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Najim, Mustafa. "Hepatitis C virus induced changes in cellular trafficking and lipid metabolism - identifying novel host factors required for HCV replication". Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/20895.
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Hepatitis C virus (HCV) infection is endemic in numerous countries and is a global health issue, placing a huge burden on health care systems and society. The high prevalence of people who are chronically infected with HCV, the disease burden and the absence of an effective vaccine reinforce the importance of HCV treatment in controlling this disease. Direct acting antivirals (DAAs), have revolutionised HCV treatment over the last few years. However, people failing DAAs typically develop antiviral resistance, so new treatments may still be needed in future. To identify novel host factors involved in HCV infection, a mass spectrometry based Stable Isotope Labelling of Amino acids in Cell culture (SILAC) screen was performed, to identify proteins that are enriched in the Endoplasmic Reticulum (ER) of HCV infected hepatocytes. This showed significant upregulation of Coatomer Protein Complex-I (COPI). Next, we examined the effect of Phosphatidylinositol 4-phosphate (PI4P) depletion on tafficking of COPI to the ER and on HCV replication, by over-expressing the PI4P phosphatase Sac1. However, due to the limitations of the model, we could neither prove or disprove this hypothesis, as we could not achieve sustained overexpression of Sac1. Next, we evaluated the consequence of silencing heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNPC1/C2) and transmembrane 6 superfamily member 2 (TM6SF2) on HCV replication. We found that hnRNPC1/C2 inhibition has no effect on HCV replication, while TM6SF2 is required for HCV replication. In summary, these findings identify a range of essential host proteins that could be targeted by novel host-targeting antiviral drugs. Finally, our findings provide potential insights into the life cycle of other related viruses, which have far fewer treatments available.
2
Labrecque, Benoît. "Identification des résidus contribuant à l'interaction hnRNP A1- hnRNP A1". Mémoire, Université de Sherbrooke, 2003. http://savoirs.usherbrooke.ca/handle/11143/3336.
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HnRNP A1 est une protéine impliquée dans la sélection des sites 5 ' d'épissage in vitro et in vivo . Il est connu que son domaine riche en glycines est important pour médier son activité dans l'épissage alternatif. Le modèle"looping-out" a été proposé comme mécanisme d'action de A1; lorsque A1 lie des sites de haute affinité localisés dans la région intronique de part et d'autre d'un exon alternatif, l'exclusion de celui-ci est favorisé par une interaction A1-A1 impliquant le domaine riche en glycines. Les régions et les acides aminés impliqués dans l'interaction A1-A1 restent méconnus. Une mutagénèse aléatoire de l'ADNc de A1, suivie d'une approche génétique chez la bactérie basée sur la répression traductionnelle, nous a permis d'aborder l'importance du domaine riche en glycines et du domaine RRM2 dans la liaison coopérative de la protéine hnRNP A1 à PARN. La majorité des mutants identifiés montrent un défaut au niveau de l'interaction A1-A1 lorsque quantifiés dans un essai double-hybride chez la levure."--Résumé abrégé par UMI.
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Fisette, Jean-François. "Le contrôle de l'épissage alternatif par les protéines hnRNP H et hnRNP A1". Thèse, Université de Sherbrooke, 2009. http://savoirs.usherbrooke.ca/handle/11143/4275.
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Les protéines hnRNP A1 sont impliquées dans l'épissage alternatif. Un mode d'action proposé implique la formation d'homodimères entre molécules hnRNP A1 causant un réarrangement dans la structure de l'ARN pré-messager. Cette modulation de l'ARN permettrait le rapprochement de sites d'épissage 5' et 3' d'exons situés de par et d'autres d'un exon alternatif. Le domaine riche en résidus glycines est responsable, en grande partie, de l'interaction entre les deux protéines hnRNP A1. Comme la protéine hnRNP H contient aussi un domaine riche en résidus glycines, nous avons postulé que cette dernière pouvait moduler l'épissage alternatif de la même manière que hnRNP A1. Afin de vérifier cette hypothèse, nous avons utilisé un ARN pré-messager constitué de deux sites d'épissage 5' (distal et proximal) en compétition pour un seul site d'épissage 3'. En présence de sites de liaison pour hnRNP H, nous observons que le choix du site d'épissage 5' est déplacé vers le site distal. Nous avons confirmé le rôle des protéines hnRNP H dans la sélection des sites d'épissage 5' in vitro et avons déterminé que le domaine riche en résidus glycines (GRD) est important pour l'activité d'épissage de ce régulateur. Nous avons ensuite exploré la possibilité que des combinaisons de sites de liaison pour hnRNP H et hnRNP A1 puissent activer l'utilisation du site d'épissage 5' distal. Nous avons observé que des combinaisons hétérotypiques peuvent reproduire cette activité d'épissage. Finalement, nous avons utilisé la technologie BRET ("bioluminescence resonance energy transfer") pour démontrer que des interactions homotypiques entre protéines hnRNP H et hétérotypiques entre molécules hnRNP A1 et hnRNP H peuvent se former dans les cellules vivantes. Notre étude suggère que les protéines hnRNP H et hnRNP A1 peuvent changer la conformation de l'ARN pré-messager et affecter le choix du site d'épissage.
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Moran-Jones, Kim. "hnRNPs A2 and A3 : nucleic acid interactions /". St. Lucia, Qld, 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17983.pdf.
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Jansen, Lara [Verfasser], i Christian [Akademischer Betreuer] Haass. "Generation and functional analysis of the ALS associated HNRNPA zebrafish mutants / Lara Jansen ; Betreuer: Christian Haass". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/122243654X/34.
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Barral, Paola. "Characterization of a novel hnRNP : E1B-AP5". Thesis, University of Birmingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403905.
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Le, Bras Morgane. "Rôle des protéines de liaison à l'ARN hnRNP H et hnRNP F dans les régulations traductionnelles dans les glioblastomes". Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30277.
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Le glioblastome multiforme (GBM) est une tumeur cérébrale extrêmement agressive associée à un mauvais pronostic. C'est pourquoi, il apparaît nécessaire d'identifier les mécanismes moléculaires participant au développement des GBM ainsi qu'à leurs résistances aux traitements afin de développer de nouvelles approches thérapeutiques. Récemment, il a été montré que les régulations traductionnelles jouent un rôle fondamental dans les propriétés agressives du GBM. Les protéines de liaison à l'ARN (RBP) sont des acteurs majeurs de ces régulations dont l'expression/activité est altérée dans les GBM. Les RBP hnRNP HF (HF) font partie des RBP les plus surexprimées dans les GBM et leur contribution dans la régulation traductionnelle des GBM n'a encore jamais été investiguée. Nous avons émis l'hypothèse que hnRNP H et hnRNP F soient au centre d'un réseau de régulations post-transcriptionnelles impactant la machinerie traductionnelle qui contrôle le développement tumoral et la résistance aux traitements des GBM. Nos résultats montrent qu'HF régulent la prolifération et la réponse aux traitements car leur perte d'expression (i) diminue la prolifération des GBM (modèle cellulaire, sphéroïde et xénogreffes in vivo), (ii) active les voies de réponse aux dommages à l'ADN et (iii) sensibilise les cellules de GBM aux irradiations. De plus, nous avons identifié un nouveau rôle pour HF en tant que régulateurs de la traduction. En effet, nos données montrent que les hnRNP HF contrôlent la traduction d'un ensemble d'ARNm en régulant l'expression et l'activité de facteurs d'initiation ainsi qu'en collaborant avec des ARN hélicases partenaires en ciblant des ARNm impliqués dans des processus reliés au développement tumoral et la résistance aux traitements possédant des structures secondaires G-quadruplexe dans leurs 5'UTR. Les données que nous avons générées suggèrent que hnRNP H et hnRNP F sont des régulateurs traductionnels essentiels au développement tumoral et à la résistance aux traitements des GBMGlioblastoma multiforme (GBM) is one of the most aggressive brain tumors with poor prognosis. Understanding the molecular mechanisms involved in the development and resistance to treatments of gliomas could improve treatment efficiency. Recently, it has been demonstrated that translational regulations play a key role in the GBM aggressivity. RNA binding proteins (RBP) are major regulators of these processes and have altered expression / activity in GBM. The RBP hnRNP H and hnRNP F (HF) are among the most overexpressed RBP in GBM and their role in GBM translational regulation has never been investigated yet. We hypothesize that HF are at the core of a post-transcriptional regulation network which impacts the translational machinery that controls GBM tumor development and resistance to treatment. We have demonstrated that hnRNP H and hnRNP F regulate proliferation and response to treatment because their depletion (i) decreases the GBM proliferation (cell line model, spheroid and in vivo xenografts), (ii) activates the DNA damage response pathways and (iii) sensitizes the GBM cells to irradiation. We have identified HF as new regulators of GBM translation. Indeed, our data show that hnRNP H and hnRNP F control mRNA translation by regulating expression/activity of initiation factors and in collaboration with RNA helicases by targeting mRNA involved in oncogenic processes and containing secondary structures called G-quadruplex in their 5'UTR. The data that we have generated suggest that HF are essential translational regulators involved in tumor development and resistance to treatment in GBM
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Santerre, Maryline. "Étude de l'action sur l'épissage de protéines nucléaires se liant à la région de l'ARN du virus VIH-1 contenant le site d'épissage A7 et role de ces protéines sur d'autres sites accepteurs d'épissage de VIH-1". Thesis, Nancy 1, 2010. http://www.theses.fr/2010NAN10115/document.
