Molecular architecture of LSM 14 interactions involved in the assembly of mRNA silencing complexes
2018; Springer Nature; Volume: 37; Issue: 7 Linguagem: Inglês
10.15252/embj.201797869
ISSN1460-2075
AutoresTobias Brandmann, Hana Fakim, Zoya Padamsi, Ji‐Young Youn, Anne‐Claude Gingras, Marc R. Fabian, Martin Jínek,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle6 March 2018free access Source DataTransparent process Molecular architecture of LSM14 interactions involved in the assembly of mRNA silencing complexes Tobias Brandmann Department of Biochemistry, University of Zurich, Zurich, Switzerland Search for more papers by this author Hana Fakim orcid.org/0000-0002-9751-7908 Department of Oncology, McGill University, Montreal, QC, Canada Segal Cancer Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Zoya Padamsi Department of Oncology, McGill University, Montreal, QC, Canada Segal Cancer Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Ji-Young Youn Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Anne-Claude Gingras Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Marc R Fabian Corresponding Author [email protected] orcid.org/0000-0003-3700-7604 Department of Oncology, McGill University, Montreal, QC, Canada Segal Cancer Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Martin Jinek Corresponding Author [email protected] orcid.org/0000-0002-7601-210X Department of Biochemistry, University of Zurich, Zurich, Switzerland Search for more papers by this author Tobias Brandmann Department of Biochemistry, University of Zurich, Zurich, Switzerland Search for more papers by this author Hana Fakim orcid.org/0000-0002-9751-7908 Department of Oncology, McGill University, Montreal, QC, Canada Segal Cancer Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Zoya Padamsi Department of Oncology, McGill University, Montreal, QC, Canada Segal Cancer Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Ji-Young Youn Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Anne-Claude Gingras Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Marc R Fabian Corresponding Author [email protected] orcid.org/0000-0003-3700-7604 Department of Oncology, McGill University, Montreal, QC, Canada Segal Cancer Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Martin Jinek Corresponding Author [email protected] orcid.org/0000-0002-7601-210X Department of Biochemistry, University of Zurich, Zurich, Switzerland Search for more papers by this author Author Information Tobias Brandmann1,‡, Hana Fakim2,3,‡, Zoya Padamsi2,3, Ji-Young Youn4,5, Anne-Claude Gingras4,5, Marc R Fabian *,2,3 and Martin Jinek *,1 1Department of Biochemistry, University of Zurich, Zurich, Switzerland 2Department of Oncology, McGill University, Montreal, QC, Canada 3Segal Cancer Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada 4Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada 5Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +1 514 340 8222; E-mail: [email protected] *Corresponding author. Tel: +41 44 635 5572; E-mail: [email protected] EMBO J (2018)37:e97869https://doi.org/10.15252/embj.201797869 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The LSM domain-containing protein LSM14/Rap55 plays a role in mRNA decapping, translational repression, and RNA granule (P-body) assembly. How LSM14 interacts with the mRNA silencing machinery, including the eIF4E-binding protein 4E-T and the DEAD-box helicase DDX6, is poorly understood. Here we report the crystal structure of the LSM domain of LSM14 bound to a highly conserved C-terminal fragment of 4E-T. The 4E-T C-terminus forms a bi-partite motif that wraps around the N-terminal LSM domain of LSM14. We also determined the crystal structure of LSM14 bound to the C-terminal RecA-like domain of DDX6. LSM14 binds DDX6 via a unique non-contiguous motif with distinct directionality as compared to other DDX6-interacting proteins. Together with mutational and proteomic studies, the LSM14-DDX6 structure