Structural and functionnal studies of the actors of mRNAs decapping in yeast Saccharomyces cerevisiae.

La régulation fine des mécanismes d’élimination des ARN messagers (ARNm) au sein des cellules contribue au contrôle de l’expression génétique ainsi qu’à l’adaptation rapide des niveaux de transcrits en réponse à divers événements cellulaires ou stimuli externes. Elle intervient ainsi dans différents aspects de la physiologie cellulaire : différentiation, prolifération, homéostasie, inflammation ou encore défense anti-parasitaire. Les ARNm eucaryotes matures sont protégés d’une dégradation incontrôlée par une coiffe et une queue poly(A), à chacune de leurs extrémités. Le premier événement amorçant la dégradation des ARNm est le raccourcissement de la queue poly(A) par le complexe CCR4/Not par un processus appelé déadénylation. Ensuite, la coiffe 5’ est éliminée pendant l’étape de « decapping » qui est considérée comme une étape cruciale, irréversible et extrêmement contrôlée, nécessaire à la dégradation rapide du corps du messager par Xrn1. L’étape de “decapping” est effectuée via le recrutement d’un complexe protéique formé de l’enzyme Dcp2 et de son co-activateur essentiel Dcp1. Cependant, ce complexe n’est que peu actif et nécessite de nombreux co-facteurs pour être pleinement efficace. Ces facteurs comprennent l’anneau LSm1-7 qui reconnaît l’extrémité 3’ des ARNm déadénylés et interagit avec Pat1, une protéine plateforme qui recrute l’hélicase Dhh1 et les protéines activatrices du decapping Edc1-2-3. Tous ces facteurs sont organisés au sein d’un réseau d’interaction complexe et dynamique qui, dans certaines conditions, colocalise dans les P-bodies, des foyers cytoplasmiques impliqués dans la dégradation des ARNm et dans la répression de la traduction.Même si de nombreuses études ont révélé l’importance des interactions protéine/protéine dans le processus de decapping, peu d’informations sont disponibles sur les mécanismes moléculaires du recrutement et d’activation de Dcp2 par ses différents co-facteurs. De même, en raison de l’absence de structure de Dcp2 en complexe avec un ARNm coiffé, les détails moléculaires de la reconnaissance et du clivage de la coiffe sont inconnus. Mon projet de thèse a pour but de répondre à ces questions par l’étude fonctionnelle et structurale des acteurs du decapping, en utilisant les protéines de la levure Saccharomyces cerevisiae comme système modèle, puisque la plupart des acteurs du decapping sont conservés au sein des eucaryotes. Dans ce but, j’ai exprimé par génie génétique et isolé la majorité des facteurs impliqués dans le “decapping” et reconstitué plusieurs sous complexes comprenant Dcp2 et ses différents cofacteurs.MRNA decay is a highly regulated process allowing cells to rapidly adapt their abundance of transcripts to environmental conditions. Eukaryotic mRNAs are protected from uncontrolled decay by a cap structure (m7GpppX) and a poly(A) tail at their 5’ and 3’ ends, respectively. The first event initiating the 5’ to 3’ degradation pathway is the shortening of the poly(A) tail by the CCR4/Not complex through a process known as deadenylation. Then the 5’ cap is degraded during the decapping step, which is considered as a crucial and irreversible step before rapid degradation of RNAs. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its activator Dcp1. However, this complex has a low intrinsic decapping activity and requires several accessory factors to be fully efficient. These include the Lsm1-Lsm7 complex that binds to the 3’ end of deadenylated mRNAs and promotes decapping. This complex binds to Pat1, a scaffolding protein recruiting other accessory proteins such as Dhh1 and Edc1-3 proteins (Enhancer of Decapping), which favor decapping. After efficient removal of the cap, Xrn1 (the major cytoplasmic 5’-3’ exonuclease) is recruited and degrades the resulting uncapped RNAs. Interestingly, all these proteins are part of dynamic and multifunctional protein assemblies that, under conditions, localize into cytoplasmic foci known as P-bodies.Although many studies have revealed the importance of these protein/protein interactions, little is known concerning the mechanisms of recruitment and activation of the decapping enzyme by its numerous co-factors. Moreover, in the absence of Dcp2 in complex with a capped RNA, molecular details of cap recognition and cleavage are lacking. My thesis project aims at answering these open questions with the structural and functional studies of the decapping machinery, using yeast Saccharomyces cerevisiae as a model organism, as most of decapping actors are well conserved among eukaryotes. For this purpose, I expressed and purified the majority of the decapping factors and reconstituted several sub-complexes including Dcp2 and its cofactors

mRNA decapping: finding the right structures

International audienceIn eukaryotes, the elimination of the m7GpppN mRNA cap, a process known as decapping, is a critical, largely irreversible and highly regulated step of mRNA decay that withdraws the targeted mRNAs from the pool of translatable templates. The decapping reaction is catalysed by a multi-protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor, a holoenzyme that is poorly active on its own and needs several accessory proteins (Lsm1-7 complex, Pat1, Edc1-2, Edc3 and/or EDC4) to be fully efficient. Here, we discuss the several crystal structures of Dcp2 domains bound to various partners (proteins or small molecules) determined in the last couple of years that have considerably improved our current understanding of how Dcp2, assisted by its various activators, is recruited to its mRNA targets and adopts its active conformation upon substrate recognition. We also describe how, over the years, elegant integrative structural biology approaches combined to biochemistry and genetics led to the identification of the correct structure of the active Dcp1-Dcp2 holoenzyme among the many available conformations trapped by X-ray crystallography.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'

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

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

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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

The C-Terminal Domain from S. cerevisiae Pat1 Displays Two Conserved Regions Involved in Decapping Factor Recruitment

International audienceEukaryotic 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 59 and 39 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

RNA Mimicry by the Fap7 Adenylate Kinase in Ribosome Biogenesis

<div><p>During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.</p></div