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L'épissage est une étape clef de la multiplication du VIH-1. Par utilisation de 4 sites donneurs et 8 sites accepteurs d'épissage, plus de 40 ARNm différents sont produits. Une approche protéomique nous a permis d'identifier de nouvelles protéines interagissant avec la région de l'ARN viral contenant le site A7. Nous avons démontré l'interaction directe avec l'ARN viral de 5 des protéines identifiées (nucléoline, hnRNP A1/B, hnRNP H et hnRNP K). Nous avons montré que hnRNP K a plusieurs sites de fixation dans la région du site A7 et que hnRNP A1et hnRNP K se lient de façon coopérative. Nous avons montré un effet inhibiteur de hnRNP K sur l'épissage au site A7. Comme la protéine hnRNP A1 est un régulateur négatif de plusieurs sites accepteurs d'épissage (A1, A2, A3, A7), nous avons testé si la protéine hnRNP K pouvait renforcer l'inhibition à ces sites. En fait, hnRNP K active l'épissage in vitro des introns entre le site donneur D1 et les sites accepteurs A1, A2 et A3. Nous avons montré que la protéine hnRNP K renforce fortement l'activité de ASF/SF2 au site A2, ce qui indique que selon le contexte, la protéine hnRNP K peut être activatrice ou inhibitrice de l'épissage du VIH-1. J'ai observé de plus que la surexpression de la protéine hnRNP K dans des cellules HeLa, transfectées avec le plasmide p PSP contenant le virus VIH-1 dépourvu de ses capacités d'encapsidation, produit un changement très marqué de l'épissage alternatif de l'ARN PSP, ce qui confirme la forte influence de hnRNP K sur l'épissage alternatif du VIH-1. L'augmentation de la concentration cellulaire de hnRNP K dans les cellules HeLa conduit aussi à une diminution de la protéine virale Nef. La protéine hnRNP K intervient donc non seulement dans la régulation du site A7, mais aussi dans celle de la majorité des sites d'épissage régulés de l'ARN du VIH. L'action de cette protéine sur plusieurs des sites d'épissage montre que la protéine hnRNP K est probablement un régulateur général de l'épissage de VIH-1HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 transcripts in HeLa cell nuclear extracts by affinity chromatography to identify new associated proteins. We showed that, in addition to the well known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. We demonstrated that hnRNP K has multiple binding sites in the vicinity of site A7 and that binds cooperatively to hnRNP A1 to the A7 RNA region and limits the A7 utilization in vitro. As hnRNP A1 is a negative regulator of several HIV-1 splicing sites (A1, A2, A3), we tested whether hnRNP K may also reinforce hnRNP A1 inhibition at these sites. Surprisingly, hnRNP K activated in vitro splicing of the D1-A1, D1-A2 and D1-A3 introns. Interestingly, hnRNP K was found to reinforce strongly the ASF/SF2 activity at site A2, which indicates that depending on the splicing site hnRNP K can be a splicing activator or inhibitor. To test how hnRNP K influences the relative utilization of HIV-1 splicing sites in cellulo, we used plasmid p PSP containing all the HIV-1 splicing sites and tested the effect of over-expression in HeLa cells on alternative splicing of the PSP RNA. Doubling the amount of hnRNP K in HeLa cells led to a drastic change of the PSP RNA alternative splicing, which confirms the strong influence of hnRNP K on alternative splicing. Moreover, increase of cellular concentration of hnRNP K strongly decrease the viral Nef protein production. hnRNP K protein affects A7 splicing regulation but also regulates the majority of regulated splicing sites of HIV. By extension of the study of hnRNP K effect to other HIV-1 splicing sites, we discovered that hnRNP K is a general regulator of HIV-1 splicing
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Paradis, Caroline. "Rôles de SRp30c et hnRNP I/PTB dans le contrôle de l'épissage alternatif du pré-ARN messager de hnRNP A1". Mémoire, Université de Sherbrooke, 2007. http://savoirs.usherbrooke.ca/handle/11143/3857.
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L'épissage alternatif des pré-ARN messagers est un mécanisme qui permet de générer une très grande diversité protéique chez les eucaryotes supérieurs. La sélection des sites d'épissage permet ainsi de produire certains isoformes protéiques plutôt que d'autres dans des conditions précises. Cette modulation implique généralement la participation d'une multitude de facteurs aux propriétés parfois synergiques et/ou antagonistes. Dans le cas du pré-ARN messager hnRNP A1, au moins trois éléments distincts renforcent l'exclusion de l'exon 7B. Par contre, l'élément intronique conservé de 38 nt (CE9) situé en aval de l'exon 7B permet la répression du site d'épissage 3', ce qui entraînerait l'inclusion de l'exon 7B. La portion 5' de l'élément CE9 est liée par la protéine SRp30c et cette interaction est importante pour permettre l'activité de CE9 in vitro. Afin de déterminer les composantes essentielles à l'activité de répression de SRp30c, des sites de liaison de haute affinité pour cette protéine ont été identifiés à l'aide de la procédure"SELEX". Les résultats obtenus indiquent que plusieurs sites de haute affinité reproduisent l'activité de l'élément CE9 complet dans un essai d'épissage où deux sites d'épissage 3' sont en compétition pour un seul site d'épissage 5'. De plus, les résultats suggèrent une contribution de la portion 3' de CE9, en plus de la partie 5', pour la liaison de la protéine SRp30c. En utilisant la séquence complète de CE9 pour effectuer une chromatographie d'affinité, la protéine hnRNP UPTB a été isolée et identifiée par spectrométrie de masse. Cependant, nous avons été surpris de constater que la protéine recombinante PTB agit comme activateur du site d'épissage 3' en diminuant l'activité de répression de CE9. Ces résultats suggèrent donc un nouveau rôle pour la protéine PTB, c'est-à-dire comme anti-répresseure de l'activité d'inhibition de SRp30c. PTB pourrait donc être un nouveau facteur capable de contrôler l'épissage alternatif du pré-ARN messager de hnfP A1.
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Söderberg, Malin. "Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase Gene". Doctoral thesis, Uppsala University, Department of Pharmaceutical Biosciences, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6137.
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Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented.
Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA.
Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. Trans-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs.
In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.
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Christian, Kyle. "The role of hnRNP A1 and hnRNP C1/C2 in the regulation of the stress responsive genes Cyp2a5/2A6 and p53". Doctoral thesis, Uppsala University, Department of Pharmaceutical Biosciences, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8722.
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The family of proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs) is large and diverse. Often, one and the same hnRNP will perform multiple cellular functions, leading to their description as “multifunctional proteins”. The two hnRNPs known as hnRNP A1 and hnRNP C1/C2 are multifunctional proteins found to affect the transcription, splicing, stability, and translation of specific genes’ mRNA. They are implicated in carcinogenesis, apoptosis, and DNA damage response mechanisms.
The aims of this thesis were to study the hnRNP A1 and hnRNP C1/C2 dependent regulation of two highly stress responsive genes, the tumor suppressor p53 and the cytochrome P450 enzyme Cyp2a5/CYP2A6. We identified hnRNP C1/C2 as a DNA damage induced binding protein towards the coding region of p53 mRNA, and found that while a specific cis binding site appears to have a positive function in p53 expression, interaction of hnRNP C1/C2 with this site represses the expression. The data suggest that two distinct molecular mechanisms exist for the down-regulation of p53 by hnRNP C1/C2. One mechanism, active during transcriptional stress, is dependent upon the aforementioned site, and the other, independent. We discuss how hnRNP C1/C2 dependent repression of p53 may play a role in apoptosis.
The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine Cyp2a5 gene are linked via hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a trans factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, CYP2A6, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, via changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR.
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Price, Robert Jordan. "The role of histone variants and hnRNPs in early Xenopus laevis development". Thesis, University of Portsmouth, 2011. https://researchportal.port.ac.uk/portal/en/theses/the-role-of-histone-variants-and-hnrnps-in-early-xenopus-laevis-development(a938531c-ccba-4dfe-ba77-b8144278de30).html.