reveals that LSM14 has adopted a divergent mode of binding DDX6 in order to support the formation of mRNA silencing complexes and P-body assembly. Synopsis This study uses structural, proteomic and functional approaches to elucidate how the translational repressor LSM14 interfaces with a number of mRNA silencing factors, including 4E-T, DDX6 and EDC4. Structures of LSM14-4E-T and LSM14-DDX6 complexes highlight the role of short linear interacting motifs (SLiMs) in organizing multiprotein complexes in mRNA metabolism. A conserved C-terminal motif of 4E-T forms a bipartite motif that wraps around the LSM14 N-terminal LSM domain. LSM14 uses non-contiguous motifs to bind DDX6 RecA-C domain with reverse directionality, as compared to EDC3, PATL1 and 4E-T binding. Proteomic analysis of LSM14-interacting proteins identifies EDC4 as a novel interactor. The evolutionarily conserved LSM14 FFD motif is required for LSM14 to associate with EDC4. Introduction Translational repression and mRNA turnover are central processes in post-transcriptional regulation of eukaryotic gene expression. Shortening of the 3′ poly(A) tail of eukaryotic mRNAs is the initial and rate-limiting step of mRNA turnover and is carried out by the CCR4-NOT deadenylase complex (Chen & Shyu, 2011). The deadenylated 3′-terminus subsequently serves as a binding platform for numerous protein factors promoting translational repression of the mRNA and facilitating the irreversible removal of the 5′-terminal cap structure by the Dcp1–Dcp2 decapping complex. Ultimately, decapped mRNAs are degraded by the 5′–3′ exonuclease XRN1 and removed from the translational pool (Chowdhury & Tharun, 2009). The recruitment of the decapping complex to the 5′-terminal cap structure is orchestrated by an intricate, dynamic network of protein–protein interactions involving various decapping factors and translational repressors. These include the LSM1-7 complex, PATL1, enhancer of decapping 3 (EDC3), EDC4, the eIF4E-binding protein 4E-T, Like Sm14 (LSM14), and the DEAD-box RNA helicase DDX6 (Arribas-Layton et al, 2013; Nishimura et al, 2015). Most of the translational repressors and decapping activators have a modular architecture in which conserved globular protein domains are embedded within low-complexity sequence stretches that are predicted to be unstructured (Jonas & Izaurralde, 2013). Intriguingly however, these intrinsically disordered protein regions often contain highly conserved short linear sequence motifs (SLiMs) that mediate protein complex assembly, as well as the formation of ribonucleoprotein (RNP) granules such as processing bodies (P-bodies; Eulalio et al, 2007a). One of the key components in the assembly of the mammalian gene silencing complexes is LSM14 (also known as RAP55), a member of the like-Sm (LSM) protein family that is highly conserved from yeast to humans (Marnef et al, 2009). The N-terminal LSM domain of LSM14 is a common fold among decapping factors and is also found in the LSM1-7 proteins and EDC3. However, in contrast to LSM1-7 proteins, whose LSM domains interact to form a heteroheptameric ring-like structure with RNA-binding properties, the LSM domains in LSM14 and EDC3 diverge functionally and serve as interaction platforms for other protein factors (Tritschler et al, 2007, 2008). In addition to its N-terminal LSM domain, LSM14 also contains a central phenylalanine–aspartate–phenylalanine (FDF) motif, which interacts with DDX6, and C-terminal RGG motifs that are methylated by the protein arginine methyltransferase 1 (PRMT1; Matsumoto et al, 2012). Additionally, two highly conserved sequence stretches downstream of the FDF motif, the FFD and TFG motifs, have been identified (Fig 1A; Albrecht & Lengauer, 2004). The roles of these latter motifs in LSM14 function, however, remain to be established. Figure 1. Structure of the N-terminal LSM domain of LSM14 in complex with a conserved C-terminal fragment of 4E-T Schematic diagram of LSM14. The