Structure and RNA-binding properties of the Not1–Not2–Not5 module of the yeast Ccr4–Not complex

Abstract

The Ccr4–Not complex is involved in several aspects of gene expression, including mRNA decay, translational repression and transcription. We determined the 2.8-Å-resolution crystal structure of a 120-kDa core complex of the Saccharomyces cerevisiae Not module comprising the C-terminal arm of Not1, Not2 and Not5. Not1 is a HEAT-repeat scaffold. Not2 and Not5 have extended regions that wrap around Not1 and around their globular domains, the Not boxes. The Not boxes resemble Sm folds and interact with each other with a noncanonical dimerization surface. Disruption of the interactions within the ternary complex has severe effects on growth in vivo. The ternary complex forms a composite surface that binds poly(U) RNA in vitro, with a site at the Not5 Not box. The results suggest that the Not module forms a versatile platform for macromolecular interactions.

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Figure 1: Structure of a yeast Not1–Not2–Not5 core complex.
Figure 2: Not1 interacts with extended regions of Not2 and Not5.
Figure 3: The globular domains of Not2 and Not5 contain divergent Sm folds.
Figure 4: Analysis of mutants targeting interaction surfaces of the Not module.
Figure 5: Not1c–Not2–Not5c binds poly(U) RNA.

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Acknowledgements

We thank the MPI Biochemistry Crystallization Facility and Core Facility. We thank F. Bonneau and C. Basquin (MPI Biochemistry) for help with biochemical assays and for Supplementary Figure 5b; F. Lacroute, F. Gabriel and M.C. Daugeron (Centre de Génétique Moléculaire) for yeast strains; the staff of the Swiss Light Source synchrotron for assistance during data collection; and members of our laboratories for discussions and for critical reading of the manuscript. E.C. acknowledges support from the Max Planck Gesellschaft, the European Research Council (ERC Advanced Investigator Grant 294371, Marie Curie Initial Training Network RNPnet 289007) and the Deutsche Forschungsgemeinschaft (DFG SFB646, SFB1035, GRK1721, FOR1680, CIPSM). B.S. acknowledges support from the Centre Européen de Recherche en Biologie et en Médecine (CERBM)-IGBMC, the CNRS and the Ligue Contre le Cancer (Equipe Labellisée 2011).

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V.B. and J.B. carried out the structure determination and the in vitro experiments; V.R. carried out the in vivo experiments; K.S. and H.U. carried out the MS analysis; E.C. and B.S. supervised the project; and E.C., V.B. and B.S. wrote the manuscript.

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Correspondence to Elena Conti.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of the core of the S. cerevisiae Not1-Not2-Not5 interaction.

The 12% SDS PAGE gel shows in lane 1 the larger complex that we initially purified (Not1 starting at residue 1348, Not2 f.l. and Not5 f.l.). Lane 2 shows the result of the limited proteolysis of the complex in lane 1 with elastase. Lane 3 shows the protease alone as a control. Lane 4 shows the complex reconstituted with the minimal interacting regions of Not1, Not2 and Not5 that yielded diffracting crystals (Not1c, Not2 and Not5c).

Supplementary Figure 2 Structure-based sequence alignments of Not1c, Not2 and Not5c.

The sequence alignments include the polypeptides of S. cerevisiae (Sc) Not1, Not2 and Not5 we crystallized in a complex and their orthologues from H. sapiens (Hs) and D. melanogaster (Dm). Not5Sc is a homologue of Not3. Secondary structure elements are shown above the sequences and colored in yellow for Not1 (a), magenta for Not2 (b) and green for Not5 (c). Straight lines refer to extended regions and dotted lines refer to disordered regions in the structure of the S. cerevisiae complex. Sequence conservation is highlighted in shades of gray.

Supplementary Figure 3 The HEAT and Sm folds of Not1–Not2–Not5.

(a) Comparison of the HEAT–repeat architecture in the C–terminal arm of Not1 (on the right) and the N–terminal arm of Not1 (on the left, PDB code 4B8B1). The MIF4G–like folds are shown in gray. The longer HEAT–repeat units perpendicular to the MIF4G–like folds are shown in yellow and red for the C–terminal and N-terminal arms, respectively. (b) Dimerization properties of Not–box domains. The subcomplex of SmF and SmE (PDB code 2Y9A2) is shown on the left in gray. The β4 strand of one monomer (SmE) interacts with strand β5 of the other monomer (SmF). On the right, dimerization of Not2–Not5 leaves strand β4 exposed to solvent. (c) Lattice contacts are reminiscent of Sm–Sm interactions. In the upper panel, the loop between strands β2 and β3 of a Not2 molecule (in magenta) has an extended conformation and interacts both with the β4 strand of a symmetry–related Not5 molecule (in cyan). In the lower panel, the β4 strand of a Not2 molecule (in magenta) interacts with the β–hairpin of a symmetry–related Not1 molecule (in orange).

Supplementary Figure 4 In vivo interactions of Not proteins.

(a) Immunoprecipitation of Not1 from yeast strains harbouring a TAP tagged Not1 wildtype (WT) or indicated mutants and carrying tagged chromosomal variant of Not2 and Not3 (BSY1230), or Not2 and Pop2/Caf1 (BSY1231). Co–immunoprecipitation of Not2–HA, Not3–VSV or Pop2 VSV was assayed by western blotting. As a control, the presence of the tagged protein in the starting extracts was also assayed. (b) Immunoprecipitation of Not3 from yeast strains carrying tagged chromosomal variant of Not3, Pop2/Caf1 and Not4 (BSY1240), or Not3, Caf1/Pop2 and Not2 (BSY1242). Co-immunoprecipitation of Not2–HA or Not4–HA was assayed by western blotting. As a control, the level of the tagged protein in the starting extracts was also assayed.

Supplementary Figure 5 Protein and RNA interactions at the Not boxes.

(a) Surface features of the Not2 and Not5 Not–boxes. On the left is the structure of Not2, showing Arg165 (putative ADA2–binding residue) as well as positively–charged residues at a similar position as in Not5. In the central panel is the Not–box of Not5, in the same orientation, showing the uridine–crosslinked residue Cys546 as well as the surrounding positively–charged residues (as in Fig. 5d). On the right is the structure of RNA–bound SmE (U4 snRNP, PDB code 2Y9A2) oriented in a similar view as the structures in the left and central panels (after optimal superimposition), showing a bound uridine nucleotide (in black). (b) Quantification of the RNA–binding properties of Not1c–Not2–Not5c by fluorescence anisotropy. The Kd of the Not1c–Not2–Not5c complex to 6–FAM–labeled U15 RNA under these conditions was found to be 9.47±0.95 μM.

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Bhaskar, V., Roudko, V., Basquin, J. et al. Structure and RNA-binding properties of the Not1–Not2–Not5 module of the yeast Ccr4–Not complex. Nat Struct Mol Biol 20, 1281–1288 (2013). https://doi.org/10.1038/nsmb.2686

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