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Structure and assembly of the NOT module of the human CCR4–NOT complex


The CCR4–NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4–NOT complex to mRNA targets.

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Figure 1: Domain organization of Homo sapiens (Hs) CNOT1–CNOT3 and the structure of the Ct NOT1 superfamily homology domain.
Figure 2: Structures of the CNOT2 and CNOT3 NOT-box domains.
Figure 3: Structure of the CNOT1–CNOT2–CNOT3 ternary complex.
Figure 4: The CNOT2–CNOT3 heterodimerization interface.
Figure 5: The interface between CNOT1 and the CNOT2–CNOT3 heterodimer.
Figure 6: Mutagenesis of the NOT1-NOT2-NOT3 interfaces in human and Dm S2 cells.
Figure 7: Effects on mRNA degradation.

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We are grateful to J. Su (Max Planck Institute for Developmental Biology) for providing the cDNA for the CNOT1 fragment (residues 1565–2371) and to E. Wahle (Martin Luther University Halle-Wittenberg) for the kind gift of anti–Dm NOT1–NOT3 antibodies. This work was supported by the Max Planck Society, by grants from the Deutsche Forschungsgemeinschaft (DFG, FOR855 and the Gottfried Wilhelm Leibniz Program awarded to E.I.) and the European Union Seventh Framework Program through a Marie Curie Fellowship to S.J. (FP7, 275343).

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Authors and Affiliations



A.B., Y.C., T.R. and S.J. contributed equally to this work. A.B. and Y.C. purified, crystallized and solved the structures of CNOT3 and CNOT2 oligomers and of Ct NOT1. A.B. and Y.C. cloned, expressed and established the purification protocol for the ternary complex. T.R. purified and crystallized the ternary complex. T.R. and S.J. solved the structure of the ternary complex. A.B., Y.C., T.R., S.J. and O.W. collected and analyzed diffraction data. L.W. performed pulldowns and coimmunoprecipitations in human cells. D.K.-Ö. performed coimmunoprecipitations and functional assays in S2 cells. E.I. conceived of the project. E.I. and O.W. supervised the project. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Oliver Weichenrieder or Elisa Izaurralde.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 CNOT1, CNOT2 and CNOT3 interact via their C-termini.

(a,b) Interaction between GFP-tagged CNOT2 (full-length or fragments) and HA-tagged CNOT1 (a) and CNOT3 (b). GFP-tagged MBP served as a negative control. In all panels, cell lysates were treated with RNase A. (c,d) Interaction between GFP-tagged CNOT3 (full-length or fragments) and HA-tagged CNOT2 (c) and CNOT1 (d). (e,f) Interaction between GFP-tagged CNOT1 (full-length or fragments) and HA-tagged CNOT2 (e) and CNOT3 (f). In panel (f), the interaction with CNOT3 was analyzed in the absence (lanes 2 and 6) or presence (lanes 4 and 8) of CNOT2. (g) Interaction between MBP-CNOT2 fragments and His6-tagged CNOT1 (residues 1565–2371). (h) Interaction between MBP-CNOT2 fragments and GST-CNOT3 (residues 589–753). (i) Interaction between MBP-CNOT2 (residues 344–540) and GST-CNOT3 fragments. (j) Interaction between MBP-CNOT1 (residues 1595–2376) and GST-CNOT2 (residues 344–540) or GST-CNOT3 (residues 589–753).

Supplementary Figure 2 Structure-based sequence alignment of the NOT1 superfamily homology (SH) domain.

Secondary structure elements as determined from the Hs CNOT1 structure are shown above the alignment. Residues conserved in all aligned sequences are shown with a yellow background and residues with >70% similarity are highlighted in orange. Residues interacting with CNOT2 and CNOT3 are indicated by purple and green dots, respectively. Residues involved in CNOT1 subdomain interactions are indicated by blue dots. Residues mutated in this study are marked by red asterisks. Residues substituted in mutant M1 and M5 are indicated. The species abbreviations are as follows: Hs (Homo sapiens), Dm (Drosophila melanogaster), Xt (Xenopus tropicalis), Ce (Caenorhabditis elegans), Ct (Chaetomium thermophilum) and Sc (Sacchromyces cerevisiae).

Supplementary Figure 3 CNOT2 and CNOT3 multimerize in solution.

(a,d) MALLS analysis of the CNOT3 and CNOT2 NOT-box domains. The molecular weight of the protein in solution is indicated in the elution profile. (b) Interfaces between the CNOT3 NOT-box α-helices and the β-barrel. Residues along the interfaces are shown as sticks. (c) Hydrophobic core of the CNOT3 NOT-box β-barrel. (e) Structure of the CNOT2 tetramer as observed in the crystal. The tetramer consists of two pairs of dimers (orange-yellow vs. purple-rose) with a perpendicular orientation to each other. (f) Structure-based sequence alignments of the NOT2 and NOT3 NOT-box domains. Secondary structure elements as determined from the CNOT2 and CNOT3 structures are shown above the alignment. Residues conserved in all aligned sequences are shown with a yellow background, and residues with >70% similarity are highlighted in orange. A NOT2-specific insertion is boxed in purple. Black squares mark residues that form the interface between the NOT-Box N-terminal α-helices and the β-barrel. Gray squares mark residues that form the hydrophobic core of the β-barrel. The species abbreviations are the same as those in Supplementary Fig. 2. (g) Pulldowns showing that CNOT2-C and CNOT3-C exclusively form heterodimers in solution. MBP-tagged CNOT2 and His6-tagged CNOT3 were coexpressed in E. coli. The heterodimers were copurified using MBP pulldown followed by Ni-affinity purification (lanes 1 and 2) or Ni-affinity purification followed by MBP pulldown (lanes 3 and 4).

