Régulation de la traduction des facteurs de croissance (lymph)angiogéniques et rôle de l'ARN non codant NEAT1 lors du stress hypoxique

Translation is a highly regulated step of gene expression. During cellular stress, global protein synthesis is blocked but translation of specific subsets of mRNAs is activated by alternative mechanisms. One of these mechanisms involves an RNA structure called IRES (Internal Ribosome Entry Site), that enables the recruitment of the translational machinery in a 5' cap-independent manner. IRES activity is regulated by factors called ITAF (IRES trans-acting factor). Hypoxic stress occurs in different pathologies such as cardiac ischemia and cancer. In response to stress, the cell produces (lymph)angiogenic growth factors that stimulate blood and lymphatic vessel formation, allowing reperfusion of the injured area in ischemic heart, or stimulation of tumor growth and metastatic spread in cancer. My thesis project is focused on identification of ITAF-controlled translation of (lymph)angiogenic growth factor mRNAs during hypoxia. The first part of my thesis addresses translational regulation in hypoxic cardiomyocytes. A semi-global study showed that (lymph)angiogenic genes are mostly regulated at the translational level. Furthermore IRESs of mRNAs coding (lymph)angiogenic growth factors are activated in early hypoxia while IRESs of mRNAs non-related to (lymph)angiogenesis are activated later. Finally, we have identified a new ITAF, vasohibin (VASH1), that specifically activates the FGF1 IRES, but not the other IRES tested. A PCR array study indicates however that VASH1 is able to inhibit or stimulate translation of numerous mRNAs. Identification of a global mechanism of FGF and VEGF IRES activation is the main focus of the second chapter of my thesis. Considering that VASH1 is not a common ITAF, I searched for new candidates. I started from the observation that another ITAF previously identified in the laboratory, P54nrb, is also a component of the paraspeckle, a nuclear body formed during cellular stress. We make the hypothesis that other paraspeckle components could be ITAFs, particularly the backbone of the paraspeckle, the long non-coding RNA NEAT1. I established a correlation between NEAT1 induction in hypoxia and FGF1 IRES activation in cardiomyocytes. Moreover, NEAT1 depletion leads to inactivation of the FGF1 IRES, suggesting that NEAT1 is an ITAF. I also highlighted that another paraspeckle component, PSPC1, has an ITAF function. Analysis of IRESsome composition by mass spectrometry allowed me to identify three other candidates: hnRNPM, Rps2 and nucleolin. We then expanded the study to the other IRESs studied in chapter 1 and demonstrated that P54nrb and PSPC1 are able to activate several IRES whilst the non-coding RNA NEAT1 is a positive ITAF of all tested IRES. Thus NEAT1 seems to be the key of IRES activation, and confers on the paraspeckle the novel function of assembly platform for IRESsome formation in cardiomyocytes during hypoxia. In a third chapter, we investigated the same way in other cellular types, metastatic and non metastatic mammary carcinoma 4T1 and 67NR. NEAT1 is induced in correlation with FGF1 IRES activation when the cells are subjected to hypoxia, suggesting that the role of NEAT1 in translational control can be associated to tumoral hypoxia as well as to ischemia. Thus, this work reveal the unique potential of NEAT1 as a therapeutic target.La traduction est une étape de l'expression des gènes fortement régulée. Lorsque la cellule est stressée, cela bloque la synthèse protéique globale tout en activant la traduction de certains ARNm par des mécanismes alternatifs. L'un