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An asymmetric PAN3 dimer recruits a single PAN2 exonuclease to mediate mRNA deadenylation and decay

An Erratum to this article was published on 03 December 2014

Abstract

The PAN2–PAN3 complex functions in general and microRNA-mediated mRNA deadenylation. However, mechanistic insight into PAN2 and its complex with the asymmetric PAN3 dimer is lacking. Here, we describe crystal structures that show that Neurospora crassa PAN2 comprises two independent structural units: a C-terminal catalytic unit and an N-terminal assembly unit that engages in a bipartite interaction with PAN3 dimers. The catalytic unit contains the exonuclease domain in an intimate complex with a potentially modulatory ubiquitin-protease–like domain. The assembly unit contains a WD40 propeller connected to an adaptable linker. The propeller contacts the PAN3 C-terminal domain, whereas the linker reinforces the asymmetry of the PAN3 dimer and prevents the recruitment of a second PAN2 molecule. Functional data indicate an essential role for PAN3 in coordinating PAN2-mediated deadenylation with subsequent steps in mRNA decay, which lead to complete mRNA degradation.

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Figure 1: Structure of the PAN2 catalytic unit.
Figure 2: Structure and interactions of the PAN2 Nuc domain.
Figure 3: PAN3 interacts with the PAN2 assembly unit.
Figure 4: Crystal structure of the NcPAN2 WD40 in complex with the NcPAN3 C-terminal region.
Figure 5: The PAN2 CS1 region specifies the stoichiometry of the PAN2–PAN3 complex.
Figure 6: PAN3 links PAN2-mediated deadenylation with subsequent mRNA degradation.

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Acknowledgements

We are grateful to H. Budde (Izaurralde laboratory) for generating some of the constructs used in this study and the staff of the Swiss Light Source (Villigen, Switzerland) for assistance during data collection. We thank C. Weiler and S. Helms for excellent technical assistance, D.H. Scharf and A.A. Brakhage (Hans-Knoell-Institut, Jena, Germany) for providing the N. crassa cDNA library and C. Romier (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) for the pNYC and pNEA vectors. This study was supported by the Max Planck Society, by the Gottfried Wilhelm Leibniz Program from the Deutsche Forschungsgemeinschaft (awarded to E.I.) and the European Union Seventh Framework Programme through a Marie Curie Fellowship (awarded to S.J.; FP7, no. 275343).

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Authors and Affiliations

Authors

Contributions

S.J. purified, crystallized and solved the structures of the Nc and Ct PAN2–PAN3 complexes and determined the stoichiometry of the Nc complexes. M.C. purified, crystallized and solved the structure of the NcPAN2 catalytic unit. D.P. purified and crystallized the isolated PAN2 WD40 domain. D.P. and S.J. solved the structure of the isolated PAN2 WD40 domain. M.C. and D.P. performed pulldowns and nuclease assays in vitro. S.J., M.C., D.P. and O.W. collected and analyzed diffraction data. D.B. performed tethering and complementation assays in S2 cells. B.L. generated Dm PAN2 and PAN3 mutants and performed coimmunoprecipitations in S2 cells. E.H. generated HsPAN2 fragments and performed coimmunoprecipitations in human cells. E.I. conceived of the project. E.I. and O.W. supervised the project. S.J., M.C., O.W. and E.I. wrote the manuscript. All authors corrected the manuscript.

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Correspondence to Oliver Weichenrieder or Elisa Izaurralde.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Structure-based sequence alignment of the PAN2 USP domain and ScUbp8.

Secondary structure elements are shown above and below the alignment for NcPAN2 and ScUbp8, respectively. Ubp8 residues that contact ubiquitin are boxed with a black dashed line. Residues corresponding to the catalytic triad are highlighted with a red frame. Fully conserved residues are highlighted with a dark purple background and residues with >70% similarity are shown with a light purple background. The side chains coordinating ZnA and ZnB are marked by cyan and yellow diamonds, respectively. Residues involved in main-chain or side-chain interactions are indicated by empty and filled ovals, respectively, and are colored green for contacts with the nuclease domain and pink for contacts with the CS2 region. Species abbreviations are as follows: Nc (Neurospora crassa), Dm (Drosophila melanogaster), Hs (Homo sapiens), Ce (Caenorhabditis elegans) and Sc (Saccharomyces cerevisiae).

Supplementary Figure 2 Sequence alignments of the PAN2 nuclease domain, the PAN2 CS2 region and the PAN3 C-terminal fragment.