Supplementary Figure 4 Structure of the NOT module.

(a) Time course of a limited proteolysis of the ternary complex between CNOT1 (residues 1565–2371), CNOT2 (residues 344–540) and CNOT3 (residues 607–753) by thermolysin. CNOT1 is digested into a stable C-terminal fragment corresponding to the SH and an N-terminal fragment (which comigrates with CNOT2), while CNOT2 and CNOT3 remain uncleaved. (b) Time course of a limited proteolysis of CNOT2 (residues 344–540) and CNOT3 (residues 607–753) by thermolysin in the absence or presence of CNOT1 SH (1833–2361). In the absence of CNOT1, CNOT2 and CNOT3 are digested into stable C-terminal fragments corresponding approximately to the NOT-boxes and the CSs, but they remain largely uncleaved in the presence of CNOT1 (c,d) Omit electron density map of the CNOT2 (c) and CNOT3 (d) NAR-Cs folded onto the CNOT1 surface. The electron density (black mesh, 2F0-FC of a composite omit map, calculated with Phenix.AutoBuild) is contoured at 1.0 σ. (e,f) Refined electron density of the CNOT2 (e) and CNOT3 (f) NAR-C regions in stereo view. The views correspond to panels (c) and (d), respectively. The electron density (black mesh, 2F0-FC map) is contoured at 1.0 σ. (g) Superposition of Ct NOT1 (blue) and Hs CNOT1 (gray) showing strong structure conservation. (h) Superposition of the CNOT3 NOT-box domain in isolation (gray) and in the complex (green). (i) Superposition of the NOT-box domains of CNOT2 (purple) and CNOT3 (green) as observed in the ternary complex.

Supplementary Figure 5 Structure-based sequence alignment of the CNOT2 and CNOT3.

Secondary structure elements as determined from the CNOT2 and CNOT3 structure are shown above the alignment. Symbols are as described in Supplementary Fig. 2. Residues interacting with the respective partner proteins are indicated by dots above the alignments and are colored blue for CNOT1, purple for CNOT2 and green for CNOT3. Residues forming the lock are marked with black diamonds, and residues at the junction between the symmetric and asymmetric lobes of the ternary complex are marked with pink triangles. The species abbreviations are the same as those in Supplementary Fig. 2.

Supplementary Figure 6 Surface conservation of the trimeric complex.

(a–e) The conservation scores of the individual residues are represented on the surface by color gradients from light (no conservation) to dark colors (100% conservation) for CNOT1 (blue), CNOT2 (purple) and CNOT3 (green). Conservation scores were calculated based on well-balanced multiple alignments covering all eukaryotic strata. (a) View from the CNOT1 surface that binds CNOT2 and CNOT3 (a) or the opposite surface (b). Conservation of the CNOT1 surface contacting CNOT2 and CNOT3 (c). The view is the same as that shown in Fig. 5a. (d) Conservation of CNOT2 surface residues contacting the CNOT3 connector sequence (CS). The CNOT3 residues involved in the interaction with CNOT2 are shown as sticks. (e) Conservation of CNOT3 surface contacting the CNOT2 connector sequence (CS). The CNOT2 residues involved in CNOT3 binding are shown as sticks.

Supplementary Figure 7 Mutagenesis of the NOT1-NOT2-NOT3 interfaces in human and Dm S2 cells.

(a) Interaction of GFP-CNOT1 (either wild-type or the indicated mutants) with endogenous CNOT3 and HA-CNOT2. (b,c) Interaction of GFP-CNOT1 (either wild-type or the indicated mutants) with HA-CNOT7 in human cells. (d) The decay of the adh-hsp70 mRNA was monitored in control cells (expressing MBP) and in cells expressing NOT1 (either wild-type or mutant). Adh-hsp70 mRNA levels were normalized to the levels of long-lived rp49 mRNA and plotted against time. A representative Northern blot is shown in Fig. 7a. The mRNA half-lives (t1/2) ± standard deviations calculated from the decay curves obtained from three independent experiments are indicated. (e) Interaction of GFP-tagged Dm CAF1 with wild-type NOT1 or NOT1ΔN-SD in S2 cells. F-Luc-GFP served as a negative control. (f,g) The decay of adh-hsp70 mRNA was analyzed in control cells (treated with GFP dsRNA and expressing MBP) or in cells depleted of NOT1 or NOT3 and expressing MBP or the indicated proteins. Northern blots corresponding to the decay curves are shown in Fig. 7c and 7d, respectively. Adh-hsp70 mRNA levels were normalized to the levels of rp49 mRNA and plotted against time. The mRNA half-lives (t1/2) ± standard deviations obtained from three independent experiments are indicated.

Supplementary Figure 8 Original images of gels, western and northern blots used in this study.

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Boland, A., Chen, Y., Raisch, T. et al. Structure and assembly of the NOT module of the human CCR4–NOT complex. Nat Struct Mol Biol 20, 1289–1297 (2013).

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