The C-terminal domain from S. cerevisiae Pat1 displays two conserved regions involved in decapping factor recruitment

Eukaryotic mRNA decay is a highly regulated process allowing cells to rapidly modulate protein production in response to internal and environmental cues. Mature translatable eukaryotic mRNAs are protected from fast and uncontrolled degradation in the cytoplasm by two cis-acting stability determinants: a methylguanosine (m(7)G) cap and a poly(A) tail at their 5' and 3' extremities, respectively. The hydrolysis of the m(7)G cap structure, known as decapping, is performed by the complex composed of the Dcp2 catalytic subunit and its partner Dcp1. The Dcp1-Dcp2 decapping complex has a low intrinsic activity and requires accessory factors to be fully active. Among these factors, Pat1 is considered to be a central scaffolding protein involved in Dcp2 activation but also in inhibition of translation initiation. Here, we present the structural and functional study of the C-terminal domain from S. cerevisiae Pat1 protein. We have identified two conserved and functionally important regions located at both extremities of the domain. The first region is involved in binding to Lsm1-7 complex. The second patch is specific for fungal proteins and is responsible for Pat1 interaction with Edc3. These observations support the plasticity of the protein interaction network involved in mRNA decay and show that evolution has extended the C-terminal alpha-helical domain from fungal Pat1 proteins to generate a new binding platform for protein partners

Études structurales des intéractions protéines-protéines et ARN-protéines impliquées dans l'assemblage des snoRNP à boîtes C/D

A lot of essential cellular functions like translation, splicing, ribosome biogenesis and telomere replication need the activity of non coding RNPs. The biogenesis of non coding RNPs in eukaryotes is a complex pathway involving numerous cellular factors. For instance, ribosome biogenesis requires more than 150 factors. They are important to facilitate and to control the biogenesis of this essential cellular machinery. These factors include the C/D box snoRNPs. These RNPs are involved in pre-rRNA maturation (post-transcriptional ribose methylation and endo-nucleolytic cleavages). Recently, our laboratory participated to the discovery of snoRNP assembly factors: the Rsa1p protein, R2TP complex (Rvb1p, Rvb2p, Tah1p and Pih1p) and Hit1p in the yeast Saccharomyces cerevisiae. Using a high throughput co-expression approach, we deciphered a network of interactions between RNP core proteins and the assembly factors. Coupled with a limited proteolysis strategy, the co-expression method allowed us to obtain proteins sub-complexes Snu13p/Rsa1p and Rsa1p/Hit1p which are currently studied by NMR. In collaboration with the F. Allain team (ETH Zurich), we also determined the tridimensional structure of protein Tah1p and its complex with the chaperon Hsp90 C-terminal peptide at high resolution. The data obtained reveal a particular mode of association of the Tah1p TPR domain with the Hsp90 peptideDe nombreuses fonctions cellulaires essentielles telles que la traduction, l'épissage, la biogenèse des ribosomes et la réplication des télomères font appels aux particules RNP non codantes. La biogenèse de ces dernières chez les eucaryotes est un processus très complexes qui fait intervenir de nombreux facteurs cellulaires. La biogenèse du ribosome nécessite au moins 150 facteurs. Ceux-ci sont importants pour faciliter mais également contrôler la biogenèse de cette machinerie cellulaire essentielle qu'est le ribosome. Parmi ces facteurs, nous avons les snoRNP à boîtes C/D. Ces RNP sont impliqués dans la maturation des pré-ARNr (méthylation post-transcriptionnelle des riboses et clivages endonucléolytiques). Récemment notre laboratoire a participé à la découverte de facteurs d'assemblage de ces RNP. Il s'agit entre autre des protéines Rsa1p, du complexe R2TP (Rvb1p, Rvb2p, Tah1p et Pih1p) et de Hit1p chez la levure Saccharomyces cerevisiae. En utilisant une approche de co-expression à haut débit, nos travaux ont révélé un réseau complexe d'interactions entre les protéines constitutives des snoRNP et leurs facteurs d'assemblage. Couplé à une stratégie de protéolyse ménagée, la co-expression nous a permis d'obtenir des sous-complexes protéiques Snu13p/Rsa1p et Rsa1p/Hit1p qui font actuellement l'objet d'une étude structurale par RMN. En collaboration avec l'équipe de F. Allain (ETH Zurich), nous avons également déterminé la structure tridimensionnelle de la protéine Tah1p, ainsi que de son complexe avec le peptide C-terminale de la protéine chaperonne Hsp90 à haute résolution. Ces travaux ont révélé un mode d'association particulier entre le domaine TPR de la protéine et le peptid