(a) Structure-based sequence alignment of the nuclease domain of PAN2 orthologs and HsCAF1. Secondary structure elements for PAN2 and CAF1 are shown above and below the alignment, respectively. Residues conserved in all aligned sequences are highlighted with a dark green background, and residues with >70% similarity are shown with a light green background. Filled and empty ovals denote amino acids interacting via their side- or main-chains, respectively, and are colored pink for CS2 interface residues and purple for USP interface residues. The asterisk indicates residues that are mutated in the catalytically inactive mutant. The catalytic DEDDH residues are highlighted with black boxes; the putative base-stacking residue is highlighted with a red box. Species abbreviations are as in Supplementary Fig. 1. (b) Sequence alignment of the PAN2 CS2 region. Fully conserved residues are highlighted by a dark pink background; a light pink background indicates >70% similarity. Residues involved in main-chain or side-chain interactions are indicated by empty and filled ovals, respectively, and are colored green for contacts with the nuclease domain and purple for contacts with the USP domain. (c) Sequence alignment of the PAN3 CK domain with secondary structure elements of the NcPAN3 CK structure. A dark yellow background denotes fully conserved residues, while light yellow indicates >70% similarity. Asterisks mark residues mutated in this study. Residues binding PAN2 via their main or side chains are indicated by empty or filled blue ovals, respectively. Amino acids in close proximity to the CS1 region are indicated by black ovals. The region that changes conformation upon NcPAN2 WD40 binding is indicated (switch region).

Supplementary Figure 3 PAN2 degrades oligo(A) RNA in vitro in the absence of PAN3.

(a,b) The Nc (a) and DmPAN2 (b) C-terminal fragments (catalytic unit) degrade single-stranded oligo(A)27 RNA (left panels) but not oligo(A)27 DNA (right panels). The catalytic mutants (NcPAN2 D900N and DmPAN2 D1039N, E1041Q) are inactive in this assay. (c) Gel filtration analysis of oligo(A)27 RNA in the presence (solid black line) or absence (dashed black line) of NcPAN2 C-term fragment (D900N mutant). The elution of the protein is shown by a red line. No change in the RNA elution profile was observed in the presence of the protein, indicating that the RNA does not form a stable complex with the protein. (d) The interaction of HA-tagged DmPAN3 (wildtype or mutant) with GFP-tagged DmPAN2 was analyzed in S2 cell lysates by co-immunoprecipitation using anti-GFP antibodies as described in the legend to Fig. 6d. (e) Structure of the PAN2 WD40 domain and orientation of loop L19. Superposition of the two chains from the asymmetric unit of the NcPAN2 WD40 domain structure shown in dark blue and gray for chains A and B, respectively. The movement of loop L19 is indicated. (f) Superposition of the WD40 domain of NcPAN2 as observed in the complex (light blue) and in isolation (chain B, gray). The conformational change of loop L19 is indicated.

Supplementary Figure 4 Structure-based sequence alignment of the PAN2 WD40 domain and CS1 region.

(a) Structure-based sequence alignment of the WD40 domain of PAN2 orthologs and HsGNB1 (PDB ID: 1GOT). Secondary structure elements are shown above and below the alignment for NcPAN2 and HsGNB1, respectively. The eponymous WD-repeats of GBN1 are marked by black frames. Fully conserved residues are highlighted in dark blue; residues with >70% sequence similarity are highlighted in light blue. Residues contacting PAN3 with their side chains or main chains are indicated by filled and empty orange ovals, respectively. Amino acids binding the MES molecule or loop L19' of the symmetry-related PAN2' are marked by red and cyan ovals, respectively. Residues mutated in this study are marked by black asterisks. Loop L8, which is deleted in the Δloop mutant, is indicated by a black bracket. Species are abbreviated as in Supplementary Fig. 1; Ct (Chaetomium thermophilum). (b) Sequence alignment of the PAN2 CS1 region. Colors and species abbreviations are as described in a. Residues at which the stoichiometry of the PAN2-PAN3 complex changes are indicated by a black vertical lines and labeled with the corresponding PAN2:PAN3 ratios.

Supplementary Figure 5 Assembly and stoichiometry of the PAN2–PAN3 complex.

