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Cid, Samper Fernando 1991. „Computational approaches to characterize RNP granules“. Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/668449.
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Ribonucleoprotein granules (RNP granules) are liquid-liquid phase separated
complexes composed mainly by proteins and RNA. They are responsible of many
processes involved in RNA regulation. Alterations in the dynamics of these proteinRNA complexes are associated with the appearance of several neurodegenerative
disorders such as Amyotrophic Lateral Sclerosis ALS or Fragile X Tremor Ataxia
Syndrome FXTAS. Yet, many aspects of their organization as well as the specific
roles of the RNA on the formation and function of these complexes are still unknown.
In order to study RNP granules structure and formation, we integrated several state of
the art high-throughput datasets. This includes protein and RNA composition obtained
from RNP pull-downs, protein-RNA interaction data from eCLIP experiments and
transcriptome-wide secondary structure information (produced by PARS). We used
network analysis and clustering algorithms to understand the fundamental properties
of granule RNAs. By integrating these properties, we produced a model to identify
scaffolding RNA. Scaffolding RNAs are able to recruit many protein components into
RNP granules. We found that the main protein components of stress granules (a kind
of RNP granules) are connected through protein-RNA interactions. We also analyzed
the contribution of RNA-RNA interactions and RNA post-transcriptional
modifications on the granule internal organization.
We applied these findings to understand the biochemical pathophysiology of FXTAS
disease, employing as well some novel experimental data. In FXTAS, a mutation on
the FMR1 gene produces a 5´microsatellite repetition that enhances its scaffolding
ability. This mutated mRNA is able to sequester some important proteins into nuclear
RNP granules, such as TRA2A (i.e. a splicing factor), impeding their normal function
and therefore producing some symptoms associated with the progress of the disease.
The better understanding of the principles governing granules formation and structure
will enable to develop novel therapies (e.g. aptamers) to mitigate the development of
several neurodegenerative diseases.Los gránulos ribonucleoproteicos (gránulos RNP, por sus siglas en inglés) son complejos producidos mediante separación líquido-líquido y están constituidos principalmente por proteínas y ARN. Son responsables de numerosos procesos involucrados con la regulación del ARN. Alteraciones en la dinámica de estos complejos de proteínas y ARN están asociadas con la aparición de diversas enfermedades neurodegenerativas como el ELA o FXTAS. Sin embargo, todavía se desconocen muchos aspectos relativos a su organización interna así como las contribuciones específicas del RNA en la formación y funcionamiento de estos complejos. A fin de estudiar la estructura y formación de los gránulos RNP, hemos integrado varias bases de datos de alto rendimiento de reciente aparición. Esto incluye datos sobre la composición proteica y en ARN de los RNP, sobre la interacción de proteínas y ARN extraída de experimentos de eCLIP y sobre la estructura secundaria del transcriptoma (producida mediante PARS). Todos estos datos han sido procesados para comprender las propiedades fundamentales de los ARNs que integran los gránulos, mediante el empleo de métodos computacionales como el análisis de redes o algoritmos de agrupamiento. De esta manera, hemos producido un modelo que integra varias de estas propiedades e identifica candidatos denominados ARNs de andamiaje. Definimos ARNs de andamiaje como moléculas de ARN con una alta propensión a formar gránulos y reclutar un gran número de componentes proteicos a los gránulos RNP. También hemos encontrado que las interacciones proteína-ARN conectan los principales componentes proteicos de consenso de los gránulos de estrés (un tipo específico de gránulos RNP). También hemos estudiado la contribución de las interacciones ARN-ARN y las modificaciones post-transcriptionales del RNA en la organización interna del gránulo. Hemos aplicado estos resultados para la comprensión de la fisiopatología molecular de FXTAS, empleando también algunos datos experimentales originales. En FXTAS, una mutación en el gen FMR1 produce una repetición de microsatélite en 5´ que incrementa su capacidad como ARN de andamiaje. Este mARN mutado es capaz de secuestrar algunas proteínas importantes como TRA2A (un factor de ayuste alternativo) en gránulos RNP nucleares, impidiendo su normal funcionamiento y por consiguiente produciendo algunos síntomas asociados con el progreso de la enfermedad. Una mejor comprensión de los principios que gobiernan la formación y estructura de los gránulos puede permitir desarrollar nuevas terapias (ej: aptámeros) para mitigar el desarrollo de diversas enfermedades neurodegenerativas.
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Vijayakumar, Jeshlee Cyril. „Rôle du domaine de type prion de Imp dans la régulation des granules RNP neuronaux“. Thesis, Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4099/document.
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Les ARNms des cellules eucaryotes sont liés à des protéines de liaison aux ARNs (RBPs) et empaquetés au sein d’assemblages macro-moléculaires appelés granules RNP. Dans les cellules neuronales, les granules RNP de transport sont impliqués dans le transport d’ARNms spécifiques jusqu’aux axones et dendrites, ainsi que dans leur traduction locale en réponse à des signaux externes. Bien que peu de choses soient connues sur l’assemblage et la régulation de ces granules in vivo, des résultats récents ont indiqué que la présence de domaines de type prion (PLDs) dans les RBPs facilite les interactions protéines-protéines et protéines-ARN, favorisant ainsi la condensation de complexes solubles en granules RNP. La RBP conservée Imp est un composant central de granules RNP qui sont transportés dans les axones lors du remodelage neuronal chez la drosophile. De plus, la fonction de Imp est nécessaire au remodelage des axones lors de la maturation du système nerveux de drosophile. Une analyse de la séquence de la protéine Imp a révélé qu’en plus de quatre domaines de liaison aux ARNs, Imp contient un domaine C-terminal désordonné enrichi en Glutamines et Serines, deux propriétés caractéristiques des domaines PLDs. Lors de ma thèse, j’ai étudié la fonction de ce PLD dans le contexte de l’assemblage et du transport des granules RNP. J’ai observé en culture de cellules que les granules Imp s’assemblent en absence de PLD, bien que leur nombre et leur taille soient augmentés. Des protéines présentant une séquence PLD mélangée, au contraire, s’accumulent dans des granules au nombre et à la taille normale, indiquant que l’état désordonné de ce domaine, et non sa séquence primaire, est essentiel à l’homéostasie des granules. De plus, des expériences de FRAP réalisées en culture de cellule et in vivo ont révélé que le domaine PLD de Imp favorise la dynamique des granules. In vivo, ce domaine est nécessaire et suffisant à l’accumulation axonale de Imp. Comme montré par une analyse en temps réel, l’absence de domaine PLD aboutit également à une diminution du nombre de granules axonaux motiles. Fonctionnellement, le domaine PLD de Imp est essentiel au remodelage neuronal car des protéines sans ce domaine ne sont pas capables de supprimer les défauts de repousse axonale observés après inactivation de imp. Enfin, la génération d’un variant de Imp dans lequel le domaine PLD a été déplacé en N-terminus a montré que les fonctions du PLD dans le transport des granules et dans leur assemblage sont découplées, et que la modulation des propriétés des granules Imp médiée par le domaine PLD n’est pas nécessaire au remodelage neuronal in vivo. En conclusion, mes résultats ont montré que le domaine PLD de Imp n’est pas nécessaire à l’assemblage des granules RNP Imp, mais régule leur nombre et leur dynamique. De plus, mon travail a mis en évidence une fonction inattendue pour un domaine PLD dans le transport axonal et le remodelage des neurones lors de la maturation du système nerveuxEukaryotic mRNAs are bound by RNA Binding Proteins (RBP) and packaged into diverse range of macromolecular assemblies named RNP granules. In neurons, transport RNP granules are implicated in the transport of specific mRNAs to axons or dendrites, and in their local translation in response to external cues. Although little is known about the assembly and regulation of these granules in vivo, growing evidence indicates that the presence of Prion Like domains (PLD) within RBPs favours multivalent protein–protein and protein-RNA interactions, promoting the transition of soluble complexes into RNP granules. The conserved RBP Imp is as a core component of RNP granules that are actively transported to axons upon neuronal remodelling in Drosophila. Furthermore, Imp function was shown to be required for axonal remodelling during Drosophila nervous system maturation. Analyses of the domain architecture of the Imp protein revealed that, in addition to four RNA binding domains (RBD), Imp contains a Cterminal domain showing a striking enrichment in Glutamines and Serines, which is one of the characteristics of a PLD. During my PhD, I explored the function of the PLD in the context of granule assembly and transport. In cultured cells, I observed that Imp granules assembled in the absence of the PLD, however their number and size were increased. Proteins with scrambled PLD sequence accumulated in granules of normal size and number, implying that the degree of disorder of this domain, and not its sequence, is essential for granule homeostasis. Moreover, FRAP experiments, performed on cultured cells and in vivo, revealed that Imp PLD is important to maintain the turnover of these granules. In vivo, this domain is both necessary and sufficient for efficient transport of Imp granules to axons. These defects are associated with a reduction on the number of motile granules in axons. Furthermore, mutant forms lacking the PLD do not rescue the axon remodelling defects observed upon imp loss of function. Finally, a swapping experiment in which I moved Imp PLD from the C-terminus to the N-terminus of the protein revealed that the functions of Imp PLD in granule transport and homeostasis are uncoupled, and that PLD-dependent modulation of Imp granule properties is dispensable in vivo. Together, my results show that Imp PLD of is not required for the assembly of RNP granules, but rather regulates granule number and dynamics. Furthermore, my work uncovered an unexpected in vivo function for a PLD in axonal transport and remodelling during nervous system maturation
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Pushpalatha, Kavya Vinayan. „Remodelage des condensats RNP neuronaux au cours du vieillissement chez la drosophile“. Electronic Thesis or Diss., Université Côte d'Azur, 2021. http://theses.univ-cotedazur.fr/2021COAZ6007.