Structural studies on protein-protein and RNA-protein interactions implicated in the C/D snoRNP biogenesis

De nombreuses fonctions cellulaires essentielles telles que la traduction, l'épissage, la biogenèse des ribosomes et la réplication des télomères font appels aux particules RNP non codantes. La biogenèse de ces dernières chez les eucaryotes est un processus très complexes qui fait intervenir de nombreux facteurs cellulaires. La biogenèse du ribosome nécessite au moins 150 facteurs. Ceux-ci sont importants pour faciliter mais également contrôler la biogenèse de cette machinerie cellulaire essentielle qu'est le ribosome. Parmi ces facteurs, nous avons les snoRNP à boîtes C/D. Ces RNP sont impliqués dans la maturation des pré-ARNr (méthylation post-transcriptionnelle des riboses et clivages endonucléolytiques). Récemment notre laboratoire a participé à la découverte de facteurs d'assemblage de ces RNP. Il s'agit entre autre des protéines Rsa1p, du complexe R2TP (Rvb1p, Rvb2p, Tah1p et Pih1p) et de Hit1p chez la levure Saccharomyces cerevisiae. En utilisant une approche de co-expression à haut débit, nos travaux ont révélé un réseau complexe d'interactions entre les protéines constitutives des snoRNP et leurs facteurs d'assemblage. Couplé à une stratégie de protéolyse ménagée, la co-expression nous a permis d'obtenir des sous-complexes protéiques Snu13p/Rsa1p et Rsa1p/Hit1p qui font actuellement l'objet d'une étude structurale par RMN. En collaboration avec l'équipe de F. Allain (ETH Zurich), nous avons également déterminé la structure tridimensionnelle de la protéine Tah1p, ainsi que de son complexe avec le peptide C-terminale de la protéine chaperonne Hsp90 à haute résolution. Ces travaux ont révélé un mode d'association particulier entre le domaine TPR de la protéine et le peptideA lot of essential cellular functions like translation, splicing, ribosome biogenesis and telomere replication need the activity of non coding RNPs. The biogenesis of non coding RNPs in eukaryotes is a complex pathway involving numerous cellular factors. For instance, ribosome biogenesis requires more than 150 factors. They are important to facilitate and to control the biogenesis of this essential cellular machinery. These factors include the C/D box snoRNPs. These RNPs are involved in pre-rRNA maturation (post-transcriptional ribose methylation and endo-nucleolytic cleavages). Recently, our laboratory participated to the discovery of snoRNP assembly factors: the Rsa1p protein, R2TP complex (Rvb1p, Rvb2p, Tah1p and Pih1p) and Hit1p in the yeast Saccharomyces cerevisiae. Using a high throughput co-expression approach, we deciphered a network of interactions between RNP core proteins and the assembly factors. Coupled with a limited proteolysis strategy, the co-expression method allowed us to obtain proteins sub-complexes Snu13p/Rsa1p and Rsa1p/Hit1p which are currently studied by NMR. In collaboration with the F. Allain team (ETH Zurich), we also determined the tridimensional structure of protein Tah1p and its complex with the chaperon Hsp90 C-terminal peptide at high resolution. The data obtained reveal a particular mode of association of the Tah1p TPR domain with the Hsp90 peptid

Identification of the Rps28 binding motif from yeast Edc3 involved in the autoregulatory feedback loop controlling RPS28B mRNA decay

International audienceIn the yeast Saccharomyces cerevisiae, the Edc3 protein was previously reported to participate in the auto-regulatory feedback loop controlling the level of the RPS28B messenger RNA (mRNA). We show here that Edc3 binds directly and tightly to the globular core of Rps28 ribosomal protein. This binding occurs through a motif that is present exclusively in Edc3 proteins from yeast belonging to the Saccharomycetaceae phylum. Functional analyses indicate that the ability of Edc3 to interact with Rps28 is not required for its general function and for its role in the regulation of the YRA1 pre-mRNA decay. In contrast, this interaction appears to be exclusively required for the autoregulatory mechanism controlling the RPS28B mRNA decay. These observations suggest a plausible model for the evolutionary appearance of a Rps28 binding motif in Edc3