de ces mécanismes implique des structures de l'ARNm, les IRES (Internal Ribosome Entry Sites), qui permettent un recrutement de la machinerie de traduction indépendamment de l'extrémité 5' coiffée de l'ARNm. L'activité des IRES est régulée par des facteurs appelés ITAF (IRES trans-acting factor). Le stress hypoxique survient dans différentes pathologies comme l'ischémie cardiaque et le cancer. Pour répondre rapidement à ce stress, les cellules produisent des facteurs de croissance (lymph)angiogéniques qui stimulent la formation de vaisseaux sanguins et lymphatiques. Cela permet de reperfuser la zone lésée dans le cœur ischémique, ou de stimuler la croissance tumorale et la dissémination métastatique dans le cancer. Mon projet de thèse porte sur l'identification d'ITAF contrôlant la traduction des ARNm de ces facteurs de croissance lors de l'hypoxie. La première partie de ma thèse s'est focalisée sur la régulation de la traduction dans les cardiomyocytes hypoxiques. Une étude semi-globale a montré que les gènes de la (lymph)angiogenèse sont régulés majoritairement au niveau traductionnel, alors que tous les ARNm connus pour posséder des IRES sont recrutés plus activement dans les polysomes lors du stress. Nous avons montré que les IRES d'ARNm des familles FGF (fibroblast growth factor) et VEGF (vascular endothelial growth factor) sont tous activés lors de l'hypoxie précoce alors que les IRES d'ARNm non liés à la (lymph)angiogenèse sont activés plus tardivement. Enfin, nous avons identifié un nouvel ITAF, la vasohibine (VASH1), qui active spécifiquement l'IRES du FGF1, mais pas les autres IRES testés. Une analyse par PCR array indique cependant que VASH1 est capable d'inhiber ou de stimuler la traduction de nombreux autres ARNm. La recherche d'un mécanisme global d'activation des IRES des FGF et VEGF fait l'objet du deuxième chapitre de ma thèse. VASH1 n'étant pas un ITAF commun, j'ai recherché d'autres candidats. Je suis partie de l'observation qu'un autre ITAF identifié précédemment au laboratoire, p54nrb, est un composant essentiel du paraspeckle, un corps nucléaire formé en cas de stress. Nous avons émis l'hypothèse que d'autres composants du paraspeckle pourraient être des ITAFs, en particulier le long ARN non codant NEAT1 sur lequel repose sa formation. J'ai établi une corrélation entre l'induction de NEAT1 et l'activation de l'IRES du FGF1 lors de l'hypoxie. De plus, la déplétion de NEAT1 entraine une inactivation de l'IRES, suggérant que cet ARN non codant est un ITAF. J'ai mis en évidence qu'une autre protéine du paraspeckle, PSPC1, est également un ITAF. Une analyse de la composition de l'IRESome par spectrométrie de masse m'a permis d'identifier trois candidats supplémentaires : hnRNPM, rps2 et Nucléoline. Ayant élargi cette étude aux autres IRES étudiés dans le chapitre 1, nous avons démontré que p54nrb et PSPC1 sont capables d'activer plusieurs IRES alors que l'ARN non codant NEAT1, est un ITAF activateur de tous les IRES testés. NEAT1 paraît donc être la clé de l'activation des IRES, et donne au paraspeckle la fonction nouvelle de plateforme d'assemblage de l'IRESome dans les cardiomyocytes en réponse à l'hypoxie. Dans un troisième chapitre, nous avons élargi l'étude à d'autres types cellulaires, les carcinomes mammaires métastatiques et non-métastatiques 4T1et 67NR. NEAT1 est induit en corrélation avec l'activation de l'IRES du FGF1 lorsque ces cellules sont soumises à l'hypoxie, suggérant que le rôle de NEAT1 et du paraspeckle dans le contrôle de la traduction concerne l'hypoxie tumorale comme l'ischémie. Ainsi, ces travaux de thèse révèlent le grand potentiel de NEAT1 en tant que nouvelle cible thérapeutique