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Dans la cellule, les molécules d’ARN s’assemblent avec des protéines de liaison aux ARNs pour former des assemblages macromoléculaires très dynamiques appelés granules ribonucléoprotéiques (RNP). Ces assemblages régulent l’expression génique en contrôlant le transport, la stabilité et/ou la traduction des ARNs associés. Des travaux réalisés in vitro ont montré que la formation et la composition des granules RNP reposent sur l’établissement de réseaux denses d’interactions établis entre protéines et ARN, ainsi que sur leur stoechiométrie. Comment les propriétés des granules RNP sont régulées en contexte physiologique, et en particulier lors du vieillissement, est cependant actuellement peu connu. Mon projet de thèse visait à répondre à cette question par une étude in vivo des granules RNP présents dans les cellules neuronales du cerveau de drosophile.A cette fin, j’ai analysé dans des cerveaux d’âge croissant des granules RNP caractérisés par la présence de la protéine de liaison aux ARNs Imp/ZBP1 et de la DEAD-box hélicase Me31B/DDX6. Mes travaux ont révélé une augmentation progressive de la condensation de Imp et Me31B en larges granules au cours du vieillissement. Ces granules sont dynamiques et ne co-localisent pas avec des marqueurs d’agrégation, suggérant qu’ils ne correspondent pas à des agrégats protéiques statiques. Remarquablement, la condensation de Imp et Me31B est associée à la perte des granules Me31B+ Imp- observées dans les cerveaux jeunes, et à la coalescence de Me31B et Imp pour former des granules uniques Me31B+ Imp+. De plus, ce processus est accompagné d’une inhibition spécifique de la traduction des ARNms associés aux granules, parmi lesquels profilin. Par une analyse fonctionnelle, j’ai mis en évidence qu’une modification de la concentration en Me31B est responsable de la condensation de Me31B dans les cerveaux âgés. Alors qu’une augmentation de la quantité de Me31B est observée au cours du vieillissement, enlever une copie de me31B supprime la condensation age-dépendante de ce composant. Étant donné que la condensation de Imp n’est que partiellement affectée dans ce contexte, j’ai réalisé un crible génétique afin d’identifier des régulateurs de ce processus. Ceci m’a permis de montrer que l’activité de la kinase PKA est essentielle d’une part à la condensation de Imp chez les drosophiles âgées, et d’autre part à la répression traductionnelle des ARNms associés aux granules.En conclusion, mon travail a montré pour la première fois que les propriétés des granules RNP neuronaux sont modifiées au cours du vieillissement, un phénomène qui ne reflète pas une altération générale de l’homéostasie des ARNs, mais plutôt une modulation spécifique de la concentration en composants RNP combinée à l’activité de kinase conservée. Ces résultats démontrent comment les systèmes biologiques peuvent moduler des paramètres clés initialement identifiés dans des contextes in vitro, et ouvrent de nouvelles perspectives dans le domaine de la régulation de l’expression génique au cours du vieillissementNascent mRNAs complex with RNA binding proteins (RBPs) to form highly dynamic, phase-separated organelles termed ribonucleoprotein (RNP) granules. These macromolecular assemblies can regulate gene expression by controlling the transport, decay and/or translation of associated RNA molecules. As mostly shown in vitro, RNP granule assembly and function rely on the interaction networks established by individual components and on their stoichiometry. To date, how the properties of constitutive RNP granules are regulated in different physiological context is unclear. In particular, the impact of physiological aging is unclear. My PhD project aimed at addressing this question by analyzing in vivo in long-lived neuronal cells the properties of RNP granules. To this end, I have analysed in flies of increasing age RNP granules characterized by the presence of the conserved RBP Imp/ZBP1 and DEAD-box RNA helicase Me31B/DDX6. Strikingly, a progressive increase in the condensation of Imp and Me31B into granules was observed upon aging. The large granules observed in aged flies were dynamic, contained profilin mRNA, and did not colocalize with Ubiquitin or aggregation markers, suggesting that they do not correspond to static protein aggregates. Increased condensation also associated with the loss of Me31B+ Imp- granules observed in young brains and the collapse of RNP component into a unique class of Me31B+ Imp+ granule. Furthermore, it was accompanied by a specific inhibition of the translation of granule-associated mRNAs, among which the Imp RNA target profilin. Through functional analysis, I uncovered that changes in Me31B stoichiometry trigger Me31B condensation in aged flies. While an increase in Me31B protein levels was observed upon aging, decreasing the dosage of Me31B suppressed its age-dependent condensation. As Imp condensation was only partially suppressed in this context, I performed a selective screen to identify regulators of this process. This revealed that downregulating PKA activity by different genetic means both drastically reduced Imp recruitment and prevented the age-dependent translational repression of granule-associated mRNAs. Taken together, my work thus showed for the first time in vivo that the properties of neuronal RNP granules change upon aging, a phenomenon that does not reflect general alterations in RNA homeostasis but rather specific modulation of RNP component stoichiometry and kinase activity. These results demonstrate how biological systems can modulate key parameters initially defined based on in vitro framework, and also open new perspectives in the field of age-dependent regulation of gene expression
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Shah, Khyati H. „REGULATION, COMPOSITION AND FUNCTIONS OF RNP GRANULES IN QUIESCENT CELLS OF SACCHAROMYCES CEREVISIAE“. The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1417541239.
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Formicola, Nadia. „Remodelage des granules ARN en réponse à l’activité neuronale“. Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2019. http://theses.univ-cotedazur.fr/2019AZUR6008.
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Une des questions les plus fascinantes – et les plus ouvertes – en neuroscience est de comprendre comment les cellules neuronales contribuent à la formation, le maintien puis le rappel des souvenirs. Des travaux antérieurs ont montré que la formation de la mémoire à long-terme (MLT) requiert la synthèse de novo de protéines, impliquant non seulement la traduction d’ARNs nouvellement transcrits, mais aussi la traduction locale, induite par l’expérience, d’ARNms latents transportés et stockés dans les synapses. En vue de leur transport et du contrôle de leur traduction, les ARNms sont empaquetés avec des protéines de liaison aux ARNs (RBP), qui sont majoritairement des répresseurs de traduction, dans des granules ribonucléoprotéiques (RNP). La manière dont les granules RNP neuronaux sont remodelés en réponse à l’activité neuronale pour lever la répression traductionelle des ARNms est pour l’instant peu claire. En outre, l’impact fonctionnel d’un tel remodelage sur l’établissement de la MLT reste à démontrer in vivo. L’objectif de mon doctorat était 1) d’étudier les mécanismes in vivo qui sous-tendent le remodelage des granules RNP neuronaux ; 2) de tester l’hypothèse que les granules RNP pourraient être impliqués dans les mécanismes de renforcement de la MLT en régulant l’expression génétique. Dans cette optique, j’ai utilisé comme modèle des granules RNP contenant la RBP conservée Imp chez la drosophile. Tout d’abord, j’ai étudié l’impact de l’activité neuronale sur les propriétés des granules RNP Imp, en traitant des explants de cerveau soit avec du KCl, soit avec le neuromodulateur Tyramine. Dans les deux cas, un désassemblage des granules RNP Imp - caractérisé par une dé-granulation à la fois de Imp et d’autres composants – est observé. Le désassemblage des granules RNP est réversible après retrait de la tyramine, et n’a pas été observé dans les neurones hyperpolarisés. Il ne dépend pas strictement du domaine de type prion qui se trouve à l’extrémité carboxy-terminale de Imp, un domaine connu pour être impliqué dans l’homéostasie des granules RNP. De plus, mes données suggèrent que ce désassemblage soit lié à une augmentation de la traduction des ARNms associés, ce qui est cohérent avec un modèle dans lequel le remodelage des granules RNP induit par l’activité des neurones induit une dé-répression de la traduction. Ensuite, j’ai recherché les mécanismes contrôlant le remodelage des granules RNP. Un candidat pour cette régulation était CamkII, une kinase conservée activée par le calcium, et identifiée comme partenaire de Imp dans une analyse d’immunoprécipitation-spectrométrie de masse. Au cours de mon doctorat, j’ai pu valider l’intéraction Imp-CamkII et montrer qu’elle n’est pas médiée par l’ARN, mais dépend de l’activité de CamkII. De plus, j’ai montré qu’inhiber l’activité de CamkII empêche le désassemblage des granules RNP Imp observé lors de l’activation neuronale, suggérant que CamkII pourrait être impliquée dans le remodelage des granules RNP Imp induit par l’activité neuronale. Ces résutats sont particulièrement intéressants dans le contexte de l’établissement de la MLT, car CamkII est depuis longtemps reconnue comme y étant essentielle. Plus encore, nous avons récemment démontré chez la drosophile qu’inactiver la fonction de Imp dans une population de neurones du cerveau central impliquée dans l’apprentissage et la mémoire – les neurones du Mushroom Body – altère radicalement la MLT. En conclusion, mes résultats sont cohérents avec un modèle où le remodelage des granules RNP Imp en réponse à l’activation neuronale dépend de CamkII, et pourrait contribuer à la formation de la MLT in vivoOne of the most fascinating – and still open – questions in neuroscience is how neuronal cells can form, store and then recall memories. Previous work has shown that Long-term memory (LTM) formation requires de novo protein synthesis, involving not only translation of newly transcribed RNAs, but also local, experience-induced translation of quiescent mRNAs carried and stored at synapses. For their transport and translational control, mRNAs are packaged with regulatory RNA binding proteins (RBPs), mainly translational repressors, into ribonucleoprotein (RNP) granules. To date, how neuronal RNP granules are remodelled in response to neuronal activity to relieve translation repression of mRNAs is unclear. Furthermore, the functional impact of such a remodelling in the establishment of long-term memories remains to be demonstrated in vivo. The objective of my PhD was to 1) investigate the in vivo mechanisms underlying activity-dependent remodelling of neuronal RNP granules; 2) test the hypothesis that RNPs could be involved in LTM-underlying mechanisms by regulating gene expression. To this end, I used as paradigm RNPs containing the conserved RBP Imp in Drosophila. First, I studied the impact of neuronal activity on Imp RNP properties by treating Drosophila brain explants with either KCl or the tyramine neuropeptide. In both cases, a disassembly of Imp RNPs was observed, characterized by a loss of both Imp and other RNP-component granular patterns, and a de-clustering of RNP-associated mRNA molecules. RNP disassembly could be reverted upon Tyramine withdrawal and was not observed in hyperpolarized neurons. Furthermore, my data suggest that RNP-disassembly is linked to increased translation of associated mRNAs, consistent with a model in which activity-induced RNP remodelling would lead to translational de-repression. Second, I investigated the mechanisms controlling RNP remodelling. A candidate regulator was CamkII, a conserved Ca2+ -activated kinase identified as a partner of Imp in an IP-Mass Spectrometry analysis. During my PhD, I could validate the Imp-CamkII interaction and showed that it is not mediated by RNA but depends on CamkII activity. Furthermore, I showed that inactivating CamkII function prevents the disassembly of Imp RNPs observed upon neuronal activation of brain explants, suggesting that CamkII may be involved in the activity-dependent remodelling of Imp RNP granules. These results are particularly interesting in the context of establishment of LTM, as CamkII has long been recognized as essential for LTM. Moreover, we recently showed in Drosophila that interfering with Imp function in a population of CNS neurons involved in learning and memory – the Mushroom Body γ neurons -, dramatically impairs LTM and that this effect relies on Imp C-terminal Prion-like domain, a domain known to be involved in RNP homeostasis. Altogether, my thesis work suggests a model where CamkII-dependent remodelling of Imp RNPs in response to neuronal activation might underlie LTM formation in vivo
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Agostini, Federico 1985. „Predictions of RNA-binding ability and aggregation propensity of proteins“. Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/318159.