(a) Superposition of the crystallographic dimer formed in the NcPAN2 WD40–PAN3 CK complex structure onto the isolated PAN3 PKC homodimer structure (light gray, PDB ID: 4BWX). The two symmetry-related PAN2 molecules are colored blue and teal; the corresponding PAN3 protomers are shown in yellow and orange. The crystallographic 2-fold axis is indicated by a dashed line. (b) Superposition of the NcPAN3 CK domain from the isolated PAN3 homodimer structure (dark gray, PDB ID: 4BWX) with the PAN2 WD40–PAN3 CK complex (blue and yellow). The black frame indicates the view shown in c. (c) Close-up of the superposition shown in b. Helix α2 of PAN3 CK is extended by a full turn in the complex. The side chains in the switch region that change orientation are labeled and shown as sticks (in orange for the complex and in dark gray for the isolated PAN3 structure). The arrow indicates the distance between the two conformations of W566. (d) Superposition of the NcPAN2 WD40-CS1–PAN3 PKC complex with the isolated NcPAN3 homodimer (PDB ID: 4BWX). The α-helices are shown as tubes. In the complex, the two PAN2 WD40 domains are shown in blue and cyan, and the two PAN3 chains are shown in yellow and orange. The corresponding two protomers of the isolated PAN3 dimer are shown in light and dark gray. The arrow indicates the orientation of the view shown in f. (e) View from above the PAN3 dimer, highlighting the movement of the CK domains relative to the PK domains along the central coiled coil axis. The angles between the two corresponding CK helices and the distance between the two corresponding loops are indicated. (f) Close-up view of the contacts between the PK and CK domains and the coiled coil on the closed side of the PAN3 homodimer in the PAN2–PAN3 complex (left panel) and in isolated PAN3 (right panel). The residues involved in the hydrogen-bond network are shown as sticks, and hydrogen bonds are represented as dotted lines. (g) Binding surface of the CS1 region (red dashed line) on the open half of the PAN2 WD40- CS1–PAN3 PKC complex colored as in Fig. 5a. The electron density of the 2Fo-Fc map contoured at 1σ is shown as a black mesh. The residues in proximity to the extra density are highlighted as sticks; the residues mutated in this study are underlined.

Supplementary Figure 6 PAN2 tethering causes accumulation of deadenylated mRNA decay intermediates.

(a,b) Western blot analysis showing the equivalent expression of the λN-HA-tagged proteins used in the tethering assays shown in Fig. 6e and Fig. 6f, respectively. (c) Tethering assays using the F-Luc-5BoxB reporter and the λN-HA-PAN2 (wild-type or ΔWD40 mutant) in the absence or presence of overexpressed PAN3. A plasmid expressing R-Luc served as a transfection control. The panel shows a Northern blot analysis of representative RNA samples. Note that in this experiment, the amount of transfected plasmid expressing PAN2 (wild-type or mutant) was 5-fold higher than in the experiment shown in Fig. 6e. The position of the deadenylated mRNA (A0) is indicated on the right. (d) RNA samples corresponding to lanes 1 and 2 of the Northern blot shown in c were treated with RNase H in the presence of oligo(dT). The smear observed when PAN2 was tethered collapsed to a single band after oligo(dT)-directed RNase H cleavage (lane 4 vs. 2); this band commigrated with the reporter in cells expressing λN-HA after RNase H treatment (lane 3) and with the deadenylated reporter loaded as the control (lane 5). As a control for the RNase H treatment, the mobility of the rp49 mRNA increased after oligo(dT)-directed RNase H cleavage (lanes 3 and 4 vs. 5). (e) Decay of the F-Luc-5BoxB mRNA reporter in S2 cells expressing either λN-HA peptide or λN-HA-tagged PAN2 or PAN3. The cells were treated with actinomycin D (5 μg/ml) and harvested at the indicated time points. The long-lived rp49 mRNA served as a loading control. In cells expressing PAN3, deadenylated mRNA decay intermediates are observed after 15 min, whereas in cells expressing PAN2, deadenylation is slowed. (f) The expression of the proteins in the experiment shown in e was analyzed by western blotting. (g) Western blot analysis showing expression of the PAN2 proteins used in the complementation assay shown in Fig. 6g. (h–m) Electron density maps (stereo). (h) Final 2Fo-Fc map of the NcPAN2 catalytic unit contoured at 1σ at the position of the USP-nuclease interface. Helices α13 (nuclease) and α3 (USP) are shown. (i) Simulated annealing composite omit map for the CS2 region in the catalytic unit of NcPAN2, contoured at 1σ. (j) Final 2Fo-Fc density map of the isolated NcPAN2 WD40 domain, contoured at 1σ. The β sheets β5A and β5B are shown. (k) Final 2Fo-Fc map of the NcPAN2 WD40 domain bound to the NcPAN3 C-terminus, contoured at 1σ at the position of the complex interface. The β sheets β1D (PAN2) and βa (PAN3) are shown. (l) Final 2Fo-Fc map of the structure of NcPAN2 WD40-CS1 bound to the NcPAN3 PKC domain, contoured at 1σ at the position of the kink in the PAN3 coiled coil. (m) Final 2Fo-Fc map of CtPAN2 WD40-CS1 bound to the CtPAN3 PKC domain, contoured at 1σ around the CS1 peptide-binding pocket at the interface between CK-1 and CK-2.

Supplementary Figure 7 Original images of gels, western and northern blots used in this study.

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Jonas, S., Christie, M., Peter, D. et al. An asymmetric PAN3 dimer recruits a single PAN2 exonuclease to mediate mRNA deadenylation and decay. Nat Struct Mol Biol 21, 599–608 (2014). https://doi.org/10.1038/nsmb.2837

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