Regulation of the translation of (lymph)angiogenic growth factors and role of the non-coding RNA NEAT1 during hypoxic stress

La traduction est une étape de l'expression des gènes fortement régulée. Lorsque la cellule est stressée, cela bloque la synthèse protéique globale tout en activant la traduction de certains ARNm par des mécanismes alternatifs. L'un de ces mécanismes implique des structures de l'ARNm, les IRES (Internal Ribosome Entry Sites), qui permettent un recrutement de la machinerie de traduction indépendamment de l'extrémité 5' coiffée de l'ARNm. L'activité des IRES est régulée par des facteurs appelés ITAF (IRES trans-acting factor). Le stress hypoxique survient dans différentes pathologies comme l'ischémie cardiaque et le cancer. Pour répondre rapidement à ce stress, les cellules produisent des facteurs de croissance (lymph)angiogéniques qui stimulent la formation de vaisseaux sanguins et lymphatiques. Cela permet de reperfuser la zone lésée dans le cœur ischémique, ou de stimuler la croissance tumorale et la dissémination métastatique dans le cancer. Mon projet de thèse porte sur l'identification d'ITAF contrôlant la traduction des ARNm de ces facteurs de croissance lors de l'hypoxie. La première partie de ma thèse s'est focalisée sur la régulation de la traduction dans les cardiomyocytes hypoxiques. Une étude semi-globale a montré que les gènes de la (lymph)angiogenèse sont régulés majoritairement au niveau traductionnel, alors que tous les ARNm connus pour posséder des IRES sont recrutés plus activement dans les polysomes lors du stress. Nous avons montré que les IRES d'ARNm des familles FGF (fibroblast growth factor) et VEGF (vascular endothelial growth factor) sont tous activés lors de l'hypoxie précoce alors que les IRES d'ARNm non liés à la (lymph)angiogenèse sont activés plus tardivement. Enfin, nous avons identifié un nouvel ITAF, la vasohibine (VASH1), qui active spécifiquement l'IRES du FGF1, mais pas les autres IRES testés. Une analyse par PCR array indique cependant que VASH1 est capable d'inhiber ou de stimuler la traduction de nombreux autres ARNm. La recherche d'un mécanisme global d'activation des IRES des FGF et VEGF fait l'objet du deuxième chapitre de ma thèse. VASH1 n'étant pas un ITAF commun, j'ai recherché d'autres candidats. Je suis partie de l'observation qu'un autre ITAF identifié précédemment au laboratoire, p54nrb, est un composant essentiel du paraspeckle, un corps nucléaire formé en cas de stress. Nous avons émis l'hypothèse que d'autres composants du paraspeckle pourraient être des ITAFs, en particulier le long ARN non codant NEAT1 sur lequel repose sa formation. J'ai établi une corrélation entre l'induction de NEAT1 et l'activation de l'IRES du FGF1 lors de l'hypoxie. De plus, la déplétion de NEAT1 entraine une inactivation de l'IRES, suggérant que cet ARN non codant est un ITAF. J'ai mis en évidence qu'une autre protéine du paraspeckle, PSPC1, est également un ITAF. Une analyse de la composition de l'IRESome par spectrométrie de masse m'a permis d'identifier trois candidats supplémentaires : hnRNPM, rps2 et Nucléoline. Ayant élargi cette étude aux autres IRES étudiés dans le chapitre 1, nous avons démontré que p54nrb et PSPC1 sont capables d'activer plusieurs IRES alors que l'ARN non codant NEAT1, est un ITAF activateur de tous les IRES testés. NEAT1 paraît donc être la clé de l'activation des IRES, et donne au paraspeckle la fonction nouvelle de plateforme d'assemblage de l'IRESome dans les cardiomyocytes en réponse à l'hypoxie. Dans un troisième chapitre, nous avons élargi l'étude à d'autres types cellulaires, les carcinomes mammaires métastatiques et non-métastatiques 4T1et 67NR. NEAT1 est induit en corrélation avec l'activation de l'IRES du FGF1 lorsque ces cellules sont soumises à l'hypoxie, suggérant que le rôle de NEAT1 et du paraspeckle dans le contrôle de la traduction concerne l'hypoxie tumorale comme l'ischémie. Ainsi, ces travaux de thèse révèlent le grand potentiel de NEAT1 en tant que nouvelle cible thérapeutique.Translation is a highly regulated step of gene expression. During cellular stress, global protein synthesis is blocked but translation of specific subsets of mRNAs is activated by alternative mechanisms. One of these mechanisms involves an RNA structure called IRES (Internal Ribosome Entry Site), that enables the recruitment of the translational machinery in a 5' cap-independent manner. IRES activity is regulated by factors called ITAF (IRES trans-acting factor). Hypoxic stress occurs in different pathologies such as cardiac ischemia and cancer. In response to stress, the cell produces (lymph)angiogenic growth factors that stimulate blood and lymphatic vessel formation, allowing reperfusion of the injured area in ischemic heart, or stimulation of tumor growth and metastatic spread in cancer. My thesis project is focused on identification of ITAF-controlled translation of (lymph)angiogenic growth factor mRNAs during hypoxia. The first part of my thesis addresses translational regulation in hypoxic cardiomyocytes. A semi-global study showed that (lymph)angiogenic genes are mostly regulated at the translational level. Furthermore IRESs of mRNAs coding (lymph)angiogenic growth factors are activated in early hypoxia while IRESs of mRNAs non-related to (lymph)angiogenesis are activated later. Finally, we have identified a new ITAF, vasohibin (VASH1), that specifically activates the FGF1 IRES, but not the other IRES tested. A PCR array study indicates however that VASH1 is able to inhibit or stimulate translation of numerous mRNAs. Identification of a global mechanism of FGF and VEGF IRES activation is the main focus of the second chapter of my thesis. Considering that VASH1 is not a common ITAF, I searched for new candidates. I started from the observation that another ITAF previously identified in the laboratory, P54nrb, is also a component of the paraspeckle, a nuclear body formed during cellular stress. We make the hypothesis that other paraspeckle components could be ITAFs, particularly the backbone of the paraspeckle, the long non-coding RNA NEAT1. I established a correlation between NEAT1 induction in hypoxia and FGF1 IRES activation in cardiomyocytes. Moreover, NEAT1 depletion leads to inactivation of the FGF1 IRES, suggesting that NEAT1 is an ITAF. I also highlighted that another paraspeckle component, PSPC1, has an ITAF function. Analysis of IRESsome composition by mass spectrometry allowed me to identify three other candidates: hnRNPM, Rps2 and nucleolin. We then expanded the study to the other IRESs studied in chapter 1 and demonstrated that P54nrb and PSPC1 are able to activate several IRES whilst the non-coding RNA NEAT1 is a positive ITAF of all tested IRES. Thus NEAT1 seems to be the key of IRES activation, and confers on the paraspeckle the novel function of assembly platform for IRESsome formation in cardiomyocytes during hypoxia. In a third chapter, we investigated the same way in other cellular types, metastatic and non metastatic mammary carcinoma 4T1 and 67NR. NEAT1 is induced in correlation with FGF1 IRES activation when the cells are subjected to hypoxia, suggesting that the role of NEAT1 in translational control can be associated to tumoral hypoxia as well as to ischemia. Thus, this work reveal the unique potential of NEAT1 as a therapeutic target