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RNA-binding proteins (RBPs) control the fate of a multitude of coding and non-coding transcripts. Formation of ribonucleoprotein (RNP) complexes fine-tunes regulation of post-transcriptional events and influences gene expression. Recently, it has been observed that non-canonical proteins with RNA-binding ability are enriched in structurally disordered and low-complexity regions that are generally involved in functional and dysfunctional associations. Therefore, it is possible that interactions with RNA protect unstructured protein domains from aberrant associations or aggregation. Nevertheless, the mechanisms that prevent protein aggregation and the role of RNA in such processes are not well understood. In this work, I will describe algorithms that I have developed to predict protein solubility and to estimate the ability of proteins and transcripts to interact. I will illustrate applications of computational methods and show how they can be integrated with high throughput approaches. The overarching goal of my work is to provide experimentalists with tools that facilitate the investigation of regulatory mechanisms controlling protein homeostasis.Las proteínas de unión de ARN son responsables de controlar el destino de una multitud de transcriptos codificantes y no codificantes. De hecho, la formación de complejos de ribonucleoproteínas (RNP) afina la regulación de una serie de eventos post-transcripcionales e influye en la expresión génica. Recientemente, se ha observado que las proteínas con capacidad no canónica de unión al ARN se enriquecen en las regiones estructuralmente desordenadas y de baja complejidad, que son las que participan generalmente en asociaciones funcionales y disfuncionales. Por lo tanto, es posible que interactuar con el ARN pudiera ser una manera de proteger las proteínas no estructuradas de asociaciones aberrantes o de agregación. Sin embargo, los mecanismos que impiden la agregación de proteínas y la función del ARN en tales procesos no están bien descritas. En este trabajo, se describen los me ́todos que he desarrollado para predecir la solubilidad de proteínas y para estimar la capacidad de transcriptos y proteínas de interactuar. De otra parte, voy a ilustrar sus aplicaciones y explicar como los métodos de bajo rendimiento han evolucionado a un mayor rendimiento. El objetivo final es proporcionar instrumentos a los investigadores experimentales que se pueden utilizar para facilitar la investigación de los mecanismos reguladores que controlan la homeostasis molecular.
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Al-Sailawi, Majid. „Investigating RNA granules formation during caliciviruses infection“. Thesis, University of Surrey, 2015. http://epubs.surrey.ac.uk/809289/.
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Human norovirus (HuNV) is a member of the calicivirus family and is a major cause of viral gastroenteritis worldwide. Due to the absence of a suitable cell culture system, HuNoV replication mechanisms are poorly understood, but two animal caliciviruses, Feline calicivirus (FCV) and Murine Norovirus (MNV) provide models to increase our understanding of norovirus biology. Unlike cellular mRNAs, the calicivirus RNA genome does not possess a 5' cap structure but instead has a 13–15 kDa viral protein, genome linked (VPg) directing translation, hijacking the host protein synthesis machinery. The viral life cycle requires separated events occurring at different times since viral transcripts are used as the template both for translation (mRNA) and replication (genomic RNA). Therefore mechanisms are required to control the viral RNA fate. In eukaryotes, during stress conditions, mRNAs can be stored in subcellular compartments such as stress granules to stall their translation or in processing bodies to be degraded. Recent evidence indicates that these compartments also play an important role during the viral life cycle. Therefore, using immunofluorescence microscopy we set out to investigate how FCV and MNV infection regulate the formation of G3BP1- and PABP-1-containing stress granules and DCP-1-containing processing bodies to address whether these cytoplasmic granules could play a role during the viral life cycle. We have now shown that FCV has the ability to prevent stress granules formation during infection and that this is important for replication in CRFK and FEA cells. Using FCV-free supernatant from infected CRFK cells and immunofluorescence microscopy, we have also shown that during infection, the formation of stress granules is induced in a paracrine manner in uninfected cells via a messenger molecule released from infected cells. We hypothesize that this could reflect a new antiviral role for stress granules. Furthermore, MNV and FCV infection also led to the disruption of processing-bodies assembly. Overall, this study revealed that caliciviruses modulate the RNA granules during infection and that this could be part of viral mechanism to counteract the antiviral response.
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Oh, Seong-Wook. „Functional Analysis of RIG-I and RNP Complexes in the Antiviral Interferon System“. 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/215973.
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Pizzinga, Mariavittoria. „Granules of translation factor mRNAs and their potential role in the localisation of the translation machinery to regions of polarised growth“. Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/granules-of-translation-factor-mrnas-and-their-potential-role-in-the-localisation-of-the-translation-machinery-to-regions-of-polarised-growth(9cb42e69-3c8c-4f10-b79f-ba8261be4430).html.
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The subcellular localisation of mRNA is a widespread mechanism to determine the fate of mRNAs in eukaryotes. Translationally repressed mRNAs localise to P-bodies and stress granules where their decay and storage, respectively, are directed. In a study from the Ashe lab, specific mRNAs were identified to localise, in actively growing S. cerevisiae, to cytoplasmic granules that do not seem to be related to P-bodies or stress granules but appear to be associated with active translation (Lui et al., 2014).It is possible that this might represent a strategy to co-regulate the expression of proteins from the same pathway. In the work of this thesis, microscopy techniques to visualise RNAs in live cells were used to extend the localisation analysis to several mRNAs encoding translation factors. The investigated transcripts were all found to localise to mostly one or two cytoplasmic granules per cell and would sometimes overlap with other transcripts, suggesting that each granule contains a mixture of mRNAs. Granules tend to migrate to the bud tip and may provide the daughter cell with a "start-up kit" of transcripts essential for rapid growth. A similar pattern can be observed in yeast cells growing undergoing filamentous growth, with granules harbouring translation factor transcripts often found in the apical quarter of the elongated cell. Although the mechanism by which the granules form and their protein composition are not yet known, high-throughput genetic screens performed as part of this work offer some insight into factors that might be involved in granule assembly and proteins that partially overlap with the granules. We propose that granules containing translation factor mRNAs might be functioning as a specialised factory for the translation machinery and are possibly being directed to the point in the cell where the rhythm of protein production is highest.
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Kuznicki, Kathleen. „The function of the germline rna helicase (GLH) genes in caenorhabditis elegans“. free to MU campus, to others for purchase, 2000. http://wwwlib.umi.com/cr/mo/fullcit?p9988682.
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11
Chitiprolu, Maneka. „Novel Regulatory Mechanisms of Autophagy in Human Disease: Implications for the Development of Therapeutic Strategies“. Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38441.
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The dysfunction of autophagy pathways has been linked to the development and progression of numerous human diseases, in particular neurological disorders and cancer. Investigating these pathological autophagy mechanisms is essential to gain insights into the underlying disease mechanisms, identify novel biomarkers, and develop targeted therapies. In this thesis, I present three manuscripts that investigate the regulatory mechanisms of autophagy machinery in human diseases.
In the first manuscript (Chitiprolu et al., 2018), we investigated the mechanism of p62-mediated selective autophagic clearance of RNA stress granules implicated in Amyotrophic Lateral Sclerosis (ALS). Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. By employing mass spectrometry, high resolution imaging and biochemical assays, we demonstrated that the autophagy receptor p62 associates with C9ORF72 to eliminate stress granules by autophagy. This requires p62 to associate with proteins that are symmetrically methylated on arginines. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.
The second manuscript (Guo, Chitiprolu et al., 2014) describes the mechanism by which autophagy degrades retrotransposon RNA from both long and short interspersed elements, thereby preventing new retrotransposon insertions into the genome. By employing quantitative imaging tools, we demonstrated that retrotransposon RNA localizes to RNA granules that are selectively degraded by the autophagy receptors NDP52 and p62. Mice lacking a copy of Atg6/Beclin1, a gene critical for autophagy, also accumulate both retrotransposon RNA and genomic insertions. This suggests a mechanism for the increased tumorigenesis upon autophagy inhibition and therefore a role for autophagy in tempering evolutionary change.
Finally, the third manuscript (Guo, Chitiprolu et al., 2017) examines the intersection of autophagy machinery with exosome release and function in cancer metastasis. By employing dynamic light scattering, Nanosight particle tracking, electron microscopy, super-resolution imaging and Western blotting, we robustly quantified exosome identity and purity in multiple cell lines. We demonstrated that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings delineate autophagy-independent pathways by which autophagy-related genes can contribute to metastasis.
Taken together, data presented in the three manuscripts highlight the molecular mechanisms of autophagy core machinery proteins and selective receptors such as Atg5, p62 and NDP52, in the pathogenesis of cancer and neurodegeneration. In these diseases characterized by mutations in autophagy pathways, the mechanisms we uncover provide insights into their causes and serve as potential therapeutic targets.
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Ferrier, Emilie. „Rôle et mode d'action de l'UTP : RNA Uridylyltransférase URT1 dans l'uridylation et la dégradation des ARNm chez Aradopsis thaliana“. Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ053/document.
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La dégradation des ARN est un mécanisme essentiel à la régulation de l’expression des génomes. L’importance de l’uridylation dans les mécanismes de dégradation des ARN commence juste à être appréciée. Cette thèse présente l’étudede l’UTP :RNA Uridylyltransferase 1 (URT1) et de son rôle dans la dégradation des ARN chez Arabidopsis thaliana. L’étude des propriétés catalytiques de URT1 montre que cette uridylyltransférase est intrinsèquement spécifique des UTP et distributive pour les premières uridines ajoutées. URT1 est responsable in vivo de l’uridylation des ARNm après une étape de déadénylation, protégeant leur extrémité 3’ et polarisant la dégradation de 5’ en 3’. URT1 est localisée dans le cytosol au niveau des granules de stress et des processing bodies. Le mécanisme d’adressage de URT1 dans les processing bodies implique une partie de la région N terminale prédite comme intrinsèquement désorganisée, alors que le domainenucléotidyltransférase C terminal semble suffisant pour permettre l’adressage de URT1 au niveau des processing bodies et granules de stress en réponse à un stress thermique. Ces travaux de thèse ont permis de mieux comprendre les mécanismes et les rôles de l’uridylation dans la dégradation des ARNm chez Arabidopsis. Ils ouvrent des perspectives dans l’étude d’autres fonctions de l’uridylation comme l’inhibition de la traductionRNA degradation is an essential mechanism for the regulation of genome expression. The importance of uridylation for RNA degradation is just emerging. This thesis presents the study of URT1 (UTP :RNA Uridylyltransferase 1) and its role in RNA degradation in Arabidopsis thaliana. URT1 is an uridylyltransferase intrinsically and strictly specific for UTP and is distributive for the first nucleotides added. URT1 uridylates mRNA in vivo after a deadenylation step. This uridylation protects mRNA’s3’ end from further attacks and polarise degradation in the 5’ to 3’ direction. This protection of 3’ ends by uridylation and its conferred polarity of 5’ to 3’ degradation are also detected in polysomes. Uridylation is therefore likely important in case of cotranslational degradation of mRNAs. A region in URT1’s N terminal region predicted to be intrinsically disorganised is required for addressing URT1 to processing bodies. However, following heat shock, the nucleotidyltransferase domain present in the C terminal region of URT1 is sufficient to address URT1 to both processing bodies and stress granules, This work contributes to a better understanding of the mechanisms and roles of uridylation in RNA degradation in Arabidopsis thaliana. These results also open perspectives for studying other functions of uridylation such as translation inhibition
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Findley, Seth David. „Maelstrom and Drosophila nuage /“. Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/9255.
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14
Lockwood, Donovan Blair. „TDP-43 Modulation of PABP Positive, RNA Stress Granule Formation during Oxidative Stress“. Thesis, The University of Arizona, 2015. http://hdl.handle.net/10150/579304.
