Impact d'un stress viral sur la transcription des SINE d'Arabidopsis thaliana et influence de l'ARN SINE sur la kinase GCN2

In mammals cells, adenovirus infection induces transcription of individual Alu loci. This mechanism is well-conserved and observed with other virus families. Adenovirus encodes a protein, E1A, capable of interacting with the retinoblastoma protein (RB). This interaction inactivates RB which probably leads to the transcriptional inactivation of Alu elements. Thus, in mammals, viral proteins can negatively regulate Rb and cause cell cycle deregulation, general pol III transcription and particularly SINE transcription. In plants, SINE transcriptional activation following viral stress is still to demonstrate. However, the FBNYV virus genome encodes a protein, CLINK, which contains a RB binding domain also found in the adenovirus protein E1A. The first step of this work was to analyze the CLINK protein and more precisely the impact of this protein on cell cycle deregulation, on general pol III transcription and particularly on endogenous plant SINE transcription. The second part of this study focuses on SINE elements functions within the cell. In mammals, SINE element Alu has been shown to modulate the cell physiology in response to stresses. Indeed, transcription of those elements is activated following some viral infections. Those transcripts are able to interact with the PKR protein, an eIF2α kinase. Thus, SINE elements are capable to modulate cellular functions such as translation regulation. Only one eIF2α kinase has been identified in plants: the GCN2 protein. Thus, we characterized the function of this protein in Arabidopsis. We shown mechanisms that regulate the protein function and also found some plant-specific inductors. This study has shown that GCN2 is important in plants and that this protein intervenes in some plant-specific stress pathway. Finally, SINE impact on GCN2 activity has been study in plants.Chez les mammifères, l'infection par l'adénovirus conduit à l'activation de la transcription de certains éléments SINE Alu. Il s'agit d'un mécanisme conservé car il est observé avec d'autres familles de virus. Adénovirus code pour une protéine, E1A qui est capable d'interagir avec la protéine Rétinoblastome (RB). Cette interaction provoque l'inactivation de RB ce qui provoque probablement l'activation transcriptionelle des éléments Alu. Ainsi, chez les mammifères, certaines protéines virales agissent négativement sur RB ce qui a pour conséquence de déréguler le cycle cellulaire, la transcription pol III de manière générale et la transcription des éléments SINE en particulier. Chez les plantes, l'activation de la transcription des éléments SINE à la suite d'un stress comme l'infection virale reste à démontrer. Cependant, le virus FBNYV possède au sein de son génome une protéine, CLINK, qui est entre autre constituée d'un domaine de liaison à RB similaire à celui retrouvé chez E1A d'adénovirus. La première étape de ce travail de thèse a porté sur l'analyse de la protéine CLINK et plus particulièrement l'effet de cette protéine sur le cycle cellulaire, sur la transcription pol III en général et sur la transcription des SINE endogènes de plantes. La seconde partie de cette étude porte sur la fonction des éléments SINE de plantes au sein de la cellule. Chez les mammifères, l'élément SINE Alu est capable de jouer un rôle dans la physiologie de la cellule en réponse à certains stress. En effet, la transcription de ces éléments est activée à la suite d'une infection par certaines familles de virus. Les transcrits ainsi produits sont alors capables d'interagir avec la protéine PKR, une kinase d'eIF2α. Ainsi, les éléments SINE sont capables d'intervenir dans des processus clefs de la cellule comme le mécanisme de régulation de la traduction. La seule kinase d'eIF2α identifiée chez les plantes est la protéine GCN2. Ainsi, nous avons choisit de caractériser la fonction de cette protéine chez Arabidopsis. Nous avons déterminé les mécanismes de régulation de la protéine en mettant en évidence certains inducteurs spécifiques aux plantes. Ce travail a permis de montrer l'importance de la protéine pour la plante et de découvrir des fonctions potentielles de la protéine dans des voies de stress typiques des végétaux. Enfin, l'impact des SINE de plantes sur l'activité de GCN2 a été analysé

Impact d'un stress viral sur la transcription des SINE d'Arabidopsis thaliana et influence de l'ARN SINE sur la kinase GCN2