IRES Trans-Acting Factors, Key Actors of the Stress Response

The cellular stress response corresponds to the molecular changes that a cell undergoes in response to various environmental stimuli. It induces drastic changes in the regulation of gene expression at transcriptional and posttranscriptional levels. Actually, translation is strongly affected with a blockade of the classical cap-dependent mechanism, whereas alternative mechanisms are activated to support the translation of specific mRNAs. A major mechanism involved in stress-activated translation is the internal ribosome entry site (IRES)-driven initiation. IRESs, first discovered in viral mRNAs, are present in cellular mRNAs coding for master regulators of cell responses, whose expression must be tightly controlled. IRESs allow the translation of these mRNAs in response to different stresses, including DNA damage, amino-acid starvation, hypoxia or endoplasmic reticulum stress, as well as to physiological stimuli such as cell differentiation or synapse network formation. Most IRESs are regulated by IRES trans-acting factor (ITAFs), exerting their action by at least nine different mechanisms. This review presents the history of viral and cellular IRES discovery as well as an update of the reported ITAFs regulating cellular mRNA translation and of their different mechanisms of action. The impact of ITAFs on the coordinated expression of mRNA families and consequences in cell physiology and diseases are also highlighted

Apelin modulates pathological remodeling of lymphatic endothelium after myocardial infarction

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Functional Mapping of the N-terminal Arginine Cluster and C-terminal Acidic Residues of Kir6.2 channel Fused to a G Protein-Coupled Receptor

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Extreme Arsenic Bioaccumulation Factor Variability in Lake Titicaca, Bolivia