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RNA dysregulation is a recently recognized disease mechanism in amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease characterized by muscle atrophy and death of both upper and lower motor neurons. A key feature of the disease is the mislocalization of the RNA binding protein TDP-43 and formation of TDP-43 containing cytoplasmic aggregates in motor neurons and surrounding glia. TDP-43 is known to associate with stress granules, and recent studies in mammalian cell culture have indicated that pathological TDP-43 aggregates may arise from RNA stress granules following prolonged stress. We set out to test this hypothesis by investigating the interaction between PolyA Binding Protein (PABP), a known core RNA stress granule component, and TDP-43. Here we show that PABP colocalizes with TDP-43 in a variant dependent manner. Given that the highest risk factor for ALS is aging, an attractive model is that age-related oxidative stress triggers formation of toxic cytoplasmic aggregates from TDP-43 containing stress granules. We have therefore begun investigations using a time course, and live imaging of RNA stress granules under oxidative stress to determine if this leads to an altered RNA stress granule dynamics in cultured motor neurons. These studies will yield a better understanding of the mechanisms that lead to the toxic cytoplasmic aggregates in cases of ALS.
15
Schreier, Juliane [Verfasser]. „Role of PKCε in RNA granule formation and protein translation / Juliane Schreier“. Berlin : Freie Universität Berlin, 2013. http://d-nb.info/104162090X/34.
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16
Emara, Mohamed Maged. „Analysis of the Cellular Proteins, TIA-1 and TIAR, and their Interaction with the West Nile Virus (WNV) 3' SL Minus-Strand RNA“. Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/70.
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The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.
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Lin, Sau-wah Selma. „A study of microRNA-132 and -212 in murine granulosa cells during folliculogenesis“. Click to view the E-thesis via HKUTO, 2010. http://sunzi.lib.hku.hk/hkuto/record/B43909851.
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18
Lau, Pok, und 劉博. „MicroRNAs associated with granulin-epithelin precursor in hepatocellular carcinoma“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/206753.
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Hepatocellular carcinoma (HCC) is the major type of liver cancer. In Hong Kong, thousands of deaths are related to this disease every year. Hepatitis B virus (HBV) infection is one of the major risk factors of HCC development. The high prevalence of HBV carriers in Southeast Asia including Hong Kong can account for the particularly high HCC cases in these areas.
HCC is often asymptomatic. The diagnosis and treatment are often delayed which lead to inapplicable of surgical resection. Meanwhile, conventional treatment regimes such as systemic chemotherapy were found to have limited responses. Hence, the case mortality rate of HCC is the second highest among all the cancers.
Granulin-epithelin Precursor (GEP) is a glycoprotein growth factor which regulates multiple cellular functions. Our group has demonstrated that GEP is over-expressed in more than 70% of HCC cases and GEP expression is positively correlated to tumor malignancy. Our group has also verified that suppression of GEP by monoclonal antibody leads to significant inhibition of HCC growth and reduction of malignancy. Therefore, GEP has the potential to be a novel therapeutic target of HCC.
MicroRNAs (miRNAs) are short non-coding RNAs that regulate mRNA translation. Previous studies showed that miRNA dys- regulation is closely associated with HCC progression and the high stability of miRNAs allows them to be cancer biomarkers or therapeutic targets. This project aims to investigate the miRNAs that regulate GEP and their functions in HCC.
Potential GEP-regulating miRNAs were identified by literature review and in silico prediction by bioinformatics tools. MiR-615-5p, miR-588, miR-29b, miR-195, and miR-659 were identified as the potential candidates. Quantitative polymerase chain reaction (qPCR) was utilized to examine the miRNAs’ expressions in HCC clinical samples. Only miR-29b and miR-195 were detected and hence they were selected for further study.
Our results showed that miR-29b and miR-195 expression levels were significantly decreased in HCC comparing to adjacent non‐tumor tissue (P<0.001) in more than 70% of cases.
MiR‐195 and miR‐29b were over‐expressed in Hep3B HCC cell lines by miRNA mimics and GEP protein level was significantly suppressed after miR-29b mimic transfection. The transcript level of GEP was found to be unchanged after the miR‐29b over-expression. This suggests miR‐29b does not regulate GEP protein expression by mRNA degradation.
The effects of miR‐195 and miR‐29b on HCC proliferation were also examined. The growths of HCC cells were suppressed notably after over-expression of miR‐195 (P<0.005) and miR‐29b (P<0.005) respectively.
In conclusion, miR‐195 and miR‐29b are frequently down-regulated in HCC. MiR‐29b can negatively regulate GEP expression and does not interfere with GEP mRNA level. Furthermore, miR‐195 and miR-29b can function to inhibit HCC cell growth significantly.published_or_final_version
Surgery
Master
Master of Philosophy
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Emara, Mohamed Maged. „Analysis of the Cellular Proteins, TIA-1 and TIAR, and their Interaction with the West Nile Virus (WNV) 3' SL Minus-Strand RNA“. Digital Archive @ GSU, 2008. http://digitalarchive.gsu.edu/biology_diss/38.
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The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.
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Day, Mandy Dowson. „A study of wheat endosperm development : cell and starch granule numbers and amyloplast DNA and RNA“. Thesis, University of Bath, 1987. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379338.
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21
Yatsuzuka, Kenji. „Live-cell imaging of multiple endogenous mRNAs permits the direct observation of RNA granule dynamics“. Kyoto University, 2019. http://hdl.handle.net/2433/242400.
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22
Lin, Sau-wah Selma, und 林秀華. „A study of microRNA-132 and -212 in murine granulosa cells during folliculogenesis“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B43909851.
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Wu, Yuhong. „Structural studies of Human Caprin Protein“. OpenSIUC, 2019. https://opensiuc.lib.siu.edu/dissertations/1652.
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Human Caprin-1 and Caprin-2 are prototypic members of the caprin (cytoplasmic activation/proliferation-associated protein) protein family. Vertebrate caprin proteins contain two highly conserved homologous regions (HR1 and HR2) and C-terminal RGG motifs. Drosophila caprin (dCaprin) shares HR1 and RGG motifs but lacks HR2. Caprin-1 and Caprin-2 have important and non-redundant functions. The detailed molecular mechanisms of their actions remain largely unknown.
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Soto-Rifo, Ricardo. „Translational control of HIV-1 and HIV-2 genomic RNA“. Lyon, Ecole normale supérieure, 2010. http://www.theses.fr/2010ENSL0584.
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Les virus de la immunodéficience humaine de type 1 et 2 (VIH-1 et VIH-2) sont des pathogènes qui ont un grand impact sur la santé car plus de 33 millions de personnes sont infectées dans le monde. Les mécanismes qui contrôlent les étapes post-transcriptionelles de l’ARN génomique pendant les étapes tardives du cycle réplicatif ne sont pas très connu et donc les processus moléculaires qui permettent à l’ARN génomique de s’associer aux machineries cellulaires de traduction, transport ou dégradation restent à être déterminés. Dans ces travaux, nous avons contribué à améliorer notre connaissance sur les mécanismes qui contrôlent la synthèse des protéines à partir de l’ARN génomique de VIH-1 et VIH-2. Les résultats présentés dans ces travaux montrent que la structure d’ARN TAR joue un rôle primordial dans le contrôle des interactions de l’ARN génomique et la machinerie traductionnelle de la cellule. Nous montrons des données qui suggèrent une nouvelle étape lors du cycle réplicatif du VIH-2 dans laquelle l’ARN génomique est accumulé dans des granules cytoplasmiques avec des marqueurs des granules de stress. Nous mettons en évidence un mécanisme qui permettrait à l’ARN génomique du VIH-1 d’être exporté au cytoplasme et traduit de manière efficace grâce à l’helicase à ARN DDX3Infections by Human immunodeficiency viruses type-1 and type-2 (HIV-1 and HIV-2) have an enormous impact in Human health as more than 33 million people is living with HIV/AIDS worldwide. The mechanisms controlling post-transcriptional events during the HIV life cycle have just started to capture the attention of scientists and most of the molecular processes allowing the genomic RNA to interact with the host machineries for translation, transport or decay are still obscure or in way to be determined. In this work, we contribute to the progress in the knowledge of the mechanisms controlling protein synthesis from the HIV-1 and HIV-2 genomic RNA. Results presented here provide evidence for the TAR RNA structure as a key player in controlling the interactions between the HIV-1 and HIV-2 genomic RNA with the host translational machinery. We also provide data for a new step during the HIV-2 life cycle that involves the accumulation of the genomic RNA in cytoplasmic granules containing several stress granules components. Finally, we present evidence for a potential mechanism by which nuclear export and protein synthesis are linked during the HIV-1 replication cycle. As such, we show that DEAD-box RNA helicase DDX3, previously implicated in Rev-mediated nuclear export, is absolutely required for HIV-1 genomic RNA translation. We determined the TAR structure as the viral determinant required for DDX3 function in translation. Strikingly, we also showed that DDX3 is specifically required for HIV-2 and SIV translation but not for FIV, HTLV-1, MLV or Line-1 suggesting that this function was acquired during primate lentiviruses evolution. Taken together, results obtained during this work highlight several key aspects of the HIV-1 and HIV-2 genomic RNA post-transcriptional control that may be critical for viral replication
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Chan, On-chim, und 陳安潛. „Characterization of microbial consortia in anaerobic granular sludge: a ribosomal RNA-based molecular approach“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31239924.
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De, Leeuw Frédéric. „Etude de la protéine CIRP et sa fonction dans le métabolisme des ARN messagers“. Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210577.
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La protéine CIRP (Cold Induced RNA binding Protein) est une petite protéine de liaison à l’ARN de 172 acides aminés, qui est constituée du côté amino-terminal d’un domaine de liaison à l’ARN de type RRM (RNA recognition motif), et d’une partie carboxy-terminale riche en glycine et arginine qui comprend plusieurs motifs RGG. Elle a été identifiée comme étant inductible par hypothermie mais aussi par irradiation aux UV et par hypoxie. Nous avons analysé son expression et sa localisation en réponse à différents stress cellulaires. Nous avons montré qu’un traitement à l’arsénite qui induit un stress oxydant n’altère pas l’expression de CIRP provoque sa localisation dans les granules de stress (SG). Les SG sont des structures ribonucléoprotéiques cytoplasmiques contenant des complexes de pré-initiation incompétents pour la traduction, et qui s’accumulent dans les cellules exposées à un stress. Ces structures constituent des sites de triages des ARNm, dans lesquels les ARNm sont soit stockés en attente d’une réinitiation de la traduction une fois le stress surmonté, soit destinés à être dégradés. La protéine CIRP se localise dans les SG que ce soit suite à un stress cytoplasmique ou du réticulum endoplasmique. Nous avons montré également que la localisation de la protéine CIRP dans les SG se déroule indépendamment de la présence de la protéine TIA-1 qui a été décrite comme responsable de l’assemblage des SG. De plus la surexpression de la protéine CIRP conduit à la formation de SG. Nous suggérons donc qu’il existe plusieurs voies qui mènent à l’assemblage de ces structures. En outre, nous avons analysé la localisation de mutants par délétion de la protéine CIRP et avons montré que le domaine RRM et le domaine RGG peuvent indépendamment localiser la protéine dans les SG. Par contre, la méthylation des résidus arginine du domaine RGG est une modification nécessaire à la localisation de CIRP dans les SG. Ensuite, nous avons étudié la fonction de la protéine CIRP dans le métabolisme des ARN messagers. Nous avons montré par une méthode d’adressage, que CIRP est un répresseur de la traduction des ARNm et que le domaine carboxy-terminal est nécessaire et suffisant à cette fonction.Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Martin, Sophie. „Le composant des granules de stress G3BP : caractérisation phénotypique de souris KO, et identification de son interactome ribonucléoprotéique dans le cerveau de souris“. Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20247.