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In nearly all eukaryotic cells, genomic DNA is condensed into the cell nucleus through association with histones to form a nucleoprotein complex called chromatin. The structure of chromatin affects all DNA-dependent processes, in particular transcription, replication and DNA repair. Histone variants and histone post-translational modifications can alter the structure of chromatin, which in turn governs access of regulatory factors to the DNA. Presented in this thesis is an investigation, using X. laevis, to study the functions of histone variants and two potential histone modifying proteins, hnRNP AB and hnRNP D, in the early vertebrate embryo. Spatial and temporal mRNA expression analyses of the histone variants and hnRNPs were performed using wholemount in situ hybridisation (WISH), demonstrating various mRNA levels at differing points in the embryos. At the later stages of embryogenesis the majority of the transcripts were expressed in the anterior neural tissues, which correlated with cell proliferation. To investigate the roles of two recently identified isoforms of the histone variant H2A.Z and the two hnRNP proteins, loss of function studies were performed using morpholino oligonucleotides. Loss of both hnRNP AB and hnRNP D resulted in paralysis of the embryos. Phenotypic analysis of these morphant embryos revealed a decrease in the number of primary neurons, whilst hnRNP D morphants additionally lacked blood differentiation. Inhibition of H2A.Z2 expression likewise caused paralysis. In embryos lacking H2A.Z2 however, a significant increase in primary neurons was observed, although other developmental pathways were unaffected. This increase appeared to be caused by a down-regulation of Notch expression, which rescue experiments showed to be due to the specific loss of H2A.Z2. The loss of H2A.Z1 resulted in slowly developing embryos having morphological defects. Phenotypic analysis showed that the inhibition of H2A.Z1 caused problems in the development of many tissues. A down-regulation of Xbra in these H2A.Z1 morphant embryos resulted in a decrease in mesoderm induction, shown by rescue experiments to be due to the specific loss of H2A.Z1. The effect on the control of Xbra was direct since signalling and transduction functioned normally and acetylated H2A.Z1 was seen at the Xbra promoter by ChIP experiments. The data presented here serve to further understanding of the gene regulatory roles of chromatin structure during development and shows that hnRNP AB, hnRNP D and the H2A.Z isoforms are crucial for normal embryogene
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Peebles, Katherine Anne. "HnRNP A2/B1 expression in neoplastic mouse lung cells /". Connect to full text via ProQuest. IP filtered, 2005.
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Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2005.Typescript. Includes bibliographical references (leaves 153-171). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Lodge, Anthony Paul. "Identification of a novel protein related to HnRNP-U". Thesis, University of Liverpool, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243247.
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Cordeau, Mélanie. "Implication de hnRNP A1 dans l'épissage des longs introns". Mémoire, Université de Sherbrooke, 2002. http://savoirs.usherbrooke.ca/handle/11143/3260.
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L'épissage est un processus très répandu dans la cellule. Quatre-vingt-dix-neuf pour cent des gènes subissent au moins un événement d'épissage. Environ 25% du génome correspond aux introns, soit les séquences habituellement non-codantes. Leur longueur moyenne est de 1500 paires de bases, mais certains introns ont jusqu'à plusieurs kilobases. Il est difficile de comprendre comment la cellule s'y prend pour sélectionner une paire de sites d'épissage sur une aussi grande distance. Nous avons tenté de montrer l'implication que la protéine hnRNP A1 pourrait avoir dans l'épissage des longs introns en se basant sur un modèle dérivé de son rôle dans l'épissage alternatif. Notre modèle propose que les protéines A1, après liaison à l'ARN près des sites d'épissage, interagiraient entre elles permettant de rapprocher les sites d'épissage. Le présent compte-rendu montre que la présence de sites de liaison pour A1 à l'intérieur d'un long intron augmente son efficacité d'épissage, que l'addition de plus de deux sites de liaison pour A1 allant jusqu'à quatre sites n'augmentait pas davantage l'efficacité d'épissage, mais que la position de ces deux sites à l'intérieur de l'intron l'influençait. De plus, nous avons tenté de montrer que l'augmentation de l'efficacité était bien médiée par A1 en présence de son site de haute affinité. La présence de sites de liaison pour A1 semble donc augmenter l'efficacité de son épissage, possiblement par l'intermédiaire de la formation d'une loupe permettant de rapprocher les sites d'épissage.
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Cordeau, Mélanie. "Implication de hnRNP A1 dans l'épissage des longs introns". [S.l. : s.n.], 2002.
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Shan, Jianguo. "The role of hnrnps in a2re-mediated rna trafficking in oligodendrocytes and neurons /". [St. Lucia, Qld.], 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16858.pdf.
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Landsberg, Michael. "Structural studies on the cytoplasmic RNA transport factor, hnRNP A2 /". St. Lucia, Qld, 2003. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17441.pdf.
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Seraj, Z. I. "Core proteins of rat liver heterogeneous nuclear ribonucleoprotein (hnRNP) particles". Thesis, University of Glasgow, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380388.
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Quaresma, Alexandre Jose Christino. "Estudos funcionais e estruturais da proteina humana hnRNP Q/NSAP1". [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314763.
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Orientador: Jorg KobargTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os membros da família de proteínas chamada hnRNPs (heterogenous nuclear ribonuclein proteins) apresentam importantes papeis no controle da expressão gênica e no metabolismo dos mRNAs. Os membros hnRNPD (AUF1) e hnRNPQ (NSAP1) foram alvos deste estudo. AUF1 apresenta dois domínios de ligação à RNA do tipo RRM (RNA recognition motif) e participa ativamente no processo de desestabilização de uma classe de mRNAs que apresentam um motivo rico em AU na região 3' não traduzida. Demonstramos, através do sistema de duplo híbrido em levedura, que a isoforma p37 de AUF1 interagiu com as proteínas hnRNPQ, IMP-2, NSEP1 (YB-1) e UBC9. Além disso, a proteína hnRNPQ também foi pescada num outro ensaio de duplo híbrido em levedura, que utilizou como isca a proteína humana arginina metiltransferase (PRMT1). hnRNPQ apresenta, na sua região Cterminal, um ¿motivo rico em argininas e glicinas¿ (RGG box). Demonstramos que ela é alvo de metilação pela PRMT1 in vitro e in vivo. Funcionalmente, sua metilação é importante para sua localização nuclear. NSAP1 têm uma constituição modular com um domínio ácido (AcD) no seu Nterminal, seguido por três domínios de ligação à RNA do tipo RRM e o já mencionado RGG box no seu C-terminal. Funcionalmente hnRNPQ está envolvido em vários aspectos do etabolismo de RNA, incluindo a edição do mRNA da proteína humana ApoB. Para isso, ela interage não somente com o mRNA de ApoB, mas com a enzima efetora da edição Apobec1 e com a proteína que ativadora do Apobec1 (ACF1). Mostramos que o domínio ácido, de NSAP1 é capaz de interagir com Apobec1 e que sua fosforilação in vitro pela PKC inibe esta interação. Ainda identificamos que hnRNPQ interage com proteínas da família heat shock (incluindo HSP70 e BiP), e vimos que hnRNPQ é um alvo de fosforilação principalmente pela PKCd, in vitro. A localização sub-celular de hnRNPQ é modificada pela ativação in vivo das PKCs. Em conseqüência desta ativação ou da aplicação de estresse oxidativo, térmico ou indução de estresse do reticulo endoplasmático (tratamento com tapsigargina) hnRNPQ se desloca do núcleo para o citoplasma aonde se encontra em vesículas/corpúsculos definidas. Em resumo, nossos dados sugerem que as diversas funções da hnRNPQ relacionadas ao metabolismo de mRNAs, sofrem diferentes regulações, mediadas por modificações pós-traducionais (fosforilação e metilação), que interferem tanto na sua localização celular quanto na sua afinidade por determinados proteínas parceiras
Abstract: The members of the hnRNPs family (heterogenous nuclear ribonuclein proteins) play important roles in gene expression control and mRNAs metabolism. The proteins hnRNPD (AUF1) and hnRNPQ (NSAP1) were the main targets of this study. AUF1 has two RNA recognition motifs (RRM) and participates in the process of destabilization of a class of mRNAs that contain AU-rich sequences in their 3' untranslated regions (3'-UTR). We found, using the ¿yeast two-hybrid system¿ (Y2HS), that the isoform p37 of AUF1 (AUF1p37) interacts with the proteins: hnRNPQ, IMP-2, NSEP1 (YB-1) and UBC9. Moreover, the protein hnRNPQ was also identified as a prey protein in another Y2HS screen, which used as bait the human protein Arginine methyltransferase (PRMT1). HnRNPQ presents, in its C-terminal region, an "Arginine/Glicine-rich sequence" (RGG box). We are able to show that this RGG box is a target for methylation by PRMT1 in vitro and is methylated in vivo. Functionally, this methylation is important for its nuclear localization. hnRNPQ has a modular organization with an acid domain (AcD) in its N-terminal, followed by three RNA-binding domains (RRM) and the previously mentioned RGG box in its C-terminal. Functionally, hnRNPQ is involved in diverse aspects of RNA metabolism, including editing of the mRNA encoding the human protein ApoB. It has been shown previously to interact with the mRNA of ApoB, and also with the editing enzyme Apobec1 and the Apobec1 activation protein (ACF1). Here we show that the acid domain of hnRNPQ mediates the interaction with Apobec1 and that its in vitro phosphorylation (by PKC) inhibits this interaction. Furthermore, we found that hnRNPQ interacts with members the heat shock family of proteins (including HSP70 and BiP), and demonstrated that hnRNPQ can be in vitro phosphorylated by PKCd. Finally, we discovered that the sub-cellular localization of hnRNPQ undergoes modification after activation of PKC pathways. This also occurs after application of endoplasmic reticulum stress (using tarpsigargin), oxidative or heat stress. Under all of these conditions hnRNPQ translocated from the nucleus to the cytoplasm, where it is found at defined vesicles or granules. In summary, our data suggest that the diverse functions of hnRNPQ in the context of mRNA metabolism, may suffer specific regulations, by post-translational modifications, including phosphorylation and methylation, which modify both the proteins sub-cellular localizations as well as its affinity to interacting protein partners
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular
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Richard, Travis. "ARK5 Regulates Subcellular Localization of hnRNP A1 During Hypertonic Stress". Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36212.
