Structure of the active form of Dcp1–Dcp2 decapping enzyme bound to m7GDP and its Edc3 activator

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Recruitment of the mRNA decay machineries to the 5’ end of mRNAs

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Pby1 is a direct partner of the Dcp2 decapping enzyme

International audienceMost eukaryotic mRNAs harbor a characteristic 5 m 7 GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5-3 exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 de-capping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation , both stemming from its interaction with the Dcp1-Dcp2 holoenzyme

A unique surface on Pat1 C-terminal domain directly interacts with Dcp2 decapping enzyme and Xrn1 5′–3′ mRNA exonuclease in yeast

International audienceThe Pat1 protein is a central player of eukaryotic mRNA decay that has also been implicated in translational control. It is commonly considered a central platform responsible for the recruitment of several RNA decay factors. We demonstrate here that a yeast-specific C-terminal region from Pat1 interacts with several short motifs, named helical leucine-rich motifs (HLMs), spread in the long C-terminal region of yeast Dcp2 decapping enzyme. Structures of Pat1-HLM complexes reveal the basis for HLM recognition by Pat1. We also identify a HLM present in yeast Xrn1, the main 5'-3' exonuclease involved in mRNA decay. We show further that the ability of yeast Pat1 to bind HLMs is required for efficient growth and normal mRNA decay. Overall, our analyses indicate that yeast Pat1 uses a single binding surface to successively recruit several mRNA decay factors and show that interaction between those factors is highly polymorphic between species

Differential action on coregulator interaction defines inverse retinoid agonists and neutral antagonists.

International audienceRetinoic acid receptors (RARs) are ligand-dependent transcription factors that control a plethora of physiological processes. RARs exert their functions by regulating gene networks controlling cell growth, differentiation, survival, and death. Uncovering the molecular details by which synthetic ligands direct specificity and functionality of nuclear receptors is key to rational drug development. Here we define the molecular basis for (E)-4-[2-[5,6-Dihydro-5,5-dimethyl-8-(2-phenylethynyl)naphthalen-2-yl]ethen-1-yl]benzoic acid (BMS204,493) acting as the inverse pan-RAR agonist and define 4-[5,6-Dihydro-5,5-dimethyl-8-(quinolin-3-yl)naphthalen-2-carboxamido]benzoic acid (BMS195,614) as the neutral RARalpha-selective antagonist. We reveal the details of the differential coregulator interactions imposed on the receptor by the ligands and show that the anchoring of H12 is fundamentally distinct in the presence of the two ligands, thus accounting for the observed effects on coactivator and corepressor interactions. These ligands will facilitate studies on the role of the constitutive activity of RARs, particularly of the tumor suppressor RARbeta, whose specific functions relative to other RARs have remained elusive

Synthesis and pharmacological characterization of Disila-AM80 (Disila-tamibarotene) and Disila-AM580, silicon analogues of the RARalpha-selective retinoid agonists AM80 (Tamibarotene) and AM580.

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The yeast specific residues located at the C-terminus of ScPat1C are functionally important.

<p>A. ScPat1C structure with the residues mutated in this study shown as ball and sticks. Color scheme identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096828#pone-0096828-g001" target="_blank">Fig.1A</a>. The region absent in the ScPat1ΔC68 construct is colored in grey. B. Growth analysis of <i>PAT1</i> mutants. Serial dilutions of the different strains transformed with vector, a plasmid encoding wild type ScPat1, or the indicated mutant thereof, were deposited on plates and incubated at the indicated temperatures. C. Monitoring ScPat1 interaction with Edc3, Rps28 or Scd6 using the two-hybrid assay. The wild-type or <i>edc3Δ</i> strain was transformed with the indicated pairs of vectors and interaction between the factors encoded by these two plasmids was scored by assaying β-galactosidase activity.</p

Sequence alignment of Pat1 orthologues.

<p>Alignment was performed using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096828#pone.0096828-Chenna1" target="_blank">[69]</a>. Strictly conserved residues are in white on a black background. Partially conserved amino acids are boxed. Secondary structure elements assigned from the ScPat1C structure are indicated above the alignment. Black stars below the sequences indicate residues mutated in this study. This figure was generated using the ESPript server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096828#pone.0096828-Gouet1" target="_blank">[70]</a>.</p

Structure of <i>S. cerevisiae</i> Pat1 C domain.

<p>A and B. Left panels: ribbon representation of ScPat1C crystal structure. The ARM repeats are depicted using different colors. Middle panel: Mapping of the sequence conservation at the surface of the ScPat1C domain. Coloring is from grey (low conservation) to cyan (highly conserved). The conservation score was calculated using the CONSURF server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096828#pone.0096828-Ashkenazy1" target="_blank">[67]</a> and using an alignment made from the sequences of 30 Pat1 fungal orthologues. Right panel: Mapping of the electrostatic potential at the surface of the ScPat1C domain. Positively (10 k_BT/e^-) and negatively (−10 k_BT/e^-) charged regions are colored in blue and red, respectively. The electrostatic potential was calculated using PBEQ Solver server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096828#pone.0096828-Jo1" target="_blank">[68]</a>. Orientation in B differs from A by a 180° rotation along the horizontal axis. C. Ribbon representation of the superimposition between human Pat1C domain (green; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096828#pone.0096828-Braun1" target="_blank">[36]</a>) and ScPat1C domain. The core conserved from yeast to human is colored in yellow. The fungi specific C-terminal extension from ScPat1C is colored in red. D. Surface representation of ScPat1 domain. Same color code as panel C.</p