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Les protéines capables de lier des ARNs sont essentielles pour les différentes étapes de maturation de l'ARN messager (ARNm), en dirigeant leur localisation et leur devenir dans la cellule, et en formant avec les ARNs des particules ribonucléoprotéiques (mRNPs). Les mRNPS peuvent former des structures cellulaires dynamiques qui sont adressées vers des fonctions spécifiques. Ces granules, tels que les granules de stress formés suite à un stress cellulaire, contiennent des ARNm dont la traduction est inhibée et qui sont stockés transitoirement. Ma thèse a consisté en la caractérisation fonctionnelle de G3BP (RasGAP SH3 binding protein), une RBP exprimée de façon ubiquitaire chez l'homme et la souris, et impliquée dans l'assemblage des granules de stress. Par recombinaison homologue classique, des souris knock-out pour G3BP ont été générées. Ces souris ont une espérance de vie faible et des défauts du comportement associés au Système Nerveux Central, en particulier un phénotype de type ataxie. Des expériences d'électrophysiologie ont aussi montré une altération de la plasticité synaptique dans l'hippocampe des souris KO. J'ai donc réalisé des expériences d'immunoprécipitation après cross-link (Cross-Linking and Immunoprecipitation, CLIP) pour purifier à partir de cerveau de souris un complexe stable contenant G3BP, et les ARNs associés ont été identifiés par séquençage haut débit (High-Throughput Sequencing, HITS-CLIP). De façon surprenante, la plupart des cibles de G3BP correspondent à des transcrits codants mais qui contiennent des séquences introniques, et des ARNs non codants. De plus, mes résultats ont montré que l'absence de G3BP1 affecte la stabilité de ces transcrits pré-matures spécifiquement dans le cervelet, ce qui peut être corrélé au phénotype d'ataxie des souris KO G3BP1. Cela suggère un nouveau mécanisme de régulation qui passe par la stabilisation de transcrits pré-matures, qui pourraient être convertis en transcrits matures par exemple lors d'un stress et de la séquestration de G3BP dans les granulesRNA binding proteins (RBPs) are essential in the different steps of processing of the messenger RNAs (mRNAs), directing their localization and fate within the cell, and forming with them the ribonucleoprotein particles (mRNPs). mRNPs can assemble into dynamic cellular structures in which they are routed towards specific functions. RNA granules such as stress granules (SGs) contain translationally silenced mRNPs storing transiently repressed mRNAs.My thesis work consisted in the functional characterization of G3BP (RasGAP SH3 binding protein), an RBP that is expressed ubiquitously in both humans and mice and is involved in the assembly of SGs. Using classical homozygous recombination, viable G3BP1 knock out mice were generated that demonstrated short lifespan.and behavioral defects linked to the Central Nervous System (CNS), notably an ataxia phenotype. Electrophysiology experiments showed an alteration of synaptic plasticity in the hippocampus of KO mice. Therefore, I used Cross-Linking and Immunoprecipitation (CLIP) to purify from mouse brain a stable complex containing G3BP, and performed High-Throughput Sequencing (HITS-CLIP) to identify associated RNAs. Strikingly, most of the G3BP targets correspond to intron sequence-retaining transcripts and non-coding RNAs. My results also showed that G3BP1 depletion influences the stability of these premature transcripts in the cerebellum, which can be correlated to the ataxia phenotype of the G3BP1 KO mice. This comprehensive analysis suggests a new mechanism of gene regulation based on stabilization of silenced premature transcripts which might be converted to mature transcripts under stress condition and sequestration of G3BP in SGs
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Orsborn, April Marie. „Analysis of interactions between the germline RNA helicases (GLHs) and their regulators KGB-1 and CSN-5 in Caenorhabditis elegans“. Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4499.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2006.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.
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Bauer, Karl Emory [Verfasser], und Michael [Akademischer Betreuer] Kiebler. „Live microscopy of RNA granule sorting in hippocampal neurons in space and time / Karl Emory Bauer ; Betreuer: Michael Kiebler“. München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1196529094/34.
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Eckmann, Christian R., Mark Schmid, Adam P. Kupinski, Britta Jedamzik, Martin Harterink und Agata Rybarska. „GLS-1, a novel P granule component, modulates a network of conserved RNA regulators to influence germ cell fate decisions“. PLOS Genetics, 2009. https://tud.qucosa.de/id/qucosa%3A28993.
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Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.
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Eckmann, Christian R., Mark Schmid, Adam P. Kupinski, Britta Jedamzik, Martin Harterink und Agata Rybarska. „GLS-1, a novel P granule component, modulates a network of conserved RNA regulators to influence germ cell fate decisions“. Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-184095.
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Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.
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Goulet, Isabelle. „New Roles for Arginine Methylation in RNA Metabolism and Cancer“. Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20293.
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Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.
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Budkina, Karina. „The role of an mRNA-binding protein YB-1 in formation of stress granules and translation“. Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL006.
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Au cours de la vie de l'ARNm dans la cellule, l'ARNm existe en complexe avec des protéines et n'est jamais libre. Dans le cytoplasme, l'ARNm actif est associé aux ribosomes pour former les polyribosomes tandis que les ARNm réprimés s’associent avec certaines protéines de liaison à l'ARN (RBP) pour former des mRNP. Les mRNP réprimés sont généralement isolés dans le cytoplasme mais ils peuvent également être trouvés dans des compartiments appelés granules d’ARN, notamment lors d'un stress cellulaire. Ces granules d’ARN sont des organelles non membranaires contenant principalement de l'ARNm inactif et coexistent avec des polysomes. Selon les conditions environnementales, il y a un changement dans le ratio des ARNms trouvés dans les granules d’ARN ou dans les polysomes. De plus, il existe des différences dans la teneur en ARNm des différents types de ces organelles en fonction de leur localisation et de leurs fonctions. Actuellement, les granules de stress présentent un grand intérêt pour les chercheurs en raison de leur relation avec certaines maladies neurologiques. Les mutations trouvées dans certaines protéines de liaison à l'ARN telles que TDP43 et FUS sont directement liées à certaines maladies neurodégénératives telles que la sclérose latérale amyotrophique (SLA), la démence frontotemporale (FTLD) et la maladie d'Alzheimer (MA). Dans les neurones affectés, TDP-43 et FUS forment des agrégats cytoplasmiques alors que ces protéines se trouvent généralement dans le noyau dans des conditions physiologiques. Comme elles ont également été trouvées dans les granules de stress cytoplasmiques, les granules de stress peuvent servir d'intermédiaires pour la formation d'agrégats de FUS et TDP-43. En outre, FUS et TDP-43 contiennent des régions intrinsèquement désordonnées (IDR) qui contribuent à leur agrégation.La formation de granules de stress est stimulée par l'exposition à différents facteurs internes et / ou externes. Les granules de stress servent de lieu de stabilisation des ARNm et à les maintenir inactifs jusqu'à ce que les facteurs de stress disparaissent. On considère que les structures secondaires de l'ARNm jouent un rôle important dans l'assemblage des granules de stress. De telles structures servent aussi de sites de liaison pour les RBP, qui les stabilisent davantage (par exemple G3BP). La protéine de liaison Y-box 1 (YB-1) a également été identifiée comme un marqueur pour les granules de stress. YB-1 est une protéine de liaison à l'ARN qui accompagne l'ARNm dès sa synthèse dans le noyau jusqu’à sa dégradation dans le cytoplasme. YB-1 contient un domaine de choc froid (CSD) avec deux motifs de reconnaissance d'ARN (RNP-1 et RNP-2), ainsi qu'un domaine CTD non structuré similaire aux IDR. Pour la plupart des protéines impliquées dans la formation des granules de stress, leur activité stimulante de l'IDR dans ce processus a été démontrée. Dans le même temps, il existe quelques controverses concernant le rôle de YB-1 dans l'assemblage des granules de stress. Selon certains modèles, il y a lieu de le considérer comme un régulateur négatif dans la formation des granules de stress. Selon d'autres, YB-1 présente les propriétés d'un agent favorisant de l'assemblage de granules de stress. Par ailleurs, peu de travaux ont n'a été faits pour déchiffrer l'action de la protéine sur la traduction sous stress oxydatif. Ici, notre objectif était de démêler les mécanismes structuraux par lesquels YB-1 peut réguler négativement la formation de granules de stress et de clarifier son influence sur la traduction dans des conditions de stressDuring mRNA life in cell mRNA exists in complex with proteins and is never free. In the cytoplasm, active mRNA is associated with ribosomes to form polyribosomes while repressed mRNAs in association with RNA-binding proteins forms mRNPs. Repressed mRNPs are generally isolated in the cytoplasm but they can also be found in compartments called mRNP granules, notably during cellular stress. Such mRNP granules are non-membrane organelles contains mostly translationally inactive mRNA and coexist with polysomes. Depending on the environmental conditions, there is a change in the ratio of mRNA found in these types of granules or in polysomes. In addition, there are differences in the mRNA content of the different types of such organelles depending on their localization and functions. Currently, stress granules are of great interest to researchers due to their relation to some neurological diseases. Mutations of some RNA-binding proteins such asTDP43 and FUS are directly linked to some neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTLD), and Alzheimer's disease (AD). In the affected neurons, TDP-43 and FUS form cytoplasmic aggregates while these proteins are generally found in the nucleus under physiological conditions. As they were also found in cytoplasmic stress granules, stress granules may serve as intermediates for the formation of FUS and TDP-43 aggregates. In addition, FUS and TDP-43 contain intrinsically disordered regions (IDRs) which contribute to their aggregation. The formation of stress granules is stimulated by exposure to different internal and/or external factors. Stress granules serve as a place for mRNA stabilization and keeping it inactive until stress factors disappear. It is considered that secondary structures of mRNA play a significant role in the assembly of stress granules. Such structures serve as binding sites for RBPs, which further stabilize them (e.g. G3BP). The Y-box binding protein 1 (YB-1) was also identified as a marker for stress granules. YB-1 is an RNA-binding protein that accompanies mRNA from its synthesis in the nucleus to degradation in the cytoplasm. YB-1 contains a cold shock domain (CSD) with two RNA-recognition motifs (RNP-1 and RNP-2), as well as an unstructured CTD domain similar to IDRs. For most of the proteins involved in the formation of stress granules, their stimulating activity of IDR in this process has been shown. At the same time, there are some controversies regarding the role of YB-1 in the assembly of granules. According to some sources, there is reason to consider it as a negative regulator. According to others, YB-1 exhibits the properties of an inducer during the assembly of stress granules. At the same time, no attempts were made to decipher the mechanism of action of the protein under oxidative stress.Here our aim was to unravel the structural mechanisms by which YB-1 can negatively regulate the formation of stress granules and to clarify its influence on translation in stress conditions
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Ko, Hae Kyung. „Exploring the Role of FUS Mutants from Stress Granule Incorporation to Nucleopathy in Amyotrophic Lateral Sclerosis: A Dissertation“. eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/799.
