Composition et dynamique des complexes protéiques impliqués dans le "nonsense-mediated mRNA decay" chez la levure Saccharomyces cerevisiae

Nonsense-Mediated mRNA Decay (NMD) detects and degrades RNA for which translation ends prematurely. It affects a large diversity of cytoplasmics RNAs; it is the major decay pathway for aberrants RNAs.In yeast, NMD targeted RNAs have a short open reading frame and a long 3’-UTR. How these two features lead to an efficient degradation through NMD and what are the steps of this mechanism is still unclear.From 112 affinity purifications of NMD factors followed by an analysis using quantitative mass spectrometry, we identified two distinct complexes. Those complexes were mutually exclusive and both contained Upf1, the major NMD protein. A first complex, named Detector, might have a role in the NMD substrates recognition whereas the second one, named Effector, would initiate the degradation through a direct interaction with the decapping machinery.The factors involved in our new model are all conserved throughout eukaryotes and the steps we describe have potential equivalents in other species. Our data suggest a new paradigm for the NMD mechanism that would be organised around a shared universal base to which specific steps could be added in certain organisms or for certain types of RNA substrates.Le Nonsense-Mediated mRNA Decay (NMD) détecte et dégrade les ARNs qui ont une terminaison de la traduction précoce. Il affecte une grande diversité d’ARNs cytoplasmiques, c’est la voie majeure de dégradation des ARNs aberrants.Les ARNs ciblés par le NMD chez la levure ont une phase ouverte de lecture courte et un long 3’-UTR. Comment ces caractéristiques permettent une dégradation efficace par le NMD et quelles sont les étapes du mécanisme reste peu clair. À partir de 112 purifications d’affinités suivies d’une analyse en spectrométrie de masse quantitative, nous avons identifié deux complexes distincts impliqués dans le NMD. Ces deux complexes, mutuellement exclusifs, s’articulent autour d’UPF1, la protéine majeure du NMD. Le premier complexe, nommé Détecteur, aurait un rôle dans la reconnaissance des cibles alors que le deuxième complexe, nommé Effecteur, initierait la dégradation des ARNs par une interaction directe avec la machinerie de décapping.Les facteurs impliqués dans ce modèle sont tous conservés à travers les eucaryotes et les étapes décrites sont transposables d’après la littérature. Cela semble indiquer un nouveau paradigme pour le mécanisme du NMD qui s’articulerait autour d’une base universelle commune à laquelle pourrait s’ajouter des étapes spécifiques des organismes ou des types d’ARNs

Composition and dynamic of Nonsense-mediated mRNA decay complexes in yeast

Le Nonsense-Mediated mRNA Decay (NMD) détecte et dégrade les ARNs qui ont une terminaison de la traduction précoce. Il affecte une grande diversité d’ARNs cytoplasmiques, c’est la voie majeure de dégradation des ARNs aberrants.Les ARNs ciblés par le NMD chez la levure ont une phase ouverte de lecture courte et un long 3’-UTR. Comment ces caractéristiques permettent une dégradation efficace par le NMD et quelles sont les étapes du mécanisme reste peu clair. À partir de 112 purifications d’affinités suivies d’une analyse en spectrométrie de masse quantitative, nous avons identifié deux complexes distincts impliqués dans le NMD. Ces deux complexes, mutuellement exclusifs, s’articulent autour d’UPF1, la protéine majeure du NMD. Le premier complexe, nommé Détecteur, aurait un rôle dans la reconnaissance des cibles alors que le deuxième complexe, nommé Effecteur, initierait la dégradation des ARNs par une interaction directe avec la machinerie de décapping.Les facteurs impliqués dans ce modèle sont tous conservés à travers les eucaryotes et les étapes décrites sont transposables d’après la littérature. Cela semble indiquer un nouveau paradigme pour le mécanisme du NMD qui s’articulerait autour d’une base universelle commune à laquelle pourrait s’ajouter des étapes spécifiques des organismes ou des types d’ARNs.Nonsense-Mediated mRNA Decay (NMD) detects and degrades RNA for which translation ends prematurely. It affects a large diversity of cytoplasmics RNAs; it is the major decay pathway for aberrants RNAs.In yeast, NMD targeted RNAs have a short open reading frame and a long 3’-UTR. How these two features lead to an efficient degradation through NMD and what are the steps of this mechanism is still unclear.From 112 affinity purifications of NMD factors followed by an analysis using quantitative mass spectrometry, we identified two distinct complexes. Those complexes were mutually exclusive and both contained Upf1, the major NMD protein. A first complex, named Detector, might have a role in the NMD substrates recognition whereas the second one, named Effector, would initiate the degradation through a direct interaction with the decapping machinery.The factors involved in our new model are all conserved throughout eukaryotes and the steps we describe have potential equivalents in other species. Our data suggest a new paradigm for the NMD mechanism that would be organised around a shared universal base to which specific steps could be added in certain organisms or for certain types of RNA substrates

Nonsense‐mediated mRNA decay involves two distinct Upf1‐bound complexes

International audienceNonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that NMD operates through successive Upf1-bound Detector and Effector complexes in other species. This model can be extended to accommodate steps that are missing in yeast, and thus serve for mechanistic studies of NMD in all eukaryotes. Running title: The Detector/Effector NMD mode

Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH

TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit

Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH

TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved “topological regions” that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with Xeroderma pigmentosum and Trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit