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. 2007 Dec;27(24):8600-11.
doi: 10.1128/MCB.01506-07. Epub 2007 Oct 8.

A divergent Sm fold in EDC3 proteins mediates DCP1 binding and P-body targeting

Felix Tritschler  1 , Ana Eulalio, Vincent Truffault, Marcus D Hartmann, Sigrun Helms, Steffen Schmidt, Murray Coles, Elisa Izaurralde, Oliver Weichenrieder
Affiliations

A divergent Sm fold in EDC3 proteins mediates DCP1 binding and P-body targeting

Felix Tritschler et al. Mol Cell Biol. 2007 Dec.

Abstract

Members of the (L)Sm (Sm and Sm-like) protein family are found across all kingdoms of life and play crucial roles in RNA metabolism. The P-body component EDC3 (enhancer of decapping 3) is a divergent member of this family that functions in mRNA decapping. EDC3 is composed of a N-terminal LSm domain, a central FDF domain, and a C-terminal YjeF-N domain. We show that this modular architecture enables EDC3 to interact with multiple components of the decapping machinery, including DCP1, DCP2, and Me31B. The LSm domain mediates DCP1 binding and P-body localization. We determined the three-dimensional structures of the LSm domains of Drosophila melanogaster and human EDC3 and show that the domain adopts a divergent Sm fold that lacks the characteristic N-terminal alpha-helix and has a disrupted beta4-strand. This domain remains monomeric in solution and lacks several features that canonical (L)Sm domains require for binding RNA. The structures also revealed a conserved patch of surface residues that are required for the interaction with DCP1 but not for P-body localization. The conservation of surface and of critical structural residues indicates that LSm domains in EDC3 proteins adopt a similar fold that has separable novel functions that are absent in canonical (L)Sm proteins.