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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by preferential motor neuron death in the brain and spinal cord. The rapid disease progression results in death due to respiratory failure, typically within 3-5 years after disease onset. While ~90% of cases occur sporadically, remaining 10% of ALS cases show familial inheritance, and the number of genes linked to ALS has increased dramatically over the past decade.
FUS/TLS (Fused in Sarcoma/ Translocated to liposarcoma) is a nucleic acid binding protein that may regulate several cellular functions, including RNA splicing, transcription, DNA damage repair and microRNA biogenesis. More than 50 mutations in the FUS gene are linked to 4% of familial ALS, and many of these may disrupt the nuclear localization signal, leading to variable amounts of FUS accumulation in the cytoplasm. However, the mechanism by which FUS mutants cause motor neuron death is still unknown.
The studies presented in this dissertation focused on investigating the properties of FUS mutants in the absence and presence of stress conditions. We first examined how ALS-linked FUS mutants behaved in response to imposed stresses in both cell culture and zebrafish models of ALS. We found that FUS mutants were prone to accumulate in stress granules in proportion to their degree of cytoplasmic mislocalization under conditions of oxidative stress, ER stress, and heat shock.
However, many FUS missense mutants are retained predominantly in the nucleus, and this suggested the possibility that these mutants might also perturb one or more nuclear functions. In a human cell line expressing FUS variants and in human fibroblasts from an ALS patient, mutant FUS expression was associated with enlarged promyelocytic leukemia nuclear bodies (PML-NBs) under basal condition. Upon oxidative insult with arsenic trioxide (ATO), PML-NBs in control cells increased acutely in size and were turned over within 12-24 h, as expected. However, PML-NBs in FUS mutant cells did not progress through the expected turnover but instead continued to enlarge over 24 h. We also observed a persistent accumulation of the transcriptional repressor Daxx and the 11S proteasome regulator in association with these enlarged PML-NBs. Furthermore, the peptidase activities of the 26S proteasome were decreased in FUS mutant cells without any changes in the expression of proteasome subunits.
These results demonstrate that FUS mutant expression may alter cellular stress responses as manifested by (i) accumulation of mutant FUS into stress granules and (ii) inhibition of PML-NB dynamics. These findings suggest a novel nuclear pathology specific to mutant FUS expression that may perturb nuclear homeostasis and thereby contribute to ALS pathogenesis.
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Ko, Hae Kyung. „Exploring the Role of FUS Mutants from Stress Granule Incorporation to Nucleopathy in Amyotrophic Lateral Sclerosis: A Dissertation“. eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/799.
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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by preferential motor neuron death in the brain and spinal cord. The rapid disease progression results in death due to respiratory failure, typically within 3-5 years after disease onset. While ~90% of cases occur sporadically, remaining 10% of ALS cases show familial inheritance, and the number of genes linked to ALS has increased dramatically over the past decade.
FUS/TLS (Fused in Sarcoma/ Translocated to liposarcoma) is a nucleic acid binding protein that may regulate several cellular functions, including RNA splicing, transcription, DNA damage repair and microRNA biogenesis. More than 50 mutations in the FUS gene are linked to 4% of familial ALS, and many of these may disrupt the nuclear localization signal, leading to variable amounts of FUS accumulation in the cytoplasm. However, the mechanism by which FUS mutants cause motor neuron death is still unknown.
The studies presented in this dissertation focused on investigating the properties of FUS mutants in the absence and presence of stress conditions. We first examined how ALS-linked FUS mutants behaved in response to imposed stresses in both cell culture and zebrafish models of ALS. We found that FUS mutants were prone to accumulate in stress granules in proportion to their degree of cytoplasmic mislocalization under conditions of oxidative stress, ER stress, and heat shock.
However, many FUS missense mutants are retained predominantly in the nucleus, and this suggested the possibility that these mutants might also perturb one or more nuclear functions. In a human cell line expressing FUS variants and in human fibroblasts from an ALS patient, mutant FUS expression was associated with enlarged promyelocytic leukemia nuclear bodies (PML-NBs) under basal condition. Upon oxidative insult with arsenic trioxide (ATO), PML-NBs in control cells increased acutely in size and were turned over within 12-24 h, as expected. However, PML-NBs in FUS mutant cells did not progress through the expected turnover but instead continued to enlarge over 24 h. We also observed a persistent accumulation of the transcriptional repressor Daxx and the 11S proteasome regulator in association with these enlarged PML-NBs. Furthermore, the peptidase activities of the 26S proteasome were decreased in FUS mutant cells without any changes in the expression of proteasome subunits.
These results demonstrate that FUS mutant expression may alter cellular stress responses as manifested by (i) accumulation of mutant FUS into stress granules and (ii) inhibition of PML-NB dynamics. These findings suggest a novel nuclear pathology specific to mutant FUS expression that may perturb nuclear homeostasis and thereby contribute to ALS pathogenesis.
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Bolinger, Cheryl Giles. „Study of translation control by a RNA helicase A-responsive post-transcriptional control element in Retroviridae“. The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1226513076.
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Palud, Amandine. „Liquid-liquid phase separation mediated by low complexity sequence domains promotes stress granule assembly and drives pathological fibrillization“. Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066560/document.
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Il a été observé que l’altération des fonctions des granules de stress, entités cytoplasmiques non-membranaires composées d’ARN et de protéines liant l’ARN (RBPs), peut conduire au développement de maladies telles que la sclérose latérale amyotrophique, la démence fronto-temporale, la myopathie à inclusions et la maladie de Paget des os. Ces pathologies sont caractérisées par un dépôt cytoplasmique d’inclusions solides enrichies en RBPs et comprenant des fibrilles. Une connexion génétique a été suggérée entre la persistance des granules de stress et l’accumulation de ces inclusions pathologiques dans le cytoplasme des patients. Dans mon manuscrit de thèse, il est mis en évidence le fait que la protéine hnRNPA1, dont les mutations entrainent les maladies mentionnées plus haut, subit une séparation de phases entre deux liquides connue également sous l’appellation « Séparation de Phases Liquide-Liquide » (LLPS) dans des gouttelettes enrichies en protéines. Bien que le domaine composé d’une séquence à faible complexité (Low Complexity sequence Domains ou LCD) soit suffisant pour obtenir cette séparation de phases, les domaines de liaison à l’ARN y contribuent également en présence d’ARN. Cela a permis d’envisager l’existence de plusieurs mécanismes intervenant dans la régulation de l’assemblage de ces granules. Un autre résultat a mis en exergue le fait que la formation de fibrilles n’est pas une obligation pour permettre la séparation de phases mais que les gouttelettes, enrichies en protéines, entrainent, par ailleurs, une augmentation de la formation de ces fibrilles. La séparation de phases liquide-liquide induite par le domaine composé d’une séquence à faible complexité semble contribuer à l’assemblage des granules de stress et à leurs propriétés liquides. Finalement, cette étude propose d’établir une réelle corrélation entre la formation des granules de stress qui deviennent persistants et l’accumulation d’inclusions pathologiques dans le cytoplasme des patientsStress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, inclusion body myopathy, Paget’s disease of bone and frontotemporal dementia; these diseases are characterized by solid, fibrillar, cytoplasmic inclusions that are rich in RNA binding proteins (RBPs). Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. In this thesis manuscript, I demonstrate that the disease-related RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the folded RNA recognition motifs contribute to LLPS in the presence of RNA, potentially giving rise to several mechanisms for regulating assembly of stress granules. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. I suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties, and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease
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Twyffels, Laure. „Nucleo-cytoplasmic transport of TIS11 proteins and stress granule assembly: two potential new roles for Transportins“. Doctoral thesis, Universite Libre de Bruxelles, 2013. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209423.
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The nucleo-cytoplasmic compartmentalization enables eukaryotic cells to develop sophisticated post-transcriptional regulations of gene expression. However, managing the exchanges of macromolecules between the two compartments also represents a formidable challenge for the cells. Nucleo-cytoplasmic exchanges rely on specialized soluble carriers and take place at nuclear pore complexes that span the nuclear envelope. Active nucleo-cytoplasmic transport of proteins, in particular, is performed mainly by a family of carriers called karyopherins, which includes about twenty members in mammals. Some of them, called importins, recognize nuclear localization signals (NLSs) in their substrates and convey them into the nucleus. Others, called exportins, recognize nuclear export signals (NESs) in their substrates and bring them back to the cytoplasm. Many RNA-binding proteins (RBPs) shuttle between the nucleus and the cytoplasm, where they can often fulfill different functions. RBPs also frequently localize into specialized microdomains that are not delimited by a membrane but in which specific factors are concentrated. Those include processing bodies and stress granules, which are cytoplasmic foci associated with mRNA decay, storage and translational repression. Post-transcriptional regulations mediated by RBPs can therefore be modulated rapidly and efficiently through changes in the localization of RBPs.
The first part of this work focuses on the subcellular localization and nucleo-cytoplasmic transport of the Drosophila RBP dTIS11. Like its mammalian and yeast homologues, dTIS11 binds AU-rich elements in the 3’UTR of its target mRNAs, and stimulates their rapid deadenylation and decay. Here, we have observed that although dTIS11 appears to be located mostly in the cytoplasm, it is constantly shuttling in and out of the nucleus. We show that the export of dTIS11 from the nucleus depends on the CRM1 exportin and is mediated by a hydrophobic NES that encompasses residues 101 to 113 in dTIS11 sequence. We also identify a cryptic Transportin-dependent PY nuclear localization signal (PY-NLS) in the tandem zinc finger region of dTIS11 and show that it is conserved across the TIS11 protein family. This PY-NLS partially overlaps the second zinc finger (ZnF2) of dTIS11. Importantly, mutations disrupting the capacity of the ZnF2 to coordinate a Zn2+ ion unmask dTIS11 and TTP PY-NLS and promote nuclear import. Taken together, our results indicate that the nuclear export of Drosophila and mammalian TIS11 proteins is mediated by CRM1 through diverging NESs, while their nuclear import mechanism might rely on a conserved PY-NLS whose activity is negatively regulated by ZnF2 folding.