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

International audienc

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

International audienc

Cursos e profissões:: Conhecendo possibilidades para a escolha profissional

O trabalho apresenta reflexões sobre um projeto de extensão realizado em um Instituto Federal de Educação, Ciência e Tecnologialocalizado no Rio Grande do Sul. O projeto objetivou fornecer, aos estudantes concluintes do Ensino Médio da rede estadual do município, informações sobre os cursos ofertados pela instituição, visando a auxiliá-los no processo de escolha profissional e reduzir índices de evasão relacionados à falta de conhecimentosobre o curso escolhido. Participaram da ação oito das nove escolas definidas como público-alvo, totalizando 459 estudantes. O projeto oportunizou espaços para reflexão e qualificação do processo de escolha profissional, colocando em pauta cursos, possibilidades de formação e carreiras profissionais. Acredita-se que as ações desenvolvidas ampliaram o conhecimento local da instituição e possibilitaram maior congruência entre as concepções e expectativas dos estudantes e a realidade dos cursos ofertados, favorecendo escolhas mais conscientes e, consequentemente, maior adequação e satisfação com o curso e com a profissão escolhidaEl trabajo presenta reflexiones sobre un proyecto de extensión realizado en un Instituto Federal de Educación, Ciencia y Tecnología localizado en Rio Grande do Sul. El proyecto tuvo como objetivo subministrar a los estudiantes concluyentes de la educación secundaria de la red estadual de la municipalidad informaciones sobre los cursos ofrecidos por la institución, teniendo en vista poder prestar auxilio en el proceso de la elección profesional y reducir índices de evasión relacionada a falta de conocimiento del curso elegido. Participan de la acción ocho de las nueve escuelas definidas como público clave, con una totalidad de 459 estudiantes. El proyecto ofreció espacio para la reflexión y calificación del proceso de elección profesional, poniendo en pauta cursos, posibilidades de formación y carreras profesionales. Se cree que las acciones desarrolladas van a ampliar el conocimiento local de la institución y se cree que posibilitarán mayor congruencia entre las concepciones y expectativas de los estudiantes y la realidad de los cursos ofrecidos, favoreciendo elecciones más conscientes y como consecuencia una mayor adecuación y satisfacción con el curso y con la profesión elegida.The study presents reflections about an extension project performed in a Federal Institute of Education, Science and Technology of Rio Grande do Sul. This project aimed to provide graduating high school students from the city´s municipalitly schools, information about the courses offered by the institution, in order to assist them in the professional selection process and reduce the dropout rates related to lack of knowledge of the chosen course. This program was attended by eight of the nine schools defined as the target public, totalizing 459 students. The project provides reflection and qualification spaces for the professional selection process, by discussing about courses, training opportunities and professional careers. The actions developed expanded the local knowledge of the institution, allowed greater congruence between the conceptions and expectations of students and the reality of courses offered, encouraging more conscious choices and consequently greater adaptation and satisfaction with the course and the chosen occupatio

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

Characterization of the interaction between protein Snu13p/15.5K and the Rsa1p/NUFIP factor and demonstration of its functional importance for snoRNP assembly

The yeast Snu13p protein and its 15.5K human homolog both bind U4 snRNA and box C/D snoRNAs. They also bind the Rsa1p/NUFIP assembly factor, proposed to scaffold immature snoRNPs and to recruit the Hsp90-R2TP chaperone complex. However, the nature of the Snu13p/15.5K-Rsa1p/NUFIP interaction and its exact role in snoRNP assembly remained to be elucidated. By using biophysical, molecular and imaging approaches, here, we identify residues needed for Snu13p/15.5K-Rsa1p/NUFIP interaction. By NMR structure determination and docking approaches, we built a 3D model of the Snup13p-Rsa1p interface, suggesting that residues R249, R246 and K250 in Rsa1p and E72 and D73 in Snu13p form a network of electrostatic interactions shielded from the solvent by hydrophobic residues from both proteins and that residue W253 of Rsa1p is inserted in a hydrophobic cavity of Snu13p. Individual mutations of residues in yeast demonstrate the functional importance of the predicted interactions for both cell growth and snoRNP formation. Using archaeal box C/D sRNP 3D structures as templates, the association of Snu13p with Rsa1p is predicted to be exclusive of interactions in active snoRNPs. Rsa1p and NUFIP may thus prevent premature activity of pre-snoRNPs, and their removal may be a key step for active snoRNP production