N-terminal LSM domain and FDF, FFD, and TFG motifs are indicated (not to scale). Schematic diagram of full-length 4E-T and 4E-T fragments used for MBP pull-down experiments in Fig 2B. Coordinates are indicated on the left and right of each fragment. The indicated regions (not to scale) are as follows: eIF4E, binding site for eIF4E; CHD, cup homology domain; LSM14, LSM14-interacting site. Crystal structure of the N-terminal LSM domain of human LSM14 in complex with a conserved C-terminal 4E-T fragment. The diagram depicts the likely solution structure obtained by combining two fragments of 4E-TC that undergo crystallization-induced domain swapping, as indicated by dashed line. Left: Cartoon representation of the complex. Right: LSM domain of LSM14 shown in surface representation and 4E-TC residues critical for complex formation are shown as sticks. Secondary structure elements are labeled according to (D) and (E). Sequence alignment of conserved amino acids within the LSM domains of human (Hs) LSM14, Drosophila melanogaster (Dm) TraI, yeast (Sc) SCD6, Caenorhabditis elegans (Ce) CAR1, and human (Hs) EDC3. Secondary structure elements with corresponding numbering are indicated above the sequence. Sequence alignment of conserved amino acids within the C-terminal motifs of human (Hs), Xenopus laevis (Xl), zebrafish (Dr), and D. melanogaster (Dm) 4E-T proteins. Secondary structure elements with corresponding numbering are indicated above the sequence. Download figure Download PowerPoint The yeast LSM14 homolog SCD6 has been reported to enhance mRNA decapping in vitro. In addition, SCD6 and the Xenopus homolog, xRAP55a, have been reported to repress translation (Yang et al, 2006; Fromm et al, 2012; Rajyaguru et al, 2012), and SCD6 has been shown to bind eIF4G via its methylated RGG motifs (Rajyaguru et al, 2012). Whether this interaction is conserved in higher eukaryotes, however, is not known. Metazoan LSM14 orthologs interact with a number of multiple mRNA silencing factors, including 4E-T and DDX6, which are involved in translational repression and decay mRNAs targeted by the CCR4-NOT complex (Coller & Parker, 2005; Tritschler et al, 2009; Carroll et al, 2011; Matsumoto et al, 2012; Sweet et al, 2012; Nishimura et al, 2015). We recently reported that the LSM domain of LSM14 directly interacts with two independent regions of 4E-T (middle-motif: residues 335–490; C-terminal motif: residues 948–985) via its LSM domain (Nishimura et al, 2015). DDX6 serves as a protein interaction hub for several translational repressors and enhancers of mRNA decapping, such as LSM14, 4E-T, PATL1, and EDC3 (Tritschler et al, 2008, 2009; Sharif et al, 2013; Ozgur et al, 2015). LSM14 employs its FDF motif to interact with the C-terminal RecA domain of DDX6 (Tritschler et al, 2009). Notwithstanding these data, it is currently unclear as to exactly how LSM14 interfaces with both 4E-T and DDX6. Here we set out to obtain structural and biochemical insights into how human LSM14 interfaces with the mRNA silencing machinery. To this end, we solved the crystal structures of LSM14 bound to 4E-T and to DDX6. Our study shows that the evolutionarily conserved C-terminus of 4E-T wraps around the LSM domain of LSM14. Furthermore, we show that LSM14 employs both its FDF and TFG motifs to bind the C-terminal RecA domain of DDX6 via a bipartite interaction. We show that both interactions are underpinned by extensive hydrophobic interactions between evolutionarily conserved sequence motifs and common structural domains. Finally, we reveal that the unique architecture of the LSM14–DDX6 interaction serves to present the highly conserved FFD motif of LSM14 as an additional recruitment platform for the decapping activator EDC4. Results Extensive hydrophobic interactions anchor 4E-T to the N-terminal LSM domain of LSM14 We recently showed that the N-terminal LSM domain of LSM14 directly interacts with two conserved sites in the eIF4E-binding protein 4E-T: a central