In the second part, we present preliminary results which implicate the nucleo-cytoplasmic transport machinery in the assembly of stress granules (SGs) in mammalian cells. SGs contain silenced mRNPs which resemble stalled initiation complexes, and they form transiently in response to acute stress, concomitantly with a global arrest of translation. While their exact role remains undefined, it seems clear that SGs are able to exchange mRNPs with polysomes and with PBs, and that they are connected to post-transcriptional and translational regulations of gene expression during stress. Here, we show that inhibition of Transportin-1 expression or function does not affect the translational status of cells but impairs the assembly of stress granules. Finally, we show that Transportin-1 and -2B, but not -2A, localize into stress granules in response to several stresses.
In conclusion, we suggest two potential new roles for Transportins, in the nucleo-cytoplasmic traffic of TIS11 proteins on the one hand and in the assembly of stress granules on the other hand.
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Le compartimentage nucléo-cytoplasmique permet aux cellules eucaryotes de réguler l’expression génétique par des mécanismes post-transcriptionnels élaborés. Les ARN messagers subissent plusieurs étapes de maturation dans le noyau avant d’être exportés vers le cytoplasme où ils sont traduits et dégradés. Ces processus sont effectués via des protéines de liaison à l’ARN, ou RBPs. Beaucoup de RBPs exercent des fonctions différentes dans le noyau et dans le cytoplasme, et leur activité peut dès lors être rapidement modulée par une modification de leur localisation.
Le transport nucléo-cytoplasmique actif des protéines s’effectue à travers les pores nucléaires et fait majoritairement appel à des transporteurs solubles de la famille des karyophérines. Ceux-ci reconnaissent au sein des protéines à transporter une séquence-passeport appelée NLS (nuclear localization signal) ou NES (nuclear export signal) selon la direction nécessitée.
Le présent travail comporte deux parties. La première porte sur la localisation subcellulaire et le transport nucléo-cytoplasmique des protéines de la famille TIS11, et plus particulièrement de dTIS11 qui est le seul représentant de cette famille chez la Drosophile. Comme ses homologues dans d’autres espèces, dTIS11 est une RBP qui favorise la déadénylation et la dégradation de ses ARN messagers cibles. Nos résultats démontrent que dTIS11 fait la navette entre le noyau et le cytoplasme. L’export de dTIS11 hors du noyau est réalisé par la karyophérine CRM1 et fait appel à un NES différent de celui présent chez les protéines TIS11 mammaliennes. Nous identifions également un NLS cryptique au sein du domaine à deux doigts de zinc avec lequel dTIS11 lie l’ARN. Ce NLS correspond partiellement au signal consensus reconnu par la Transportine. Il est démasqué par la mutation du second doigt de zinc ;dans ces conditions, il permet l’import de dTIS11 par la Transportine. Enfin, nous montrons qu’il est conservé dans d’autres protéines de la famille TIS11.
Dans la seconde partie, nous nous intéressons aux granules de stress, qui sont des microdomaines cytoplasmiques dans lesquels se concentrent des RBPs et des ARN messagers non traduits en réponse à un stress cellulaire. Nous montrons que les karyophérines appartenant à la sous-famille des Transportines sont présentes dans ces granules et que l’inhibition de l’expression ou de la fonction des Transportines réduit la formation de ces granules en réponse à divers stress cellulaires. Nous écartons la possibilité que ce résultat soit un effet indirect d’un ralentissement du métabolisme traductionnel. Nos résultats suggèrent donc une implication des Transportines dans la formation des granules de stress.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Bonnet, Agnès. „Étude de l'expression des gènes au cours des stades précoces de la folliculogenèse ovarienne chez les mammifères de rente (brebis)“. Toulouse 3, 2013. http://thesesups.ups-tlse.fr/2306/.
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Le bon déroulement de la folliculogenèse ovarienne basale, est une étape clé qui conditionne la fertilité des femelles à l'âge adulte. Pourtant, les mécanismes moléculaires contrôlant ce processus sont encore mal connus et les études d'expression limitées par la difficulté d'obtention du matériel biologique. Les données actuelles proviennent essentiellement d'études réalisées chez les rongeurs (espèces poly-ovulantes). L'objectif de ce travail est de décrire, pour les deux compartiments folliculaires (ovocytes et cellules de granulosa), les spécificités d'expression et les gènes impliqués dans le dialogue moléculaire entre ces compartiments ainsi qu'aux différents stades de développement folliculaire basal " in vivo " chez une espèce mono-ovulante : la brebis. Nous avons développé, dans un premier temps, une méthode d'analyse du transcriptome pour les stades précoces qui combine microdissection à capture laser, amplification des ARN et microarrays. Puis, nous avons présenté, par la technique RNA-seq, et pour la première fois, un répertoire de 15349 gènes décrivant le profil d'expression de ces gènes dans chaque compartiment folliculaire et en fonction de leur stage de développement. L'analyse statistique révèle une différence d'expression entre compartiments folliculaires (5130 gènes) ainsi qu'une dynamique d'expression au cours du développement (3015 gènes). Nous avons également identifié 161 et 55 gènes présentant une expression préférentiellement enrichie dans l'ovocyte et dans les cellules de granulosa, respectivement. L'analyse fonctionnelle " in silico " combinée avec les données d'expression mettent en évidence des voies de signalisation (IGF1, VEGF, FGF, NOTCH) susceptibles d'être impliquées dans le dialogue moléculaire entre les deux compartiments folliculaires. Enfin, dans nos conditions expérimentales, nous montrons d'importantes variations d'expression au cours du développement folliculaire basal, lors de la transition du follicule primaire en secondaire chez la brebis. La cinétique d'expression de gènes impliqués dans des voies de signalisation tels que BMP et WNT a été précisément décrite. Un jeu de 25 gènes a été sélectionné en tant que biomarqueurs expressionnels des différentes classes folliculaires. Il pourra être utilisé pour évaluer la croissance folliculaire basaleCorrect achievement of early ovarian folliculogenesis is a crucial step which determines female fertility in adulthood. However, molecular mechanisms controlling this development are not well characterized. Furthermore, gene expression studies are restricted by the difficulty to collect biological material. Moreover, available data are mainly derived from rodent models (poly-ovulating species). In this project, we intended to describe, in both follicular compartments (oocytes and granulosa cells), the specificity of expression, to point out genes involved in molecular dialog and during early follicular development in sheep species (mono-ovulating species). First, we developed a transcriptome methodology to study early folliculogenesis that combined laser capture microdissection, RNA amplification and microarrays. Then, we described for the first time, the gene expression profile of 15349 genes for each follicular compartment during early follicular development using RNA-seq technology. Statistical analysis underlined differential expression between compartments (5120 genes) and during development (3015 genes). We identified 161 and 55 genes with a preferentially enriched expression in oocytes and granulosa cells respectively. "In silico" fonctional analysis combined with gene expression data underlined signaling pathways as IGF1, VEGF, FGF,and NOTCH that may be involved in molecular dialog between the two follicular compartments. Last, in our experimental conditions we showed important gene expression changes occurred during primary to secondary follicular transition in sheep. The expression profiles of genes involved in pathways as BMP and WNT were precisely described. A set of 25 genes was selected as follicular class biomarkers that may be used to evaluate early follicular growth
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Kaushansky, Laura J. „Investigating the Effects of Mutant FUS on Stress Response in Amyotrophic Lateral Sclerosis: A Thesis“. eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/792.
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During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress.
The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics.
I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.
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Kaushansky, Laura J. „Investigating the Effects of Mutant FUS on Stress Response in Amyotrophic Lateral Sclerosis: A Thesis“. eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/792.
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During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress.
The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics.
I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.
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Leplus, Alexis. „Study of factors implicated in small ribosomal subunit biogenesis under differents growth conditions“. Doctoral thesis, Universite Libre de Bruxelles, 2010. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210189.
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La biogenèse du ribosome est un processus complexe et dynamique qui nécessite de nombreuses étapes de maturation et de modification des ARNr ainsi que l’assemblage et le transport des RNPs précurseurs. Un ribosome mature contient une centaine de pièces, ARN et protéines confondus, mais son assemblage requiert l’intervention de plus de 400 facteurs de synthèse. De part le coût énergétique important de ce processus, plusieurs voies de régulation interviennent pour contrôler la biogenèse des ribosomes en fonction des conditions nutritives. L’une des voies les plus connue est la voie TOR (Target of rapamycin). Cette voie de régulation agît principalement au niveau de la transcription des différents intervenants de la biogenèse :les ARNr, les protéines ribosomiques mais aussi les facteurs de synthèse. Ces facteurs, ayant une action transitoire dans la maturation des ribosomes, sont, par économie, recyclés pour la synthèse de nouveaux ribosomes. Nous nous sommes donc intéressés au devenir de ces facteurs, plus particulièrement de ceux intervenants dans la biogenèse de la petite sous unité, lorsque les conditions environnementales sont inadaptées à la croissance cellulaire. Ainsi, nous avons pu montré, pour quatre facteurs particuliers :Dim2, Rrp12, Hrr25 et Fap7, que leur localisation est dépendante de la synthèse ribosomique. Ainsi, lors de carence en sources nutritives, l’inhibition de la synthèse et de l’activité ribosomique entraîne un confinement de ces facteurs ribosomiques dans le nucléole ou dans des corps cytoplasmiques. En outre, la localisation particulière des facteurs ribosomiques Hrr25 et Fap7 dans les P-bodies en phase de croissance saturée laisse penser que ces corps cytoplasmiques sont le lieu de dégradation des pré-ribosomes lorsque les carences nutritives perdurent.Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Vautrot, Valentin. „Recherche des mécanismes impliqués dans les dérégulations de l'épissage alternatif à l'origine de la progéria et étude du rôle de l'étape d'épissage dans les changements globaux d'expression des gènes en réaction au choc thermique“. Thesis, Université de Lorraine, 2013. http://www.theses.fr/2013LORR0321/document.