Chez les plantes, l'activation de la transcription des éléments SINE à la suite d'un stress comme l'infection virale reste à démontrer. Cependant, le virus FBNYV possède au sein de son gènome une protéine CLINK, qui est entre autre constituée d'un domaine de liaison à RB. La première étape de ce travail de thèse a porté sur l'analyse de la protéine CLINK et plus particulièrement l'effet de cette protéine sur le cycle cellulaire, sur la transcription pol III en général et sur la transcription des SINE endogènes de plantes. La seconde partie de cette étude porte sur la fonction des éléments SINE de plantes au sein de la cellule. La seule kinase d'eIF2alpha identifiée chez les plantes est la protéine GCN2. Nous avons choisi de caractériser la fonction de cette protéine chez Arabidopsis. Nous avons déterminé les mécanismes de régulation de la protéine en mettant en évidence certains inducteurs spécifiques aux plantes. Ce travail a permis de montrer l'importance de la protéine pour la plante et de découvrir des fonctions potentielles de la protéine dans des voies de stress typiques des végétaux. Enfin, l'impact des SINE sur l'activité de GCN2 a été analyséCLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

International audienceThe stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction

Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

International audienceThe stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn- substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd- substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd- substitutions enhance YKD-KD interactions in vitro, whereas Gcn- substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd- substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD

The Nanovirus-Encoded Clink Protein Affects Plant Cell Cycle Regulation through Interaction with the Retinoblastoma-Related Protein

Nanoviruses, multicomponent single-stranded DNA plant viruses, encode a unique cell cycle link protein, Clink, that interacts with retinoblastoma-related proteins (RBR). We have established transgenic Arabidopsis thaliana lines that conditionally express Clink or a Clink variant deficient in RBR binding. By controlled induction of Clink expression, we demonstrated the capacity of the Clink protein to alter RBR function in vivo. We showed that transcription of both S-phase-specific and G(2)/M-phase-specific genes was up-regulated depending on the RBR-binding proficiency of Clink. Concomitantly, ploidy levels increased in a substantial fraction of leaf cell nuclei. Also, leaf epidermis cells of transgenic plants producing Clink were smaller and more numerous, indicating additional cell divisions in this tissue. Furthermore, cytogenetic analyses following induction of Clink expression in mature leaves revealed the presence of metaphasic and anaphasic nuclei, clear evidence that Clink-mediated RBR inactivation is sufficient to induce quiescent cells to reenter cell cycle progression and, for at least a fraction of them, to pass through mitosis. Expression of Clink had no effect on genes transcribed by RNA polymerases I and III, suggesting that, in contrast to its mammalian homologue, A. thaliana RBR is not involved in the repression of polymerase I and polymerase III transcription. The results of these in vivo analyses firmly establish Clink as a member of the diverse class of multifunctional cell cycle modulator proteins encoded by small DNA viruses

Arabidopsis eIF2α kinase GCN2 is essential for growth in stress conditions and is activated by wounding

International audiencePhosphorylation of eIF2alpha provides a key mechanism for down-regulating protein synthesis in response to nutrient starvation or stresses in mammalian and yeast cells. However, this process has not been well characterized in plant

<it>Arabidopsis </it>eIF2α kinase GCN2 is essential for growth in stress conditions and is activated by wounding

Abstract Background Phosphorylation of eIF2α provides a key mechanism for down-regulating protein synthesis in response to nutrient starvation or stresses in mammalian and yeast cells. However, this process has not been well characterized in plants Results We show here that in response to amino acid and purine starvations, UV, cold shock and wounding, the Arabidopsis GCN2 kinase (AtGCN2) is activated and phosphorylates eIF2α. We show that AtGCN2 is essential for plant growth in stress situations and that its activity results in a strong reduction in global protein synthesis. Conclusion Our results suggest that a general amino acid control response is conserved between yeast and plants but that the plant enzyme evolved to fulfill a more general function as an upstream sensor and regulator of diverse stress-response pathways. The activation of AtGCN2 following wounding or exposure to methyl jasmonate, the ethylene precursor 1-Aminocyclopropane-1-carboxylic acid (ACC) and salicylic acid, further suggests that this enzyme could play a role in plant defense against insect herbivores.</p