region spanning residues 335–490 and a C-terminal region comprising residues 948–985 (Fig EV1A; Nishimura et al, 2015). To gain structural insights into these interactions, we determined the crystal structure of the minimal LSM14–4E-T complex composed of the N-terminal LSM domain of human LSM14 (LSM14LSM; residues 1–84) and a C-terminal 4E-T fragment (4E-TC; residues 954–985; Fig 1A and B). The structure was solved at a resolution of 2.6 Å by single-wavelength anomalous diffraction (SAD) using a selenomethionine-substituted 4E-TC construct in which an additional methionine residue (V963M) was introduced by site-directed mutagenesis (Table 1). The crystal structure reveals a tetrameric complex with 2:2 stoichiometry in which each LSM14 is simultaneously bound by two 4E-T protomers (Fig EV1B). To ascertain whether the observed quaternary structure occurs in solution, we made use of multi-angle light scattering (MALS) to determine the molecular weight of the LSM14LSM–4E-TC complex. MALS analysis indicated a combined molecular weight of 12.9 kDa (Fig EV1C) corresponding to a 1:1 complex, which suggests that the 2:2 tetramer is a result of crystallization-induced domain swapping. To reconstruct the likely solution structure of the LSM14LSM–4E-TC complex, we combined the fragments from the two 4E-TC molecules (corresponding to residues 954–973 and 974–985) that contact each LSM14LSM molecule in a single composite model (Fig 1C). Click here to expand this figure. Figure EV1. Structural analysis of the LSM14LSM–4E-TC complex Sequence alignment of conserved amino acids within the C-terminal and middle motifs of human (Hs), Xenopus laevis (Xl), zebrafish (Dr), and Drosophila melanogaster (Dm) 4E-T proteins. Crystal structure of the N-terminal LSM domain of LSM14 in complex with a conserved C-terminal 4E-T fragment reveals a tetrameric complex with 2:2 stoichiometry. Two perpendicular views shown in cartoon representation. Each LSM14 molecule (blue) is simultaneously bound by two 4E-T molecules (green). Analysis of purified LSM14LSM–4E-TC complex by size exclusion chromatography coupled to MALS. The molar mass distribution (left ordinate, black line) indicates a molar mass of 12.9 kDa, which corresponds to a 1:1 complex in solution. Structural comparison of the LSM domains of human LSM14 (blue), Drosophila TraI (yellow), and human EDC3 (cyan). The structures were superimposed using the DALI server (Holm & Laakso, 2016) and are shown in identical orientation. Structural comparison of the LSM domains of human LSM14 (blue), human EDC3 (cyan), and human SmD3 (gray, PDB ID: 1D3B-A). The structures were superimposed using the DALI server (Holm & Laakso, 2016) and are shown in identical orientation. ITC binding isotherms of 500 μM 4E-TC peptide (left) and a W958A mutant (right) titrated into 50 μM LSM14LSM. Data were fitted to a single-binding site model, and the dissociation constant (Kd) was determined based on three independent experiments. Data analysis, fitting, and Kd calculation were performed using Origin7. Representative Western blot analysis of expressed λNHA-tagged proteins showing comparable expression of the respective proteins. Proteins were resolved by SDS–PAGE and probed with HA and actin antibodies. Download figure Download PowerPoint Table 1. Crystallographic data collection and refinement statistics Dataset DDX6C–LSM14FDF-TFG LSM14LSM–4E-TC Native SeMet SAD X-ray source SLS X06DA (PXIII) SLS X06DA (PXIII) Space group P6 1 22 P4212 Cell dimensions a, b, c (Å) 92.15, 92.15, 149.90 64.89, 64.89, 61.67 α, β, γ (°) 90, 90, 120 90, 90, 90 Wavelength (Å) 0.979340 0.979090 Resolution (Å)aa Values in parentheses denote highest resolution shell. 46.07–3.03 (3.14–3.03) 45.88–2.62 (2.72–2.62) R merge aa Values in parentheses denote highest resolution shell. 0.108 (1.118) 0.090 (0.657) CC1/2aa Values in parentheses denote highest resolution shell. 0.999 (0.875) 1.000 (0.982) I/σIaa Values in parentheses denote highest resolution shell. 