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Le syndrome de Hutchinson-Gilford, ou progéria, est une pathologie génétique rare qui se caractérise par des symptômes assimilés à un vieillissement prématuré. Les mutations à l'origine de la progéria affectent le gène LMNA, codant la lamine A, qui joue un rôle majeur dans la formation, la maintenance et la résistance du noyau. Ces mutations activent l'utilisation de sites 5' alternatif ou cryptique d'épissage présents dans l'exon 11 du pré-ARNm LMNA en amont du site normalement utilisé. Nous avons révélé un effet des mutations sur la structure secondaire de l'ARN aux alentours des mutations, qui permet l'augmentation de l'utilisation des sites d'épissage mutants. De plus, nous avons montré l'implication de plusieurs protéines SR (SRSF1, SRSF5 et SRSF6) dans la régulation de l'utilisation des différents sites d'épissage. D'autre part, il a déjà été observé que les noyaux des cellules des patients atteints de progéria contiennent des granules de stress, les nSB, situés dans les régions péricentromériques des chromosomes et contenant des ARN dits satellite III et des facteurs d'épissage. Des nSB similaires sont formés dans les cellules saines suite à divers stress, comme le stress thermique. Il est possible que ces nSB séquestrent ces facteurs d'épissage afin de réguler le profil d'épissage alternatif des cellules pendant la régénération après un stress. Nous avons purifié les protéines associées aux ARN satellite III in vitro afin de trouver de nouveaux composants des nSB et analysé, par emploi de puces jonction-exon, le transcriptome de cellules soumises à un choc thermique, pour mieux comprendre à terme comment la formation des nSB peut affecter l'épissage alternatifThe Hutchinson-Gilford syndrome, also called progeria, is a rare genetic disease, characterized by symptoms that can be assimilated to accelerated natural ageing. Mutations that cause progeria affect the LMNA gene, which codes the lamin A that plays a major role in the shaping, maintenance and resistance of the nucleus. These mutations lead to the activation of alternative or cryptic 5' splice sites located within the exon 11 of LMNA pre-mRNA upstream from the normal 5' splice site. Our work revealed an effect of the mutations on the 2D RNA structure of the splice sites, which contributes to the increased use of the mutant sites. On top of it, we showed the impact of several SR proteins, (SRSF1, SRSF5 and SRSF6) on the regulation of the use of the exon 11 5' splice sites. On the other hand, it was previously observed that cells from progeria patients contain nuclear stress bodies (nSB), located in chromosomal pericentromeric regions and containing satellite III RNAs and several splicing regulatory proteins. Similar bodies are formed in healthy cells submitted to various stresses such as heat shock. A work hypothesis is that those nSBs sequester splicing factors in order to regulate the global alternative splicing profile in cells during the recovery period after stress. We purified proteins associated with satellite III RNAs in vitro, to find new components of the nSBs, and analyzed the transcriptome of cells subjected to heat shock using exon junction microarrays, in order to eventually understand how nSB formation can affect alternative splicing
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Pattabiraman, Sundararaghavan. „Vimentin protects differentiating stem cells from stress“. Doctoral thesis, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0005-151B-6.
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Hrbková, Pavlína. „Lidské proteiny z rodiny 4E ve stresových granulích a jejich další charakterizace“. Master's thesis, 2018. http://www.nusl.cz/ntk/nusl-380436.
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Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...
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Chang, Wei-Lun, und 張瑋倫. „Characterization of the role of transportin in cytoplasmic RNA granules“. Thesis, 2009. http://ndltd.ncl.edu.tw/handle/40375101175145495591.
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博士國防醫學院
生命科學研究所
98
The distribution of proteins is likely to change in response to cellular stresses triggered signaling pathways. Since importin-beta family members are essential for nucleocytoplasmic transport of macromolecules, we attempted to explore whether importin-beta family proteins change their cellular localization in response to environmental change. Among the importin-beta members we analyzed, only transportin (TRN) was shown to detect in a subset of cytoplasmic processing bodies (P-bodies) under normal cell conditions and apparently translocate to stress granules (SGs) upon arsenite, heat shock or FCCP treatment of the cells. Both cytoplasmic granules contain translationally silenced mRNAs. SGs are site for accumulation of mRNA suffered stress-induced translational arrest and PBs are the sites for degradation of a subset of mRNAs and also for siRNA or miRNA-mediated gene expression silencing. Fluorescence recovery after photobleaching analysis revealed that TRN moved rapidly in and out of cytoplasmic granules. Depletion of TRN enhanced P-body formation but did not affect the number or size of SGs, suggesting that TRN or its cargo(es) participates in cellular function of P-bodies. Accordingly, TRN associated with the AU-rich element (ARE)-binding protein, tristetraprolin (TTP), as well as its associated mRNAs. Depletion of TRN increased the number of P-bodies and stabilized ARE-containing mRNAs, as observed with knockdown of the 5’-3’ exonuclease Xrn1. Moreover, depletion of TRN retained TTP in P-bodies and meanwhile reduced the fraction of mobile TTP to SGs, indicating that TRN probably plays a role in trafficking of TTP between the cytoplasmic granules and whereby modulates the stability of ARE-containing mRNAs. Furthermore, we observed that TRN associated with microRNPs, which suggested a role of TRN in microRNA biogenesis or microRNA-mediated mRNA regulation. Taken together, our data indicated novel roles of TRN in cytoplasmic mRNA metabolism, primarily including mRNP trafficking to RNA granules and mRNA stability control.
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Weng, Jui-Hsia. „RNA-binding protein BC1 in RNA stability and stress granule formation“. 2005. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-2607200514472800.
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Weng, Jui-Hsia, und 翁瑞霞. „RNA-binding protein BC1 in RNA stability and stress granule formation“. Thesis, 2005. http://ndltd.ncl.edu.tw/handle/63442526177139931548.
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碩士國立臺灣大學
生化科學研究所
93
AU-rich element (ARE) is found in the 3’UTR of many short-lived mRNAs such as cytokines, and oncogenes. Many RNA-binding proteins that selectively recognize and bind to this ARE sequence are called AU-rich binding protein (AUBP) and can modulate stability and/or translation of ARE-containing mRNAs. Eukaryotic cells shut down protein synthesis and reprogram their translational machinery in response to environmental stress for conserving anabolic energy to repair of the stress-induced damage. In stressed-cells, mRNA is in a dynamic equilibrium between polysomes and stress granules (SGs). SGs are cytoplasmic foci at which stalled translation initiation complexes accumulate. Many RNA-binding protein such as TIA-1,TIAR, and HuR localized at stress granules and it has been proposed the carboxyl terminus of TIA1, the prion-related domain PRD, mediates the formation of SGs. In this study, we investigated the binding ability of BC1 to homo-polynucleotides. Our data demonstrated that BC1 is an RNA-binding protein and possesses strong binding activity toword the distinct sequence. The YTH domain of BC1, a putative RNA-binding domain, harbors ARE-binding activity by using the electrophoretic mobility shift assay (EMSA). RT-PCR and Northern blot analysis showed that BC1 does not alter the stability of its binding target RNAs, but we also prove that BC1 have tendency to promote gene expression at translational level. When cells encounter stress, BC1 is colocalized with TIA-1 and HuR in SGs. We also found that BC1 interacts with TIA-1 under stress in GST-pull down experiment. In deletion analysis, we found that the PRD domain of TIA-1 and the Extensin-like domain of BC1 are responsible for SG formation. Collectively, we report a novel RNA-binding protein BC1 which may exert its role in promoting translation of ARE-containing mRNAs and is involved in formation of SGs.
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Trengrove, Chelsea Brais. „Autophagy and stress granules: the merging of two pathways in Parkinson's disease“. Thesis, 2016. https://hdl.handle.net/2144/14619.
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Autophagy is compromised in Parkinson’s disease (PD) with a number of PD-associated genetic mutations leading to its dysregulation. Leucine-rich repeat kinase (LRRK2) mutations, causative of PD, aberrantly enhance autophagy. Our lab elucidated a LRRK2 gene regulatory network identifying transcripts showing coordinated expression level changes associated with PD. Histone deacetylase 6 (HDAC6) was found to be an important interactor with LRRK2, regulating many of the same transcripts. The majority of these transcripts associate with autophagy and the lysosomal complex. I hypothesized that LRRK2 interacts with HDAC6 to regulate autophagy. Silencing of HDAC6 in SH-SY5Y normalized the autophagosomal size altered by expression of PD-linked LRRK2 mutants. This work identified a key role for HDAC6 in mediating the autophagic dysfunction induced by the mutant LRRK2.
In addition to autophagy, stress granule (SG) formation has emerged as a compelling mechanism in the pathogenesis of PD. RNA-binding proteins (RBPs), such as T-cell intracellular antigen-1 (TIA-1), are major component of SGs. I observed TIA-1 translocating from the nucleus to the cytoplasm in PD cortex without forming SGs. Hu antigen D (HuD) also showed changes, with the RBP more present in the cytoplasm than the nucleus in PD with no SGs observed. These preliminary studies lead to the hypothesis that low levels of SGs result from an inhibition by alpha-synuclein (syn), or hyperactive autophagy. For that purpose, brain tissues from a mouse model of PD (A53T-syn transgenic mouse) were examined by immunohistochemistry. There was no difference in TIA-1 expression in control and A53T-syn expressing mouse brains, or SG formation in primary neurons after treatment with recombinant A53T fibrils. To determine whether the lack of SGs in PD brain was due to activation of autophagy, BE-M17 cells were treated with rapamycin, an autophagy activator, which decreased SGs by 50%. Overexpression of TIA-1 in BE-M17 cells under arsenite treatment also increased autophagosomal size by 50%, indicating co-regulation of SGs and autophagy. My work indicates that the pathophysiology of PD is associated with a loss of SGs due to elevated activity of autophagy, presumably due to PD-linked LRRK2 mutations. This co-regulatory network may be a potential therapeutic target of PD.
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LeBlang, Chelsey Jenna. „Modulation of neuroinflammation and tauopathy by RNA-binding protein TIA1 in the P301S mouse model of tauopathy“. Thesis, 2020. https://hdl.handle.net/2144/41112.
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Tauopathies are a class of neurodegenerative diseases characterized by aggregation of hyperphosphorylated microtubule associated protein tau (phospho-tau), resulting in neuroinflammation and neurodegeneration. Neuroinflammatory processes play an integral role in the exacerbation and progression of pathology in these disorders, leading to increased levels of neurodegeneration. The RNA binding protein (RBP) T-cell Intracellular Antigen 1 (TIA1) is an important regulator of the innate immune response in the periphery, dampening cytotoxic inflammation and apoptosis during cellular stress, however its role in central neuroinflammation is unclear. We have recently shown that TIA1 regulates tau pathophysiology and toxicity in part through the binding of phospho-tau oligomers into pathological stress granules. Haploinsufficiency of TIA1 in the P301S mouse model of tauopathy results in reduced accumulation of toxic tau oligomers, pathologic stress granules, and the development of downstream pathological features of tauopathy. The putative role of TIA1 as a regulator of the peripheral immune response led us to characterize the role of TIA1 in neuroinflammation, and determine its relationship with neurodegeneration in the context of tauopathy, a chronic stressor in the neural environment. Here, we evaluated indicators of neuroinflammation (reactive microgliosis and phagocytosis, pro-inflammatory cytokine release, and oxidative stress), and neurodegeneration (gross hippocampal atrophy, neuronal loss, synapse loss, and phospho-tau load) in wildtype and P301S transgenic mice expressing TIA1+/+, TIA1+/-, and TIA1-/- in both early (5 month) and advanced (9 month) disease states through biochemical, ultrastructural, and histological analyses. Our data show that both TIA1 haploinsufficiency and TIA1 knockout exacerbate neuroinflammatory processes in advanced stages of tauopathy, suggesting that TIA1 dampens the immune response in the central nervous system during chronic stress. TIA1 haploinsufficiency and knockout do not reduce neurodegeneration in advanced disease, and importantly, TIA1 knockout exacerbates neuron and synapse loss in hippocampal regions. With both increased levels of neuroinflammation and neurodegeneration, P301S animals with TIA1 knockout are distinct from age-matched P301S and wildtype mice. This study demonstrates that TIA1 plays an important role in the regulation of innate immune response in neurodegenerative disease, and its expression significantly impacts the progression of tauopathy.