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During cellular stress, the regulation of protein synthesis is a key adaptive mechanism used by cells to survive. In response to various stresses, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an RNA binding protein principally found within the nucleus, is phosphorylated and consequently accumulates in the cytoplasm. Among other roles, cytoplasmic hnRNP A1 functions as an auxiliary translation factor for internal ribosome entry site (IRES)-mediated translation of specific mRNA, including the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL). To identify which kinases control the cytoplasmic accumulation of hnRNP A1, an RNAi-based kinome-wide screen was performed in hypertonically stressed U2OS cells, from which AMPK-related kinase 5 (ARK5) was identified as a potential regulator of hnRNP A1’s localization. Here we show that ARK5 directly phosphorylates hnRNP A1 and that the inhibition of ARK5 expression blocks the stress induced cytoplasmic accumulation of hnRNP A1, modulates expression of Bcl-xL protein and increases cell viability. Our data points to a novel role for ARK5 and provides further insight into the mechanisms regulating cellular stress response.
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Cornella, Nicola. "Characterization of the hnRNP RALY in RNA transcription and metabolism". Doctoral thesis, Università degli studi di Trento, 2017. https://hdl.handle.net/11572/369300.
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The heterogeneous nuclear ribonucleoproteins (hnRNPs) form a large family of RNA-binding proteins (RBPs) that exert numerous functions in RNA metabolism. For example, soluble hnRNPs bind to RNAs to mediate their maturation, processing, and shuttling from the nuclear compartment to the cytoplasm. Additionally, hnRNPs might interact with chromatin to regulate the transcription and the post-transcriptional modification of nascent transcripts.
RALY is a member of the hnRNP family that binds poly-U rich elements within several RNAs and regulates the expression of specific transcripts. RALY is upregulated in different types of cancer and its downregulation has been shown to impair cell proliferation. In my PhD project, I characterized RALY to interact with transcriptionally active chromatin in a transcription-dependent manner and to cause a global decrease of RNA Polymerase II (RNAPII)-mediated transcription when downregulated, without affecting RNAPII elongation rate. Through microarray analysis of RALY-downregulated HeLa cells, I detected an altered expression of numerous genes involved in transcription promotion and cell cycle regulation, including the E2F transcription factors family. Due to its relevant role in regulating the cell cycle, I focused on the proliferation-promoting factor E2F1. I demonstrated that the stability of E2F1 mRNA is reduced in cells lacking RALY expression, with a resulting reduction of E2F1 protein levels. As a consequence of RALY knock-out, HeLa cells present a slower cell proliferation compared to control cells. Finally, by crossing the list of RALY targets with the list of genes affected by RALY downregulation, I propose a positive role of RALY in regulating the fate of specific transcripts.
Taken together, my results highlight the importance of RALY expression for transcription and cell proliferation.
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Cornella, Nicola. "Characterization of the hnRNP RALY in RNA transcription and metabolism". Doctoral thesis, University of Trento, 2017. http://eprints-phd.biblio.unitn.it/2625/2/Disclaimer_Cornella.pdf.
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The heterogeneous nuclear ribonucleoproteins (hnRNPs) form a large family of RNA-binding proteins (RBPs) that exert numerous functions in RNA metabolism. For example, soluble hnRNPs bind to RNAs to mediate their maturation, processing, and shuttling from the nuclear compartment to the cytoplasm. Additionally, hnRNPs might interact with chromatin to regulate the transcription and the post-transcriptional modification of nascent transcripts.
RALY is a member of the hnRNP family that binds poly-U rich elements within several RNAs and regulates the expression of specific transcripts. RALY is upregulated in different types of cancer and its downregulation has been shown to impair cell proliferation. In my PhD project, I characterized RALY to interact with transcriptionally active chromatin in a transcription-dependent manner and to cause a global decrease of RNA Polymerase II (RNAPII)-mediated transcription when downregulated, without affecting RNAPII elongation rate. Through microarray analysis of RALY-downregulated HeLa cells, I detected an altered expression of numerous genes involved in transcription promotion and cell cycle regulation, including the E2F transcription factors family. Due to its relevant role in regulating the cell cycle, I focused on the proliferation-promoting factor E2F1. I demonstrated that the stability of E2F1 mRNA is reduced in cells lacking RALY expression, with a resulting reduction of E2F1 protein levels. As a consequence of RALY knock-out, HeLa cells present a slower cell proliferation compared to control cells. Finally, by crossing the list of RALY targets with the list of genes affected by RALY downregulation, I propose a positive role of RALY in regulating the fate of specific transcripts.
Taken together, my results highlight the importance of RALY expression for transcription and cell proliferation.
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Folci, A. C. "HNRNP K: A NEW PROTEIN IN NEURON DEVELOPMENT AND FUNCTION". Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/171964.
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Regulation of actin polymerization is crucial for neuronal morphogenesis, in particular in neurite elongation and branching, in synaptogenesis and in synaptic plasticity.
The heterogeneous nuclear ribonucleoprotein (hnRNP) K protein is an RNA-binding protein characterized by three K homology domains that mediate RNA-binding.
hnRNP K is involved in a host of processes that comprise gene expression, such as chromatin remodeling, transcription, pre-mRNA splicing, mRNA export and translation.
Moreover in heterologous cells hnRNP K regulates negatively N-WASP, a protein that interacts with Arp2/3 complex .
The main aim of this project was the characterization of hnRNP K function in neurons, we analyzed its role on neuron morphology, subsequently on synaptic function.
We found that hippocampal neurons overexpressing hnRNP K siRNA show normal spine morphology, but reduced spine density than control neurons, moreover the knockdown of hnRNP K increases the spine turnover.
Furthermore we observed a significant decrease in PSD95 and GluA2 in silenced neurons while no effect was detected on VGAT and VGLUT expression; moreover the hnRNP K knockdown impairs the postsynaptic compartment in excitatory synapse, while the presynaptic compartment was not affected. Since the involvement of GluA2 subunit, we performed electrophysiological recordings in order to investigate functional changes in AMPARs physiology. Preliminary results show a dramatic decrease in excitatory, AMPARs mediated, miniature postsynaptic currents (mEPSCs) frequency in hnRNP K-silenced neurons. The importance of hnRNP K in synaptic activity was confirmed by its increase after chemical LTP induction; moreover the role of hnRNP K in LTP was also demonstrated by the impairment of this phenomena in silenced neurons. Now we are investigating the molecular mechanism underling these alterations studying the interaction with N-WASP. We demonstrated the directly interaction between hnRNP K and N-WASP in neurons by coimmunoprecipitation.
The alterations observed in hnRNP K silenced neurons were described from a morphological and a functional perspective, evidencing a strong impairment of excitatory synaptic structures. Besides, our data suggest a strong link between this protein and actin dynamics, suggesting that these effects could eventually be consequent to the actin cytoskeleton rearrangements caused by hnRNP K loss.
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Courteau, Lynn. "Regulation of hnRNP A1 Cellular Localization by Protein Kinases and its Biological Impact". Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32078.
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Human Rhinoviruses (HRVs) utilize Internal Ribosome Entry Sites (IRES) to drive viral protein synthesis. IRESs are specialized RNA elements present within the 5’ UTR of mRNAs that recruit ribosomes independently of the 5’ m7G cap structure. hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), a multifunctional RNA binding protein, is required for the IRES-dependent translation of many specific RNAs within the cell cytoplasm. The phosphorylation of hnRNP A1 is
required for its cytoplasmic accumulation. I have identified and validated the role of HK2 in hnRNP A1 cellular localization by immunofluorescence microscopy, by analysis of HRV infection and by siRNA-based screening. These studies show that decreased HK2 protein levels lead to decreased cytoplasmic accumulation of hnRNPA1 during osmotic shock and HRV infection, to a decrease in HRV-infected cells and to decreased caspase activation in osmotically stressed and HRV-infected
cells. Thus, HK2 may regulate hnRNP A1 cytoplasmic localization following HRV infection.
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Long, Jennifer Connie. "Identification of in vivo RNA tragets of the RNA-binding proteins Acinus and hnRNP A1". Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4227.
