The level to which genes are expressed depends on the rate at which the mRNA is generated, and the rate at which it is utilised and destroyed. Almost all eukaryotic mRNAs contain a stretch of adenosine nucleotides known as the poly(A) tail. The removal of the polyA tail from an mRNA, a process called deadenylation, is an important mechanism of gene expression regulation. It is the first step in the decay of the transcript, and is also linked to repression of translation. Deadenylation is predominantly catalysed by a conserved multi-protein complex called Ccr4-Not. While the poly(A) tail is a feature of almost all mRNAs, cells control the rate at which each undergoes decay by the precise targeting of Ccr4-Not in both a gene-dependent and a context-dependent fashion. Substrate-selective deadenylation is therefore a central biochemical process to the control of gene expression. It plays a pivotal role in most cellular processes including differentiation, cell cycle control and adaptation to environmental change. The inflammatory response and embryogenesis are two systems in which deadenylation has been well studied. The subject of this dissertation is the biochemical mechanisms by which mRNAs are selected for deadenylation by Ccr4-Not. Despite its importance, intact Ccr4-Not has not previously been obtained in sufficient quantity and purity for rigorous biochemical and structural analysis. Here I present the purification of recombinant Ccr4-Not. An experimental system was devised to quantify the rate and pattern of the deadenylation reaction that it catalyses in vitro. Two models of Ccr4-Not regulation were characterised in detail: the recruitment of Ccr4-Not by RNA-binding adaptor proteins, and the effect of the protein Pab1, which binds to the poly(A) tail. These have yielded insight into the features of the proteins and RNA sequences that are critical to deadenylation. In addition, a structural study of the Ccr4-Not complex was performed using electron cryomicroscopy and single-particle analysis.

Poly(A)-specific ribonuclease (PARN) is a deadenylase that degrades the poly(A) tail of eukaryotic mRNA. PARN also interacts with the 5’-cap structure of the mRNA. The binding of the cap structure enhances the deadenylation rate. PARN has previously been described as a dimer. We have studied PARN with size exclusion chromatography to investigate the oligomeric composition and revealed oligomeric compositions of PARN that are larger than dimeric PARN. Deadenylation assays have been used to measure the cap stimulated activity of PARN. The deadenylation assays showed that the cap stimulated activity of PARN correlated with the abundance of oligomers corresponding in size to tetrameric PARN. We present a model for tetrameric PARN and propose a mechanistic model for how the cap stimulates PARN mediated deadenylation.

The CCR4-NOT complex is a global regulator of gene expression which controls mRNA levels by removing the poly-(A) tail, a step known as deadenylation and one that constitutes the rate-limiting step in mRNA decay. The human complex is comprised of eight stably associated CNOT subunits where CNOT1 forms the scaffold onto which CNOT2-11 bind. Although much has been learnt about the CCR4-NOT complex, questions still remain. Thus, this study focused on a number of sub-complexes of CNOT subunits and associated proteins to determine the mode of interaction with a hope to explore the mechanism of deadenylation by the CCR4-NOT complex. Firstly, the complex of CNOT10:CNOT11, found only in higher eukaryotes, was reconstituted for the first time using recombinant proteins. Crystallisation trials, limited proteolysis and mass spectrometry were used to isolate novel interaction regions between CNOT10 and CNOT11 which may provide direction for future structural and functional studies. Secondly, the interaction between CNOT9 and TTP was characterised. TTP is a RNA-binding protein which targets inflammatory mRNAs for deadenylation by recruiting the CCR4-NOT complex. This study highlights novel interactions between TTP and both CNOT2 and CNOT9. Moreover, BioLayer interferometry (BLI), peptide arrays and site-directed mutagenesis identified that TTP interacts with CNOT9 in a tryptophan-mediated manner. These findings change the known interface between TTP and the CCR4-NOT complex. Lastly, the MultiBac system was used to reconstitute a number of human CNOT sub-complexes, one of which was shown to effectively degrade a poly-(A) substrate, demonstrating it is enzymatically active. This achievement provides a tool for the future study of the CCR4-NOT complex. In summary, this study highlights novel interactions and characterises previously unknown binding mechanisms between CCR4-NOT subunits which expands our current understanding of the complex.

No description available.