31.5 (3.5) 54.4 (8.6) Observationsaa Values in parentheses denote highest resolution shell. 147,658 (15,066) 212,500 (21,151) Unique reflectionsaa Values in parentheses denote highest resolution shell. 7,824 (762) 4,287 (418) Multiplicityaa Values in parentheses denote highest resolution shell. 18.9 (19.7) 49.6 (50.1) Completeness (%)aa Values in parentheses denote highest resolution shell. 99.8 (99.4) 99.7 (99.1) Refinement Resolution (Å) 46.07–3.03 45.88–2.62 No. reflections 7,824 (762) 4,279 (418) R work /R free 0.204/0.242 0.221/0.245 No. atoms Protein 1,734 844 Ligands 10 Water 1 B-factors Mean 71.5 68.0 Protein 71.3 68.0 Ligands 102.8 Water 46.5 R.m.s. deviations Bond lengths (Å) 0.003 0.003 Bond angles (°) 0.48 0.77 Ramachandran plot % Favored 97.1 97.1 % Allowed 2.9 2.9 % Outliers 0.0 0.0 a Values in parentheses denote highest resolution shell. The N-terminal LSM domain of LSM14 adopts a typical LSM domain fold comprising an open five-stranded β-barrel-like structure similar to previously reported structures of the LSM domains of the human enhancer of decapping EDC3 and Drosophila melanogaster TraI, an LSM14 homolog (Figs 1C–E and EV1D; Tritschler et al, 2007; Fromm et al, 2012), superimposing with root-mean-square deviations (RMSDs) of 1.7 and 2.4 Å, respectively (over 72 and 88 Cα atoms). Canonical SM and LSM proteins such as SmD3 and LSM1-7 oligomerize via an antiparallel interaction of the β-strands β4 and β5 (Kambach et al, 1999; Tritschler et al, 2007; Jonas & Izaurralde, 2013). In contrast, both LSM14 and EDC3 are monomeric and lack an N-terminal α-helix, a hallmark of canonical LSM proteins that contributes to their assembly in ring-shaped oligomers (Figs 1C, and EV1D and E). These features enable LSM14 and EDC3 to function as standalone protein interaction platforms, instead of forming ring-like heptamers with RNA-binding interfaces. The 4E-TC fragment wraps around the LSM domain in an extensive interaction that involves more than 70% of its residues, burying ~1,300 Å2 of solvent-accessible surface area. The N-terminal portion of 4E-TC (residues 954–974) binds in a mostly extended conformation, while the C-terminal part (residues 975–985) forms a short beta strand followed by an α-helical motif. The LSM14LSM–4E-TC interaction is mediated mainly by the insertion of conserved hydrophobic residues from 4E-TC into two hydrophobic cavities found at opposite ends of the LSM domain β-barrel (Figs 1C–E and 2A). The conserved residues Leu9554E-T, Trp9584E-T, and Phe9594E-T in the extended N-terminal part of 4E-TC occupy a cavity lined with Leu26LSM14, Ile29LSM14, Val36LSM14, Ile72LSM14, and Leu75LSM14. This region is additionally anchored by van der Waals contacts between Met9714E-T and Thr35LSM14, Ala37LSM14 and Ile66LSM14 (Fig 2A). The C-terminal helical element of 4E-TC is fixed on the LSM14LSM surface by interactions of Val9784E-T and Leu9814E-T with a hydrophobic patch formed by Tyr22LSM14, Phe62LSM14, Ile65LSM14, and Phe67LSM14 (Fig 2A). This interaction is further reinforced by hydrogen-bonding interactions between Glu9824E-T and Ser16LSM14 and Tyr22LSM14. The N- and C-terminal hydrophobic motifs in 4E-TC are connected by a short β-strand (residues Ile9764E-T–Val9784E-T) that runs parallel to LSM14 strand β4, forming hydrogen-bonding interactions to the polypeptide backbone of residues Tyr64LSM14–Ile66LSM14. Additional interactions involving Met9634E-T and Leu9684E-T with Leu26LSM14 and Ile29LSM14 stabilize a sharp turn in 4E-TC around the surface of LSM14LSM that connects the two main interacting regions. Figure 2. LSM14–4E-T complex formation is mediated by extensive hydrophobic interactions Close-up views of the interface between the LSM14 domain (light blue) and 4E-T peptide (green). Interacting side chains of LSM14 and 4E-T are shown as sticks and labeled by single letter code. Dashed lines indicate hydrogen-bonding interactions, and secondary structures elements are numbered as