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RNA-binding proteins play a central role in the post-transcriptional regulation of gene expression; however, little is known about the endogenous transcripts to which they bind. Here, I have used the ultra-violet cross-linking and immuno-precipitation (CLIP) technique to identify RNA targets directly bound to two RNA-binding proteins: Acinus and hnRNP A1. Acinus (apoptotic chromatin condensation inducer in the nucleus) contains a region that is homologous to the RNA binding domain of the Drosophila splicing regulator sex-lethal, and a serine and arginine rich region similar to that seen in the SR family of proteins, which function extensively in splicing. Furthermore it is a component of the multi-protein spliceosome complex, and I have demonstrated it can directly bind polyadenylated RNA. I have shown that Acinus displays a diffuse nuclear localisation pattern, however, overexpression of an epitope-tagged protein results in its accumulation in enlarged nuclear speckles. Together these results suggest a role in pre-mRNA splicing. Acinus is cleaved during apoptosis by caspase-3, resulting in a truncated protein with chromatin condensation inducing activity (Sahara et al., 1999). Accordingly, I have demonstrated that overexpression of epitope-tagged Acinus results in an increased number of cells exhibiting an apoptotic phenotype. The proteolytic fragment contains the RNA binding region, and to determine if the role of Acinus in apoptosis is mediated by RNA interactions I utilised CLIP to identify in vivo RNA targets. I have identified several mRNA targets of Acinus and found that the binding sites in those mRNA targets predominantly map to constitutively expressed exons. This is in agreement with the exon junction complex, of which Acinus is a component, being deposited on mRNAs after splicing. These results may indicate that Acinus is a core RNA binding factor of the exon junction complex. To complement this approach, I also performed CLIP with a known alternative splicing regulator, hnRNP A1. In this manner, the binding site preferences could be compared between the two proteins. As expected, the majority of hnRNP A1 binding sites are located in introns, corresponding with their identified role of antagonizing pre-mRNA splicing by binding intronic splicing elements. Interestingly, a number of the CLIP tags are located in, or adjacent to, alternatively spliced events suggesting a role for hnRNP A1 in the regulation of alternative splicing of these specific pre-mRNAs. In addition to pre-mRNA splicing hnRNP A1 also functions in the cellular stress response. Upon environmental stresses it relocates to the cytoplasm and accumulates in cytoplasmic foci known as stress granules (Guil et al., 2006). Here I show some of the targets identified by CLIP are regulated by hnRNP A1 in times of cellular stress. In summary, I have identified two novel subsets of RNAs, bound by Acinus or hnRNP A1 in vivo. I have shown these proteins exhibit distinct binding preferences, which correspond to their biological function. This work is consistent with hnRNP A1 acting as an alternative splicing regulator, and provides evidence for a dual role of Acinus in mRNA splicing and apoptosis. This study also demonstrates the power of the CLIP technique, as identification of in vivo RNA targets allows greater understanding of the mechanisms by which RNA-binding proteins exert their regulatory control.
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Dupuis, Sophie. "Interaction entre la protéine HnRNP A1/UP1 et les séquences télomériques humaines". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0001/MQ40578.pdf.
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Charroux, Bernard. "Contribution à l'étude fonctionnelle de la protéine hnRNP K chez Drosophila melonogaster". Aix-Marseille 2, 1997. http://www.theses.fr/1997AIX22049.
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Le travail presente dans ce memoire concerne la caracterisation moleculaire et fonctionnelle d'un nouveau gene de drosophila melanogaster, essentiel au developpement correct des appendices de la mouche, le gene twisted leg (twl). Ce gene code pour une proteine presentant de fortes homologies avec les proteines hnrnp k de vertebres. La proteine twl est detectee tout au long du developpement dans le noyau des cellules, a l'exception des cellules polaires ou elle est localisee dans le cytoplasme. Les mutations perte de fonction du gene twl revelent que ce gene est necessaire, mais non essentiel, a l'embryogenese normale. En revanche, la fonction twl#+ est critique pour le developpement correct des appendices adultes. En son absence, ces structures, ainsi que les disques imaginaux dont elles derivent, ont une taille reduite resultant d'un defaut de divisions cellulaires. Des transgenes exprimant la proteine hnrnp k de drosophile ou humaine sont capables de sauver le phenotype d'un mutant hypomorphe de twl, montrant la similarite fonctionnelle de ces deux proteines in vivo. De plus, les mutations twl interagissent genetiquement avec des mutations de deux genes importants pour la progression du cycle cellulaire, de2f et dmcdc2c. Ces resultats montrent que la proliferation des cellules imaginales est particulierement sensible a l'absence de la proteine twl. Dans la deuxieme partie du travail nous avons etudie les effets de la surexpression dans les disques imaginaux de la proteine twl de drosophile et de son homologue hnrnp k humain. Dans les regions des disques ou ces proteines sont surexprimees, on observe une mort cellulaire intense qui se traduit par l'absence des structures epidermiques adultes correspondantes. La surexpression de la proteine antiapoptotique p35 du baculovirus supprime le phenotype lie a la surexpression de twl, montrant qu'un exces de proteine hnrnp k induit principalement une mort cellulaire par apoptose. Dans ces experiences, nous avons mis en evidence une regulation negative de l'expression du gene twl endogene par la proteine hnrnp k humaine, suggerant qu'il doit exister un mecanisme d'auto-regulation negative qui controle la quantite de proteine hnrnp k synthetisee.
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Schwartz, Laura Link. "HnRNP E1 Protects Chromosomal Stability Through Post-Transcriptional Regulation of Cdc27 Expression". Kent State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=kent1445519325.
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Dupuis, Sophie. "Interaction entre la protéine HnRNP A1/UP1 et les séquences télomériques humaines". Sherbrooke : Université de Sherbrooke, 1998.
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Blanchette, Marco. "Modulation de l'épissage alternatif de hnRNP A1 éléments de contrôle et mécanismes d'action". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/NQ56990.pdf.
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Pratt, Kenny Matthew. "Novel properties of hnRNP-UL1 : its possible role in the pathogenesis of ALS". Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/6583/.
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Heterogeneous nuclear ribonucleoprotein-U like 1 (hnRNP-UL1) is a protein with numerous roles within the cell, including RNA processing and responses to DNA damage. Within this study two novel aspects of the protein are explored: the role of a putative nucleotide-binding domain and the protein's possible involvement in amyotrophic lateral sclerosis (ALS). hnRNP-UL1 is known to have a putative nucleotide-binding domain within its central region containing both a Walker A and Walker B motif. This region had not been investigated previously and was therefore of great interest in this study. The Walker A motif was shown to bind adenosine triphosphate (ATP) and the region appears to possess protein kinase activity. A biological substrate and function for these activities were not established, but these observations suggest that there are still layers of complexity to hnRNP-UL1's cellular roles to be elucidated. ALS is a late-onset neurodegenerative disease with limited treatment strategies and poor patient outcomes. Many of the proteins involved in its pathogenesis have two properties in common: they have roles in RNA-processing and possess prion-like domains (PrLDs). The properties of hnRNP-UL1 appertain to both of these and therefore it was of great interest when ALS patients were discovered with heterozygous hnRNP-UL1 mutations. Results showed that cells possessing the ALS patient mutations (R639C and R468C) had no DNA damage response (DDR) defects or mislocalisation of the protein, but their ssDNA/RNA-binding capability was markedly reduced. Whilst no direct causative links to ALS pathogenesis were shown with the hnRNP-UL1 patient mutations in this study, growing evidence implies good reason for the protein to have involvement in the disease.
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Kanhoush, Rasha. "Etude moléculaire et biochimique des interactions de la protéine hnRNP G avec l’ARN". Paris 6, 2010. http://www.theses.fr/2010PA066054.
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La protéine hnRNP G est impliquée dans l’épissage alternatif de plusieurs précurseurs d’ARN messager. Pour mieux comprendre la spécificité de cette protéine, j’ai étudié ses interactions avec les ARNs in vitro et in vivo. Un motif d’ARN structuré dans une tige-boucle qui contient la séquence GGAAA a été identifié in vitro comme une cible potentielle d’hnRNP G. Cet ARN m’a permis d’identifier un nouveau domaine d’interaction avec l’ARN dans la structure de l’hnRNP G, situé dans sa région C-terminale et désigné « C-ter RBD ». Le modèle des chromosomes en écouvillon chez l’amphibien m’a permis de montrer qu’un nouveau domaine situé au centre de la structure de la protéine et distinct de ses domaines d’interaction avec l’ARN est responsable de son recrutement au niveau des transcrits naissants des chromosomes. Ce domaine a été désigné Nascent transcripts Targeting Domain (NTD). Cette étude montre que l’hnRNP G s’associe avec la majorité des ARNs transcrits par l’ARN polymérase II tout en montrant un recrutement plus important au niveau de quelques boucles des chromosomes. En particulier, l’association de l’hnRNP G avec les transcrits du bivalent sexuel ZW chez Pleurodeles waltl, montre un recrutement hétéromorphe au niveau de certains sites de ce bivalent. Au cours de ma thèse, j’ai participé à la mise en évidence d’une activité différentielle de liaison à l’ARN liée au génotype sexuel, pour les isoformes de l’hnRNP G dans l’ovocyte de P. Waltl. Les résultats suggèrent la présence d’une famille multigénique de l’hnRNP G répartie sur un autosome et sur les chromosomes sexuels. Cette étude contribue à la caractérisation de l’hnRNP G et à la compréhension de ses fonctions.
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Blanchette, Marco. "Modulation de l'épissage alternatif de hnRNP A1 : éléments de contrôle et mécanismes d'action". Sherbrooke : Université de Sherbrooke, 2000.
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Chaumet, Alexandre. "Identification de nouveaux partenaires d'Ilf3 et de NF90, deux protéines aux ARN". Paris 6, 2009. http://www.theses.fr/2009PA066385.
