing that the MBP-tagged Nanos3 NIM peptide competes with the GST-tagged Nanos1–3 NIMs for binding to the NOT module. The MBP-Nanos 3 NIM competitor was present in 0.5-fold, onefold, and fivefold molar excess relative to the GST-NIMs. The corresponding experiment showing that the Nanos3 NIM Y10E mutant does not compete for binding is shown in Supplemental Figure 6A. (C–E) Interaction of V5-SBP-tagged Nanos1–3 (wild type or mutants) with endogenous CNOT1 and CNOT3 in HEK293T cells.
Figure 7.
Figure 7.
Mutations in the NIMs abrogate translational repression mediated by Nanos1–3. (A–I) Tethering assays using the β-globin-6xMS2bs reporter and the indicated proteins were performed as described in Figure 2. (A,D,G) Northern blots of representative RNA samples. The levels of β-globin-6xMS2bs mRNA were normalized to those of the control and set to 100 in cells expressing MS2-HA-GFP. The mean values ± standard deviations of three independent experiments are shown in B, E, and H. (C,F,I) Western blot analysis showing the equivalent expression of the MS2-HA-tagged proteins used in the corresponding tethering assays. (J–L) Tethering assays using the R-Luc-6xMS2bs-MALAT1 reporter and the indicated proteins were performed as described in Figure 4.

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