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Figures

FIG. 1.
FIG. 1.
EDC3 interacts with DCP1, DCP2, and Me31B. (A) Domain architecture of EDC3. EDC3 orthologs contain three classified globular domains: an LSm domain, an FDF domain, and a YjeF-N-type Rossman fold domain. The LSm and FDF domains are connected by a low-complexity linker region. Numbers above the protein outline represent amino acid positions at fragment boundaries for the D. melanogaster protein. The protein domains sufficient for the localization to P bodies and the interaction with DCP1 (red), DCP2 (orange), and Me31B (blue), as well as for self-association (green), are indicated. (B) Epitope HA-tagged versions of MBP, DCP1, Me31B, or Tral were transiently expressed in S2 cells. Cell lysates were immunoprecipitated using a monoclonal anti-HA antibody. Inputs (10%) and immunoprecipitates (IP) (25%) were analyzed by Western blotting using a polyclonal anti-HA antibody. The presence of endogenous EDC3 in the immunoprecipitates was tested by Western blotting with an anti-EDC3 antibody. (C) Epitope HA-tagged versions of MBP or EDC3 were transiently coexpressed in S2 cells with GFP-DCP1 and/or GFP-DCP2 as indicated. Cell lysates were immunoprecipitated using a monoclonal anti-HA antibody. Inputs (10%) and immunoprecipitates (25%) were analyzed by Western blotting using polyclonal anti-HA and anti-GFP antibodies.
FIG. 2.
FIG. 2.
EDC3 interacts with DCP1, DCP2, and Me31B through specific domains. HA-tagged EDC3 or the indicated EDC3 protein fragments were cotransfected in S2 cells with GFP fusions of DCP1 (A), DCP2 (B), or Me31B (C) as indicated. Cell lysates were immunoprecipitated using a monoclonal anti-HA antibody. HA-tagged MBP served as a negative control. Inputs (10%) and immunoprecipitates (IP) (25%) were analyzed as described for Fig. 1C.
FIG. 3.
FIG. 3.
The LSm domain of EDC3 is necessary and sufficient for P-body localization. Confocal fluorescent micrographs of fixed S2 cells expressing GFP fusions of full-length EDC3 or the indicated protein fragments are shown. Cells were stained with affinity-purified anti-Tral antibodies. The merged images show the Tral signal in red and the GFP signal in green. Bar, 5 μm.
FIG. 4.
FIG. 4.
Structures of the HsEDC3 LSm and DmEDC3 LSm domains. (A) Crystal structure of HsEDC3 LSm. (B) NMR structure of DmEDC3 LSm. (C) Crystal structure of HsSmD3 (PDB ID, 1d3b-A) according to reference . β-strands belonging to the Sm1 motif are colored in red and, β-strands belonging to the Sm2 motif are colored in yellow. Shown in gray are the N-terminal α-helix (which is absent in EDC3), the extended loop L4 from canonical (L)Sm proteins, and the iβ4 insertion that is unique to EDC3. Left, plain views of the open β-barrel with the open side on the top. Center, front views. Right, edge views.
FIG. 5.
FIG. 5.
Structure-based alignment of the EDC3 LSm domain. (A and B) Superposition of the structures of HsEDC3 LSm (blue), DmEDC3 LSm (lime), and HsSmD3 (gray) represented as tubes. (A) Plain view. (B) Front view (stereo) illustrating the different orientations of loops L3 and L4. (C) Alignment of EDC3 LSm domains with HsSmD3. Secondary structure elements are colored as in Fig. 4. The Sm1 and Sm2 motifs are indicated together with the Sm1 and Sm2 signatures (x, any amino acid; h, hydrophobic; p, polar; +, charged). Residues from the hydrophobic core that define the Sm1 and Sm2 motifs are shaded blue; conserved glycines are shaded gray. Red letters indicate invariant amino acids in EDC3 and SmD3. Gray letters indicate amino acids absent in the presented structures. Functionally interesting surface residues are shaded in yellow, and those shown to mediate DCP1 binding are shaded in green. Relevant backbone elements that are structurally conserved in EDC3 are boxed in orange. Abbreviations and accession numbers: Hs, Homo sapiens (gi:18204641 for EDC3 and gi:74007795 for SmD3); Dm, Drosophila melanogaster (gi:24665977); Gg, Gallus gallus (gi:71896289); Xl, Xenopus laevis (gi:82180413); Dr, Danio rerio (gi:125853827); Ag, Anopheles gambiae (AGAP003131); Ce, Caenorhabditis elegans (gi:17508551); Cb, Caenorhabditis briggsae (gi:39595594).
FIG. 6.
FIG. 6.
EDC3 LSm is not incorporated into the LSm1-7 ring. (A) HA-tagged versions of MBP, LSm7, LSm4, and EDC3 were transiently coexpressed in S2 cells with GFP-LSm1. Cell lysates were immunoprecipitated using a monoclonal anti-HA antibody. (B and C) HA-tagged versions of MBP, EDC3, and the indicated EDC3 protein fragments were transiently coexpressed in S2 cells with full-length GFP-EDC3 (B) or an EDC3 protein fragment encompassing residues 331 to 680 (C). Cell lysates were immunoprecipitated using a monoclonal anti-HA antibody. Inputs (10%) and immunoprecipitates (IP) (25%) were analyzed as described for Fig. 1C.
FIG. 7.
FIG. 7.
Localization of functionally relevant residues. (A) Surface representation (plain view) of the structures colored by sequence conservation, comparing eight species (Fig. 5). Color ramp by identity: orange (100%) to white (50% or less). (B and C) Tube representation with Cα carbons as spheres in plain view (B) and edge view (C). Structurally relevant backbone elements (the L4 β-turn and the β5 C-terminal extension in EDC3 versus loops L3 and L5 in canonical (L)Sm/Hfq proteins (represented by HsSmD3) are colored in orange. Functionally interesting surface residues are drawn as sticks with carbons in yellow, oxygens in red, and nitrogens in blue. Side chains (green carbons) known to mediate the interaction of EDC3 with DCP1 are located on a surface on the opposite side from the RNA-binding residues on loops L3 and L5 in canonical (L)Sm/Hfq proteins.
FIG. 8.
FIG. 8.
Mutations of conserved surface residues abolish DCP1 interaction but not P-body localization. (A) HA-tagged MBP, EDC3, or the indicated EDC3 mutants were cotransfected in S2 cells with GFP-DCP1. Cell lysates were immunoprecipitated using a monoclonal anti-HA antibody and analyzed by Western blotting as described for Fig. 1C. (B) Confocal fluorescent micrographs of fixed S2 cells expressing GFP fusions of full-length EDC3 (wt) or EDC3 mutants. Cells were stained with affinity-purified anti-Tral antibodies. The merged images show the Tral signal in red and the GFP signal in green. Bar, 5 μm.
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