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Ilf3 et NF90 sont deux protéines générées par épissage alternatif à partir du gène ILF3. Pour chacune d’entre-elles, un épissage alternatif supplémentaire permet la synthèse de deux formes, une longue et une courte, qui diffèrent par la présence ou non d’une séquence N-terminale de 13 acides aminés. En plus de ces modifications post-transcriptionnelles, Ilf3 et NF90 subissent des modifications post-traductionnelles, phosphorylation et méthylation. Ces deux protéines illustrent à la fois le phénomène de polymorphisme protéique ainsi que celui de plurifonctionnalité des protéines. En effet, au moins 20 isoformes sont générées à partir du même gène, 12 pour Ilf3 et 8 pour NF90. Ce polymorphisme pourrait être à l’origine des différences de localisation subcellulaire observées selon l’isoforme considérée mais également d’ajustements au niveau de la régulation des interactions avec leurs partenaires et de la diversité des fonctions qui leur ont été attribuées. Après avoir montré que la présence des 13 résidus N-terminaux permet la localisation nucléolaire des formes longues d’Ilf3 et de NF90, nous avons effectué une approche protéomique qui a permis de caractériser 6 nouveaux partenaires de ces deux protéines. Les fonctions décrites de ces partenaires permettent de suggérer une implication de ces deux facteurs dans le métabolisme des ARN métabolisme des ARN.
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Garcia, Cristiana Bernadelli. "Acúmulo da ribonucleoproteína heterogênea nuclear K em câncer de cabeça e pescoço: estudos mitocondriais". Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-04062014-112939/.
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A ribonucleoproteína heterogênea nuclear K (hnRNP K) é uma proteína envolvida em processos de expressão gênica e tem sido proposta como ligante de RNAs mensageiros mitocondriais. Apesar de ser considerada um marcador de pior prognóstico no câncer de cabeça e pescoço, o papel da hnRNP K nesta doença ainda é pouco conhecido. O objetivo deste trabalho foi estudar o envolvimento da hnRNP K na mitocôndria com ênfase na bioenergética e na identificação de novos potenciais ligantes de hnRNP K. As linhagens celulares utilizadas foram de carcinoma de cabeça e pescoço (HN13 e CAL 27) com silenciamento de RNA para hnRNP K e células HEK293 com super-expressão de hnRNP K. O efeito do acúmulo celular da hnRNP K na cadeia transportadora de elétrons mitocondrial foi avaliado por meio da atividade dos complexos mitocondriais I, II e V em células HN13. A redução do nível de hnRNP K usando RNA de interferência promoveu uma diminuição da atividade dos complexos nas células HN13, indicando o envolvimento da proteína na eficiência do transporte de elétrons na cadeia respiratória mitocondrial. Células HEK293 com super-expressão da hnRNP K (HEK293/hnRNP K) e as linhagens HN13 e CAL 27 com silenciamento e redução estável de hnRNP K foram utilizadas para determinar o papel de hnRNP K no potencial de membrana mitocondrial, níveis de ATP, produção de lactato e consumo de oxigênio. Células HEK293/hnRNP K comparadas ao controle apresentaram maior nível de ATP, menor potencial de membrana mitocondrial, menor consumo de oxigênio e maior produção de lactato. As células HN13 com redução da hnRNP K apresentaram níveis mais baixos de ATP, com menor liberação de lactato para o meio extracelular e maior consumo de oxigênio. Esses resultados sugerem que o acúmulo da proteína hnRNP K tem ação importante na mitocôndria por alterar o metabolismo bioenergético celular de fosforilação oxidativa para glicólise anaeróbica. A estratégia de co-imunoprecipitação usando anticorpos para hnRNP K, digestão de proteínas com tripsina e cromatografia líquida acoplada a espectrômetria de massa foi usada para encontrar novos potenciais ligantes de hnRNP K. A análise dos dados com o software SEPro identificou 57 proteínas candidatas a ligantes da hnRNP K. Três proteínas foram validadas por co-IP e Western blotting: o fator de transcrição mitocondrial PTCD3, YB1 e PSF. Propomos que a hnRNP K apresenta função na energética mitocondrial, e provavelmente, a sua interação com PTCD3 participa desta função.Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a protein involved in gene expression processes, which has been proposed to bind mitochondrial mRNAs. Despite it to be considered a prognostic marker in cancer, the hnRNPK role in this disease is unknown. We addressed the involvement of hnRNP K in mitochondria with emphasis on bioenergetics and identification of new potential ligands of hnRNP K. The cell lines used were from head and neck squamous cell carcinoma (HN13 and CAL 27) with RNA silencing for hnRNP K , and HEK293 cells with overexpression of hnRNP K. The effects of cellular accumulation of hnRNP K in mitochondrial electron chain carriers were assessed by the activity of mitochondrial complexes I, II and V in HN13 cells. Reduced levels of hnRNP K using RNA interference promoted a decrease in the activity of the complexes in HN13 cells, indicating the involvement of the protein in the efficiency of the electron transport in mitochondrial respiratory chain. HEK293 cells with overexpression of hnRNP K (HEK293/hnRNP K) and HN13 and CAL 27 cells with silencing and stable reduction of hnRNP K were used to determine the role of hnRNP K in mitochondrial membrane potential, ATP levels, lactate production and oxygen consumption. HEK293/hnRNP K, compared to control cells, showed higher levels of ATP, reduced mitochondrial membrane potential, lower oxygen consumption and higher production of lactate. HN13 cells with reduced hnRNP K had lower ATP levels, with lower release of lactate to the extracellular medium and higher oxygen consumption. These results suggest that accumulation of hnRNP K protein plays a role in mitochondria by changing the cellular energetic metabolism from oxidative phosphorylation to glycolysis. The strategy of co-immunoprecipitation using antibodies for hnRNP K, protein digestion with trypsin, and liquid chromatography, coupled to mass spectrometer, were used to search for new potential ligands of hnRNP K. Data analysis with software SEPro identified 57 candidate proteins binding to hnRNP K. Three proteins were validated by co-IP and Western blotting: the mitochondrial transcription factor PTCD3, YB1, and PSF. We propose that hnRNP K plays a role in the mitochondrial energetics, and probably its interaction with PTCD3 participates in this function.
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Silva, Vinicius Barreto da. "Modelagem molecular, síntese e avaliação da atividade biológica de potenciais antineoplásicos com a proteína hnRNP K e culturas de células tumorais". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/60/60136/tde-24092011-000316/.
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A proteína hnRNP K é conhecida por seu papel nos múltiplos processos que compõe a expressão gênica, incluindo funções nos estágios de splicing, transcrição e tradução, desempenhadas, principalmente, através da ligação de seus domínios KH a nucleotídeos. A ativação inadequada da hnRNP K tem relação direta com a gênese de alguns tipos de câncer, sobretudo de cabeça e pescoço, mama e colo-retal, evidenciando a mesma como um atrativo alvo molecular para o desenvolvimento de novos fármacos antineoplásicos. Com o auxílio de técnicas in silico, foram identificados dois compostos orgânicos, um derivado de benzimidazol e outro derivado de fenilbenzamida, capazes de impedir a ligação da proteína hnRNP K a oligonucleotídeos in vitro. Aliando as técnicas de docking, campos de interação e dinâmica molecular foi possível sugerir que tais compostos apresentam características estruturais que permitem a realização de interações na fenda de ligação do domínio KH3, principalmente com os resíduos R40 e R59, os quais são considerados chaves no reconhecimento molecular de nucleotídeos. Os derivados de benzimidazol e fenilbenzamida constituem novos compostos de partida na busca de novos antineoplásicos que tem como alvo a proteína hnRNP K. Do ponto de vista de metabolismo e toxicidade, o derivado de fenilbenzamida parece ser mais promissor quando se investiga uma molécula para aplicação terapêutica, uma vez que gerou poucos alertas críticos de toxicidade, ao contrario do derivado de benzimidazol, que apresenta maior potencial genotóxico. Apesar de se ligarem aos domínios KH da hnRNP K e impedir sua complexação a nucelotídeos, ensaios com culturas de células mostraram apenas tênue atividade antitumoral para tais compostos, com maior redução de viabilidade celular, ao redor de 18% a 8,4 M, exibida pelo derivado de fenilbenzamida. Seguindo o princípio do análogo ativo, simulações de triagem virtual na busca de análogos dos ligantes da hnRNP K revelaram 21 novos derivados de benzimidazol ou fenilbenzamida na base de dados EXPRESS-Pick, dos quais 5 foram testados in vitro com a proteína e 3 novos ligantes identificados. Com o intuito de otimizar tais derivados, foram sugeridos in silico substituintes (potenciais bioisósteros) para os anéis dioxopirrolidínicos dos ligantes já identificados, guiando, assim, a futura síntese de novas substâncias com potencial atividade antitumoral. Além disso, o trabalho foi complementado através da proposição de síntese de novos derivados benzoxazepínicos acoplados a purinas, os quais também tem aplicação como antineoplásicos, entretanto por mecanismos que não envolvem a hnRNP K. A grande limitação desses derivados é a presença de um grupo nitro aromático, o qual é reconhecido por sua toxicidade pronunciada. Com o intuito de otimizar tais derivados, foram sugeridos in silico potenciais bioisósteros capazes de substituir o grupo nitro e guiar a síntese e novos derivados com atividade antitumoral e toxicidade reduzida.hnRNP K protein is known for its role in the multiple processes that compose gene expression, including functions during splicing, transcription and translation, developed, mainly, by the binding of nucleotides to KH domains. Inadequate activation of hnRNP K induces the development of some types of cancer, including head and neck, breast and colorectal. In this way, hnRNP K is an attractive molecular target for antineoplastic drug design. Using in silico strategies, we have identified two organic compounds, a benzimidazole and a phenylbenzamide derivatives, able to prevent the natural binding of nucleotides to hnRNP K in vitro. Applying docking, molecular interaction fields and molecular dynamics simulations it was possible to propose that such compounds present structural characteristics capable to support intermolecular interactions inside KH3 domain binding cleft, mainly with R40 and R59 residues, which are extremely important during molecular recognition of nucleotides by hnRNP K. The benzimidazole and phenylbenzamide derivatives identified are novel lead compounds that can guide the design of new antineoplastic drugs targeting hnRNP K. Considering metabolic and toxicity predictions, the phenylbenzamide seems to be more promising than the benzimidazole derivative as a drug, once the benzimidazole presents genotoxic potential. Although both derivatives prevent the binding of nucleotides to hnRNP K, biological assays with tongue cancer cell lines revealed only a mild antitumoral activity for such compounds. Higher level of cell viability reduction, 18% at 8.4 M, was observed for the phenylbenzamide derivative. Following the similarity principle, virtual screening simulations were made intending to find novel benzimidazole and phenylbenzamide derivatives inside EXPRESS-Pick database. The search revealed 21 compounds, 5 of which were tested in vitro with hnRNP K, where 3 of them were active. Intending to optimize benzimidazole and phenylbenzamide derivatives in order to design more potent chemical entities, we have suggested in silico substituents as potential bioisosteric groups of the dioxopyrrolidine rings of hnRNP K ligands, guiding the future synthesis of novel compounds with enhanced antitumoral activity. Moreover, complementary work proposition was performed through the synthesis of benzoxazepin-purines, which also present antitumoral activity but not through hnRNP K pathway. The major limitation of such derivatives is the presence of a nitro aromatic group, which can be very toxic. 20 potential bioisosteric groups were proposed as fragment candidates to replace the nitro one in order to design novel antitumoral derivatives with reduced toxic potential.