in Fig 1D and E. SDS–PAGE analysis of input (lower image) and MBP pull-down experiments (upper image) using recombinant MBP-LSM14 protein (MBP-LSM14LSM, residues 1–84) immobilized on amylose-beads and incubated with recombinant GST-tagged C-terminal 4E-T fragments. MW, molecular weight markers. SDS–PAGE analysis of input (lower image) and pull-down experiments (upper image) using recombinant MBP-LSM14 protein (MBP-LSM14LSM, residues 1–84) immobilized on amylose-beads and incubated with recombinant wild-type or mutant GST-tagged C-terminal 4E-T (4E-TC, residues 954–985) protein constructs. MUT5, 4E-TC L955A/W958A/F959A/V978A/L981A. SDS–PAGE analysis of pull-down experiments using recombinant wild-type or mutant MBP-LSM14LSM constructs immobilized on amylose-beads and incubated with recombinant wild-type GST-tagged 4E-TC fragment. MBP-LSM14LSM variants are indicated above the gel. Immunoprecipitation (IP) of wild-type and mutant FLAG-LSM14A proteins from benzonase-treated HeLa cell extracts using anti-FLAG antibody. Immunoprecipitated complexes were separated by SDS–PAGE and probed with antibodies against the indicated proteins. Luciferase reporter assays using HeLa cells co-transfected with plasmids coding for RL-5BoxB and FL reporters and plasmids expressing λNHA fusions of the wild-type (WT) LSM14LSM domain or a Y22E/I29E mutant. A plasmid expressing the silencing domain of GW182 was used as a positive control (Zipprich et al, 2009; Fabian et al, 2011). λNHA-LacZ served as a negative control. Activity of RL was normalized to expression of FL. Values represent relative RL activities normalized to FL, with expression in the presence of λNHA-LacZ set as 100%. Values represent means (±SEM) from triplicate experiments. Source data are available online for this figure. Source Data for Figure 2 [embj201797869-sup-0002-SDataFig2.jpg] Download figure Download PowerPoint The structure of the LSM14LSM–4E-TC complex reveals that the interaction between LSM14 and 4E-T relies on multiple molecular contacts converging on the two hydrophobic interaction hotspots on the surface of the LSM domain of LSM14. This is supported by the observation that both the N- and C-terminal portions of 4E-TC are required for stable interaction between LSM14LSM and 4E-TC in vitro (Fig 2B). To further delineate the contributions of individual amino acid residues, we tested the binding of wild-type and mutant 4E-T proteins in a pull-down assay using recombinant maltose binding protein (MBP)-tagged LSM14LSM and glutathione S-transferase (GST)-tagged 4E-TC fragments (Fig 2C). Individual alanine substitutions of Trp9584E-T or Phe9594E-T in GST-4E-TC were sufficient to abrogate the interaction with LSM14LSM, as was the substitution of Glu9824E-T with lysine. Additionally, tandem alanine substitutions of Trp9584E-T and Leu9554E-T, as well as Val9784E-T and Leu9814E-T also led to loss of LSM14LSM binding. In contrast, alanine substitutions of serine residues Ser9704E-T or Ser9614E-T, which do not mediate specific contacts with LSM14LSM, did not affect binding. We additionally quantified the binding affinity of LSM14LSM for 4E-TC by isothermal titration calorimetry (ITC). LSM14LSM and 4E-TC interacted with a Kd of 0.3 μM, whereas a 4E-TC mutant containing an alanine substitution of Trp9584E-T was unable to bind to LSM14LSM (Table 2 and Fig EV1F). Together, these results indicate that 4E-TC residues making specific contacts with either of the two hydrophobic surfaces in the LSM14 LSM domain are required for the interaction. Corroborating this result, alanine substitutions of either Tyr22LSM14 or Ile29LSM14 in the two hydrophobic pockets in LSM14LSM that interact with 4E-TC were sufficient to abrogate interactions with 4E-TC in vitro (Fig 2D). Table 2. Equilibrium dissociation constants for LSM14 and DDX6 interactions Interaction Kd (μM) LSM14LSM–4E-TC 0.30 ± 0.06 DDX6C–LSM14FDF-TFG 1.62 ± 0.15 DDX6C–PATL1TFG-FDF 0.23 ± 0.04 DDX6C–PATL1TFGLSM14FDF 0.04 ± 0.01 