International audienceLatin America, like other areas in the world, is faced with the problem of high arsenic (As) background in surface and groundwater, with impacts on human health. We studied As biogeochemical cycling by periphyton in Lake titicaca and the mine-impacted Lake Uru Uru. As concentration was measured in water, sediment, totora plants (Schoenoplectus californicus) and periphyton growing on stems, and As speciation was determined by X-ray absorption spectroscopy in bulk and eDtA-extracted periphyton. Dissolved arsenic was between 5.0 and 15 μg L −1 in Lake Titicaca and reached 78.5 μg L −1 in Lake Uru Uru. As accumulation in periphyton was highly variable. We report the highest As bioaccumulation factors ever measured (BAFs periphyton up to 245,000) in one zone of Lake Titicaca, with As present as As(V) and monomethyl-As (MMA(V)). Non-accumulating periphyton found in the other sites presented BAFs periphyton between 1281 and 11,962, with As present as As(III), As(V) and arsenosugars. DNA analysis evidenced several taxa possibly related to this phenomenon. Further screening of bacterial and algal isolates would be necessary to identify the organism(s) responsible for As hyperaccumulation. Impacts on the ecosystem and human health appear limited, but such organisms or consortia would be of great interest for the treatment of As contaminated water. The arsenic (As) geogenic background of surface and groundwater is naturally high in South America, predominantly originating from young volcanic rocks and their weathering products in arid oxidizing conditions 1-4. As a result, about 4.5 million people in South America are chronically exposed to high levels of As (>50 µg L −1) 5 , and certain Andean populations have developed a unique capacity to adapt to As toxicity 6,7. Concerning Andean lakes, extreme As concentrations are observed in hypersaline lakes colonized by extremophile bacterial communities 8 , and lower but still significant concentrations are observed in other, less saline lakes, which are major freshwater resources 5. In many areas of the Altiplano, mining and smelting activities add to natural rock weathering processes in the As budget 9. The biogeochemical cycling of As has been studied in freshwater and marine ecosystems, and in hypersaline environments, but its trophic transfer and speciation in living organisms mainly concerns the marine environment and As contaminated freshwater systems 10. In the high altitude lakes of the Andean Altiplano (above 3500 m asl), shallow zones (<2 m) are colonized by totoras (Schoenoplectus californicus, syn Scirpus californicus). These macrophytes were used for construction purposes in traditional Andean culture. Nowadays, they are mainly used as cattle fodder and have been tested successfully in constructed wetlands in North America for the removal of metals (Zn, Cu, Cd, Pb) and nutrients from wastewater 11-14. The filtration potential of wetland plants does not rely on absorption by the plant, but on physico-chemical and biologically driven processes taking place on submerged stems and in the rhizosphere 15,16. In particular, the periphyton, an assemblage of algae and bacteria forming

Lymphatic Vasculature Requires Estrogen Receptor- Signaling to Protect From Lymphedema

International audienceObjective Estrogens exert beneficial effect on the blood vascular system. However, their role on the lymphatic system has been poorly investigated. We studied the protective effect of the 17 estradiolthe most potent endogenous estrogenin lymphedemaa lymphatic dysfunction, which results in a massive fluid and fat accumulation in the limb. Approach and Results Screening of DNA motifs able to mobilize ERs (estrogen receptors) and quantitative real-time polymerase chain reaction analysis revealed that estradiol promotes transcriptional activation of lymphangiogenesis-related gene expression including VEGF (vascular endothelial growth factor)-D, VEGFR (VEGF receptor)-3, lyve-1, and HASs (hyaluronan synthases). Using an original model of secondary lymphedema, we observed a protective effect of estradiol on lymphedema by reducing dermal backflowa representative feature of the pathology. Blocking ER by tamoxifenthe selective estrogen modulatorled to a remodeling of the lymphatic network associated with a strong lymphatic leakage. Moreover, the protection of lymphedema by estradiol treatment was abrogated by the endothelial deletion of the receptor ER in Tie2-Cre; ERlox/lox mice, which exhibit dilated lymphatic vessels. This remodeling correlated with a decrease in lymphangiogenic gene expression. In vitro, blocking ER by tamoxifen in lymphatic endothelial cells decreased cell-cell junctions, inhibited migration and sprouting, and resulted in an inhibition of Erk but not of Akt phosphorylation. Conclusions Estradiol protection from developing lymphedema is mediated by an activation of its receptor ER and is antagonized by tamoxifen. These findings reveal a new facet of the estrogen influence in the management of the lymphatic system and provide more evidence that secondary lymphedema is worsened by hormone therapy

Association between transportation noise exposure and type 2diabetes risk in the French E3N cohort

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Performance, meat quality and blood parameters in four strains of organic broilers differ according to range use

Chicken meat production in organic systems involves free-range access where animals can express foraging and locomotor behaviours. These behaviours may promote outdoor feed intake, but at the same time induce a loss of energy in exploring. More generally, the relationship of range use with metabolism, welfare, health, growth performance and meat quality needs to be better understood. We studied four strains of intermediate (JA757) to slow-growing (S757N, White Bresse and a dual-purpose strain) meat-type chickens with outdoor access. We selected 25 males high- (HR) and low-rangers (LR) per strain. Only in JA757, HR exhibited lower body weight before range access, which may have predisposed them to use the range more. Carcass weight and/or carcass yield were significantly lower in HR compared to LR, showing a negative trade-off between range use and growth performance in all strains. Breast meat yellowness was higher in HR compared to LR in JA757 and the dual-purpose strain, probably due to carotenoids intake from the grass. No relationship between range use and welfare indicators at slaughter was reported whatever the strain. Chicken metabolism differed by range use as HR and LR diverged for blood biomarkers of oxidative and metabolic status, innate and inflammatory system response