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Thompson, Peter Jeffrey. "Transcriptional silencing of endogenous retroviruses by the novel lysine methyltransferase co-repressor hnRNP K". Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/55760.
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Histone lysine methylation is essential for mammalian development and maintenance of somatic cell identity, as evidenced by a group of Mendelian diseases and cancers linked with mutations in lysine methyltransferases (KMTs). The transcriptional silencing of a class of retrotransposons known as endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs) provides a unique model system in which to investigate epigenetic regulation by the H3K9 family of KMTs and characterize novel molecular mechanisms of relevance to human biology and disease. In mESCs, class I and II ERVs are silenced by the SETDB1/KAP1 complex, which deposits histone H3K9 trimethylation (H3K9me3). In contrast, class III MERVL ERVs are silenced by the G9a/ GLP complex, which deposits H3K9me2. The molecular mechanisms governing the recruitment of these KMTs to their genomic ERV targets remain poorly understood. The goal of this work was to identify and characterize novel factors that regulate the functions of these KMTs in ERV silencing. In the first part of my thesis work, I identified the RNA-binding protein and transcription factor hnRNP K as a novel co-repressor for the SETDB1/KAP1 complex. HnRNP K coordinates recruitment of the KMT SETDB1 by KAP1 to its ERV targets. This function of hnRNP K involves a previously uncharacterized influence on the levels of chromatin protein SUMOylation. In the second part of my thesis work, I demonstrated that MERVL elements are also repressed by hnRNP K and can remain inactive in the absence of H3K9me2, likely due to the lack of transcriptional activators. HnRNP K forms a novel RNA-dependent complex with G9a/GLP, is required for global H3K9me2 and provides a repressive barrier to MERVL expression in the presence and absence of H3K9me2. Taken together my work has provided significant insights into the epigenetic repression of ERV transcription by KMTs and demonstrates that hnRNP K is a novel co-repressor for two different KMT complexes. As recent studies have linked mutations in HNRNPK to the novel Mendelian disorder Au-Kline syndrome and cancer, these insights should also guide future studies on the role of hnRNP K in regulation of KMT-mediated signaling pathways in human disease.Medicine, Faculty of
Medical Genetics, Department of
Graduate
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Silva, Vinicius Barreto da. "Estudos de modelagem molecular e relação estrutura atividade da oncoproteína hnRNP K e ligantes". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/60/60136/tde-02102008-164529/.
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O Projeto Genoma Humano do Câncer (PGHC), financiado pela FAPESP e pelo Instituto Ludwig de Pesquisa sobre o câncer, buscou identificar os genes expressos nos tipos mais comuns de câncer no Brasil. Tal projeto conseguiu identificar aproximadamente um milhão de seqüências de genes de tumores freqüentes no Brasil. A contribuição brasileira foi maior para tumores de cabeça e pescoço, mama e cólon. Uma das iniciativas mais recentes e estimuladas pelo PGHC é o projeto Genoma Clínico, o qual visa desenvolver novas formas de diagnóstico e tratamento do câncer através do estudo de genes expressos. A partir da análise molecular de tecidos saudáveis e neoplásicos em diferentes estágios, é possível identificar marcadores de prognóstico, permitindo escolhas de terapias mais adequadas e eficientes. A proteína hnRNP K foi identificada como um desses marcadores, em neoplasias da região da cabeça e pescoço, sendo objetivo deste estudo a aplicação de técnicas de bioinformática e modelagem molecular no planejamento baseado em estrutura de candidatos a fármacos antineoplásicos que bloqueiem a atividade da proteína. A proteína hnRNP K apresenta diversas funções e é encontrada nos mais diversos compartimentos celulares, interferindo, basicamente, no sistema de expressão gênica. Essa proteína apresenta 3 domínios KH, os quais são responsáveis por sua ligação à moléculas de DNA e RNA. Modelos de boa qualidade dos domínios KH foram construídos através da estratégia de modelagem molecular por homologia estrutural. Após screening virtual em bases de dados de compostos (330.000 aproximadamente) com propriedades drug-like, 15 compostos com potencial de interação com o domínio KH3 foram selecionados. Os modos de ligação para cada um dos mesmos no sítio ligante do domínio KH3 foram sugeridos por simulações de docking e apresentaram um bom encaixe espacial com os sítios receptores virtuais calculados pelos campos de interação molecular. Simulações de dinâmica molecular foram realizadas com o intuito de avaliar a estabilidade dos compostos selecionados, que também foram avaliados quanto à presença de grupamentos toxicofóricos em sua estrutura.The Brazilian Project Genoma Câncer (PGHC) supported by FAPESP and the Ludwig Institute for Cancer Research, intended to identify the genes involved in the most common cases of cancer in Brazil. In this project about a million of gene sequences were identified. The major contribution was made in breast, colorectal and head and neck cancers. The results obtained stimulated the creation of another project, called Genoma Clínico, which intend to develop new trends in treatments and diagnosis of cancer based on the study of expressed genes. Analyzing healthy and neoplasic tissues in different stages, it is possible to identify molecular markers related to the prognosis of cancer, allowing the use of more efficient therapies. The hnRNP K protein was identified as a molecular marker in head and neck cancer, where the objective of this work lies in the application of bioinformatics and molecular modeling strategies by structure-based drug design to identify potential antineoplasic drug candicates that could act against hnRNP K protein. The hnRNP K protein is encountered in all cellular compartments and act, basically, in the gene expression pathways. Its structure is composed by three KH domains that mediate interactions with DNA and RNA molecules. High quality models of KH domains were built by homology modeling. After the virtual screening simulations performed with drug-like compound databases, containing approximately 330.000 compounds, 15 were selected as potential ligands of KH3 domain of hnRNP K. The binding modes suggested for these compounds, by docking simulations, presented a good spatial fit when compared with the virtual receptor sites calculated by molecular interaction fields. Molecular dynamics simulations were performed in order to evaluate de stability of the binding modes suggested. The potential ligands were also evaluated to identify toxicophoric features in its chemical structures.
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Veyrier-Cammas, Anne. "Rôle et mode d'action du régulateur traductionnel hnRNP A1 dans les cellules tumorales mammaires". Toulouse 3, 2008. http://thesesups.ups-tlse.fr/335/.
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Les protéines de liaison aux ARNms ou mRBPs participent à la régulation, à la coordination et au couplage des étapes post-transcriptionnelles de l'expression des gènes. Des modifications de la régulation de leur expression et de leur activité dans les cancers pourraient contribuer au développement tumoral. Nos travaux se sont accès sur l'étude d'une mRBP régulatrice de la traduction, hnRNP A1, dans les cellules tumorales mammaires. Nous avons montré que l'activité traductionnelle de hnRNPA1 pouvait être régulée par sa relocalisation cytoplasmique dans certaines conditions de stress. Nous avons observé qu'une localisation cytoplasmique de hnRNPA1 était associée à des rechutes métastatiques et à un mauvais pronostic dans des tumeurs du sein, et précisé l'effet de cette relocalisation cytoplasmique sur la tumorigenèse. Cette étude suggère que la régulation de la traduction par relocalisation subcellulaire d'une mRBP puisse être déterminante dans les cancersMRNA binding proteins or mRBPs are involved in the regulation, the coordination and the coupling of post-transcriptional gene expression. Modifications in the regulation of their expression and/or activity in cancer contribute to the tumoral development. Our work focused on the study of the translational regulator, hnRNP A1,. We have shown that the translational activity of hnRNP A1 is regulated by its cytoplasmic relocalization upon different stress conditions. We have also observed that a cytoplasmic localization of hnRNP A1 is associated with metastatic relapse and bad prognosis in breast tumors, and we have initiated a study of the effects of this cytoplasmic relocalization on tumorigenesis. This work suggests that regulation of translation by subcellular relocalization of an mRBP may be determinant in cancer
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Thomas, Anoushka. "The regulation of p53 transcriptional activity by hnRNPUL-1 and the DNA damage response induced by a novel chemotherapeutic agent, ALX". Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/3945/.