DDX6C–EDC3TFG-FDF 0.41 ± 0.04 DDX6C–4E-TWFS-IEL 0.39 ± 0.10 We also disrupted the hydrophobic pockets in the LSM14LSM domain in the context of full-length LSM14 to determine whether this affects the 4E-T interaction in vivo. To this end, HeLa cell lines were generated that stably express FLAG-tagged wild-type LSM14 or the Y22E/I29E mutant. FLAG-LSM14 proteins were immunoprecipitated from benzonase-treated cell lysates with anti-FLAG antibody, and LSM14-interacting proteins were resolved by SDS–PAGE and analyzed by Western blotting (Fig 2E). Wild-type LSM14 co-precipitated both 4E-T and DDX6. In contrast, the Y22E/I29E LSM14 mutant failed to interact with endogenous 4E-T, even though DDX6 binding was not perturbed, indicating that the hydrophobic interaction hotspots in the LSM domain of LSM14 are required for the interaction with 4E-T in vivo. Together with our previous observations that the LSM domain of LSM14 interacts with both the middle and C-terminal regions of 4E-T, these results suggest that both LSM14-interaction motifs in 4E-T function redundantly and bind to LSM14 in a similar manner. A previous study demonstrated that Xenopus (x)LSM14 represses bound transcripts in oocytes, and that this repression was mediated by an N-terminal region in xLSM14 (Tanaka et al, 2006). As our data demonstrate that the N-terminal LSM domain directly binds to 4E-T, we tested whether this interaction plays a role in mRNA repression. To this end, we used the bacteriophage λN-BoxB tethering system in HeLa cells to tether the LSM14 LSM domain to a Renilla luciferase (RL) reporter mRNA. Tethering λNHA-tagged LSM14LSM (WT) to the RL-5BoxB reporter mRNA repressed RL expression ~threefold when compared to tethering LacZ (Figs 2F and EV1G). In contrast, the Y22E/I29E LSM domain mutant that cannot interact with 4E-T did not efficiently silence reporter mRNA translation. These results thus demonstrate that the LSM14–4E-T interaction plays a role in repressing mRNAs associated with LSM14. LSM14 uses conserved non-contiguous FDF and TFG motifs to bind DDX6 The C-terminal RecA-like domain of the RNA DEAD-box helicase DDX6 (DDX6C) serves as a binding platform for the CNOT1 subunit of the CCR4-NOT complex, as well as for a number of translational repressors and mRNA decapping factors including PATL1, EDC3, 4E-T, and LSM14 (Fig 3A; Tritschler et al, 2009). The FDF motif of the D. melanogaster LSM14 homolog Tral has previously been shown to be required in order for Tral to interact with DDX6C (Tritschler et al, 2009). Two other highly conserved short motifs are found downstream of the FDF, namely the FFD and TFG motifs (Figs 3B and 4A; Albrecht & Lengauer, 2004; Marnef et al, 2009). However, the functions of these LSM14 motifs are not known. We utilized conventional affinity purification followed by immunoblotting to determine whether deleting these conserved sequences impairs LSM14 interactions with known interacting proteins such as 4E-T, PATL1, and DDX6. To this end, HeLa cell lines were generated in which endogenous LSM14 was stably depleted by RNA interference and complemented with FLAG-tagged wild-type (WT) LSM14 or a LSM14 mutant lacking both the FFD and TFG motifs (LSM14ΔFFD-TFG). Affinity purification of wild-type FLAG-LSM14 resulted in co-precipitation of 4E-T, PATL1, and DDX6 (Fig 3C). In stark contrast, a LSM14 mutant lacking both the FFD and TFG motifs (LSM14ΔFFD-TFG) failed to co-precipitate DDX6, yet associated with PATL1 and 4E-T as efficiently as wild-type LSM14. Deleting just the FFD motif from LSM14 (LSM14ΔFFD) did not interfere with DDX6 association, whereas a LSM14 mutant lacking the TFG motif alone (LSM14ΔTFG) failed to interact with DDX6 (Fig 3C). Figure 3. The LSM14 FDF and TFG motifs interact with the C-terminal RecA-like domain of DDX6 Schematic diagram of the domain organization in human DDX6. N- and C-terminal RecA (DDX6N and DDX6C) domains are labeled as suc
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