<p>Degradation of mRNA is a highly regulated step important for proper gene expression. Degradation of eukaryotic mRNA is initiated by shortening of the 3’ end located poly(A) tail. Poly(A)-specific ribonuclease (PARN) is an oligomeric enzyme that degrades the poly(A) tail with high processivity. A unique property of PARN is its ability to interact not only with the poly(A) tail but also with the 5’ end located mRNA cap structure. A regulatory role in protein synthesis has been proposed for PARN based on its ability to bind the cap that is required for efficient initiation of eukaryotic mRNA translation. Here we have investigated how the cap structure influences PARN activity and how PARN binds the cap. We show that the cap activates PARN and enhances the processivity of PARN. Further we show that the cap binding complex (CBC) inhibits PARN activity through a protein-protein interaction. To investigate the cap binding property of PARN, we identified the cap binding site at the molecular level using site-directed mutagenesis and fluorescence spectroscopy. We identified tryptophan 475, located within the RNA recognition motif (RRM) of PARN, as crucial for cap binding. A crystal structure of PARN bound to cap revealed that cap binding is mediated by the nuclease domain and the RRM of PARN. Tryptophan 475 binds the inverted 7-Me-guanosine residue through a stacking interaction. Involvement of the nuclease domain in cap binding suggests that the cap site and the active site overlap. Mutational analysis showed that indeed amino acids involved in cap binding are crucial for hydrolytic activity of PARN. Taken together, we show that PARN is an allosteric enzyme that is activated by the cap structure and that the allosteric cap binding site in one PARN subunit corresponds to the active site in the other PARN subunit.</p>

Degradation of mRNA is a highly regulated step important for proper gene expression. Degradation of eukaryotic mRNA is initiated by shortening of the 3’ end located poly(A) tail. Poly(A)-specific ribonuclease (PARN) is an oligomeric enzyme that degrades the poly(A) tail with high processivity. A unique property of PARN is its ability to interact not only with the poly(A) tail but also with the 5’ end located mRNA cap structure. A regulatory role in protein synthesis has been proposed for PARN based on its ability to bind the cap that is required for efficient initiation of eukaryotic mRNA translation. Here we have investigated how the cap structure influences PARN activity and how PARN binds the cap. We show that the cap activates PARN and enhances the processivity of PARN. Further we show that the cap binding complex (CBC) inhibits PARN activity through a protein-protein interaction. To investigate the cap binding property of PARN, we identified the cap binding site at the molecular level using site-directed mutagenesis and fluorescence spectroscopy. We identified tryptophan 475, located within the RNA recognition motif (RRM) of PARN, as crucial for cap binding. A crystal structure of PARN bound to cap revealed that cap binding is mediated by the nuclease domain and the RRM of PARN. Tryptophan 475 binds the inverted 7-Me-guanosine residue through a stacking interaction. Involvement of the nuclease domain in cap binding suggests that the cap site and the active site overlap. Mutational analysis showed that indeed amino acids involved in cap binding are crucial for hydrolytic activity of PARN. Taken together, we show that PARN is an allosteric enzyme that is activated by the cap structure and that the allosteric cap binding site in one PARN subunit corresponds to the active site in the other PARN subunit.

De la synthèse des ARNm jusqu'à leur dégradation, le complexe CCR4-NOT est un régulateur essentiel de l'expression des gènes eucaryotes. CAF1 est une sous-unité catalytique qui joue un rôle central dans la fonction de ce complexe. La protéine humaine hCAF1 possède une activité déadénylase, régule la méthylation des arginines dépendante de PRMT1 et est un régulateur transcriptionnel des récepteurs nucléaires. Bien que l'ensemble des travaux publiés sur hCAF1 lui confère une place importante dans la régulation de l'expression des gènes, son mécanisme d'action et surtout les voies de signalisation qu'elle régule restent encore mal compris dans les cellules humaines. Lors de ce travail de thèse, nous avons mis en évidence une nouvelle fonction de la protéine hCAF1 comme régulateur de la voie des interférons via le contrôle du facteur de transcription STAT1 et la dégradation de ses ARNm cibles. L'identification de hCAF1 comme régulateur de l'activité de STAT1 et de la réponse aux interférons est très importante car des activations anormales de ces voies sont associées à de nombreuses pathologies telles que le cancer ou des maladies immunitaires. En parallèle, nous avons caractérisé un nouvel isoforme nommé hCAF1v2 produit par le gène humain Caf1 suite à un évènement d'épissage alternatif. Nos résultats indiquent que hCAF1v2 présente une divergence fonctionnelle vis-à-vis de hCAF1 puisqu'elle ne possède pas d'activité déadénylase intrinsèque et s'avère requise pour la régulation de la méthylation des arginines via son interaction avec l'enzyme PRMT1. L'ensemble des résultats obtenus identifient une nouvelle voie de signalisation régulée par la protéine hCAF1 dans les cellules humaines et permettent de mieux comprendre l'implication du complexe CCR4-NOT dans les mécanismes de régulation de l'expression des gènes / The multi-subunit CCR4-NOT complex has been implicated in all aspects of the mRNA life cycle, from synthesis of mRNAs in the nucleus to their degradation in the cytoplasm. The CAF1 protein is a catalytic subunit which plays a central role inside the complex. Human CAF1 is a deadenylase, modulates arginine methylation, and is a transcriptional cofactor of several nuclear receptors. The main objective of the thesis was to elucidate the molecular mechanism of hCAF1- mediated gene expression. We reported that hCAF1 is an important negative regulator of the interferon pathway and that hCAF1 is associated in the cytoplasm of resting cells with STAT1, a crucial transcription factor of this pathway. We found that hCAF1 participates in the extinction of the IFN signal via its deadenylase activity, by speeding up the degradation of some STAT1-induced mRNAs. Our findings are important because abnormal activations of this pathway are frequently associated with cancer and auto-immune diseases. In parallel, we characterized a novel isoform called hCAF1v2 produced by alternative splicing of the Caf1 gene. We reported that hCAF1v2 displays divergent functions compared with hCAF1. In fact hCAF1v2 does not have a deadenylase activity and is preferentially associated with PRMT1 to modulate arginine methylation. Altogether, our findings identify a new signalling pathway which is regulated by hCAF1, and reveal novel mechanisms utilized by the CCR4-NOT complex to control gene expression

Une détérioration des ARNm, le vecteur de l’information génétique, ou une altération des mécanismes régulant leur synthèse, leur traduction, ou leur dégradation peut être responsable de l’apparition d’une maladie. Ainsi, il est important de comprendre les mécanismes affectant les ARNm et notamment leur dégradation. Cette dernière est initiée par la réaction de désadénylation caractérisée par le raccourcissement progressif de la queue poly(A) de l’ARNm. Un complexe responsable de la désadénylation est CCR4-NOT. Les protéines BTG/Tob lient la désadénylase CAF1 de CCR4-NOT via leur domaine APRO. Dans cette thèse, l’interaction de BTG2 avec PABPC1, la protéine liant la queue poly(A), a été étudiée. Cette association augmente l’activité désadénylase de CAF1 in vitro et in cellulo. Le rôle de cette liaison sur les propriétés antiprolifératives de BTG2 et l’impact de BTG2 sur la traduction ont aussi été analysés. Un modèle expliquant l’activation de la désadénylation par BTG2 est proposé. / Damaging mRNA, the molecules carrying the genetic information, or altering the mechanisms responsible for their synthesis, their translation or their degradation can be responsible for initiating diseases. It is thus important to understand the mechanisms impacting mRNA, especially their degradation. The latter is initiated by the deadenylation reaction characterized by the progressive shortening of the mRNA 3’ poly(A) tail. One of the complexes responsible for deadenylation is CCR4-NOT. BTG/Tob proteins are able to bind the CAF1 deadenylase of CCR4-NOT via their APRO domain. In this thesis, interaction of BTG2 with PABPC1, the factor binding poly(A) tail, was analyzed. This binding stimulates CAF1 deadenylase activity in vitro and in cellulo. The role of this interaction on the antiproliferative properties of BTG2 and the impact of BTG2 on translation were also investigated. A model explaining deadenylation activation by BTG2 is proposed.

Local translation of mRNAs in dendrites is vital for synaptic plasticity and learning and memory. Tight regulation of this translation is key to preventing neurological disorders resulting from aberrant local translation. Here we find that CNOT7, the major deadenylase in eukaryotic cells, takes on the distinct role of regulating local translation in the hippocampus. Depletion of CNOT7 from cultured neurons affects the poly(A) state, localization, and translation of dendritic mRNAs while having little effect on the global neuronal mRNA population. Following synaptic activity, CNOT7 is rapidly degraded resulting in polyadenylation and a change in the localization of its target mRNAs. We find that this degradation of CNOT7 is essential for synaptic plasticity to occur as keeping CNOT7 levels high prevents these changes. This regulation of dendritic mRNAs by CNOT7 is necessary for normal neuronal function in vivo, as depletion of CNOT7 also disrupts learning and memory in mice. We utilized deep sequencing to identify the neuronal mRNAs whose poly(A) state is governed by CNOT7. Interestingly these mRNAs can be separated into two distinct populations: ones that gain a poly(A) tail following CNOT7 depletion and ones that surprisingly lose their poly(A) tail following CNOT7 depletion. These two populations are also distinct based on the lengths of their 3’ UTRs and their codon usage, suggesting that these key features may dictate how CNOT7 acts on its target mRNAs. This work reveals a central role for CNOT7 in the hippocampus where it governs local translation and higher cognitive function.

No description available.