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The tumour suppressor, p53, is a vital DNA damage response protein and its means of transcriptional regulation are vast. hnRNPUL-1 is a multifunctional protein and previous studies have implicated it in the modulation of p53 transcriptional activity, although this has been rather poorly understood. Results presented here demonstrate that hnRNPUL-1 represses p53 transcriptional activity and negatively regulates p21 levels. This is consistent with the depletion of hnRNPUL-1 leading to an increase in cells arrested in G1/S. Together these results confirm that hnRNPUL-1 acts as a p53 co-repressor with specific cellular targets. Mutations in p53 and other DNA damage response proteins not only often contribute to the onset of tumourigenesis but can also be the cause of drug resistance during treatment. The development of a novel chemotherapeutic agent, Alchemix (ALX), was based on the requirement for effective treatment in the face of resistance mechanisms. Little has been known about the mechanism of action of ALX up to now. Findings here demonstrate that ALX treatment primarily induces an ATR-dependent DNA damage response that occurs specifically in cycling cells and culminates in cell death via mitotic catastrophe. Results also show that the response elicited by ALX requires TOP2α and TOP2β, as well as its alkylating ability, but does not involve ATM, p53, or components of the MMR and FA pathway. Therefore, ALX has the potential to treat tumours that have developed resistance to conventional chemotherapeutic drugs.
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Fiset, Stéphan. "Interaction de hnRNP A1/UP1 avec les séquences télomériques humaines et l'ARN de la télomérase". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/MQ56900.pdf.
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Lévesque, Kathy. "hnRNP A2 is a protein involved in the trafficking pathway of HIV-1 genomic RNA". Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98748.
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HIV-1 genomic RNA trafficking is an important event which remain unclear. Different cellular proteins are involved in this process and the heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) is suggested to play a role by its potential implication in ribonucleoprotein complex which is related with the microtubule network. We identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) is involved in this process. To investigate the potential function of hnRNP A2 in the trafficking, protein expression was decreased using small interfering RNA and the impact on RNA localization, protein expression patterns and levels of genomic RNA in new virions was looked at. The results obtained suggested that hnRNP A2 knockdown impedes transport of the genomic RNA and causing its accumulation at the MTOC (microtubule organizing center). These data show the importance of the hnRNP A2 protein in genomic RNA trafficking and its role in HIV-1 RNA localization.
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Berglund, Frederick M. "Identification of hnRNP-U as a DNA-PK substrate phosphorylated in response to DNA damage". Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505651.
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Fiset, Stephan. "Interaction de hnRNP A1/UP1 avec les séquences télomériques humaines et l'ARN de la télomérase". Mémoire, Université de Sherbrooke, 2000. http://savoirs.usherbrooke.ca/handle/11143/3182.
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Les protéines hnRNP A1/UP1 sont en mesure d'interagir directement avec des séquences télomériques humaines. L'expression de l'une ou l'autre de ces protéines dans les cellules de mammifères provoque un allongement des télomères chez celles-ci. De plus, la forme recombinante de la protéine UP1 est en mesure de recruter l'activité télomérasique à partir d'un lysat cellulaire in vitro. Afin d'établir les bases moléculaires qui régissent les interactions entre les séquences télomériques, ces protéines et l'ARN de la télomérase, nous avons étudié les interactions de chacun des domaines de liaison à l'ARN pour leur habileté à interagir avec les acides nucléiques. Nous avons pu établir que les deux domaines sont impliqués dans la liaison à des acides nucléiques distincts. Le premier RRM est nécessaire pour interagir de façon spécifique avec les répétitions d'ADN télomériques alors que le second RRM est nécessaire pour permettre une liaison spécifique à l'ARN de la télomérase. Nos études de liaison en gel de retardement ont de plus permis de montrer que les protéines A1 et UP1 sont en mesure d'interagir avec l'ARN de la télomérase. Nous avons également pu établir, par des essais de protection, que la présence de la protéine UP1 entière est essentielle pour permettre une protection efficace contre l'action de la DNase I. Nos études de chromatographie suggèrent que les protéines A1 et UP1 peuvent interagir simultanément avec les répétitions d'ADN télomériques et l'ARN de la télomérase in vitro. Nous avons également montré que cette interaction pouvait résister à une incubation dans un extrait nucléaire complexe. Ces résultats suggèrent que l'interaction ARN de la télomérase-A1-ADN télomérique est possible dans un contexte cellulaire normal. Nos résultats supportent un modèle dans lequel la protéine A1 recruterait la télomérase aux extrémités des chromosomes de vertébrés.
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Brown, Andrew S. "Identification of a phospho-hnRNP E1 Nucleic Acid Consensus Sequence Mediating Epithelial to Mesenchymal Transition". Kent State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=kent1437943957.
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Hu, Wenjun. "Identification of DNA cleavage- and recombination-specific hnRNP co-factors for activation-induced cytidine deaminase". Kyoto University, 2015. http://hdl.handle.net/2433/200494.
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Cendron, F. "Are the heterogeneous nuclear ribonucleoproteins SQUID and Hrb87F involved in the regulation of circadian rhythmicity in Drosophila melanogaster?" Doctoral thesis, Università degli studi di Padova, 2020. http://hdl.handle.net/11577/3423282.
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The search for new genes involved in circadian rhythmicity in Drosophila melanogaster led to the identification of the RNA binding proteins Hrb87F and SQUID, as partners of Cryptochrome, the photoreceptor responsible for light-synchronization of the circadian clock. In the clock machinery, different post-transcriptional mechanisms have evolved to adjust and consolidate the oscillation of clock genes and proteins in interlocked negative feedback loops. The aim of my PhD project was to study the possible involvement of Hrb87F and SQUID in the post-transcriptional control of the circadian clock in Drosophila.
These preliminary data were confirmed by Co-Immunoprecipitation and western-blot experiments using transgenic flies expressing a tagged version of Cry (HACRY) under the control of the driver timGAL4, for SQUID, while for Hrb87F the interaction was validated by yeast two-hybrid assay, in which dCRY was challenged to Hrb87F as prey.
To achieve more information about a possible role of the clock in the expression of Squid and Hrb87F in Drosophila head, the analysis of the expression profile of the two hnRNPs (mRNAs and proteins) was performed during the 24 hours in wild type (wt) and clock mutant per0 flies. As for Hrb87F, mRNA levels showed an oscillatory trend in LD (Light-Dark) and DD (Dark-Dark) either in wt and per0 flies; the protein levels oscillate in LD and DD in wt flies, but not in per0, suggesting a potential role for the circadian clock in the translational/posttranslational control of the protein. As for Squid, neither the mRNA nor the protein showed rhythmic expression in LD and DD.
Subsequently, the involvement of SQUID and Hrb87F in the generation of the circadian rhythmicity was studied analyzing the locomotor activity pattern of flies’ mutant for each of the two genes. Mutants for both genes showed an impairment of the daily rhythmicity, with a loss of the morning anticipation in LD and low levels of rhythmicity in DD at 29°C, 23°C, 18°C and 15°C, suggesting that Squid and Hrb87F could play a role either in the generation and in the light-synchronization of the circadian behaviour. However, the analysis of locomotor activity in constant light, performed in order to further dissect the possible role of Squid in the light synchronization of the clock, revealed that the light-synchronization of the clock is not impaired in these flies.
The expression of period and timeless and the alternative splicing variants in relation to the temperature were also analyzed in wt and Squid mutant brains by using multiplex real time PCR. The results suggest that the expression of period and timeless mRNA is altered in Squid mutant compared to wt both in LD and DD at each temperature. Moreover, the quantities of per unspliced at high temperature and tim unspliced at low temperature are lower both in LD and DD in the
mutant. This suggests an increase of the splicing events and thus an involvement of SQUID in the post-transcriptional control of clock genes.
In collaboration with Dr Milena Damulewicz (Jagiellonian University of Krakow - Poland), the expression of PERIOD was analyzed in the clock neurons by immunocytochemistry in flies reared at both 18°C and 23°C in LD and DD. At 23°C and 18°C the oscillation of PER in l-LNvs clock neurons is lost, while, in s-LNvs, the accumulation of protein is delayed by 3 hours with a broader peak that reflects a low kinetic of degradation. The analysis of PDF projections from clock neurons shows a profound disorganization of PDF release, suggesting that it can, at least partially, account for the locomotor activity defect observed in the Squid mutant.
Taken together, these results highlight the possible involvement of the hnRNPs Hrb87F and SQUID in the generation and maintenance of circadian rhythmicity in Drosophila melanogaster.La ricerca di nuovi geni coinvolti nel controllo della ritmicità circadiana in Drosophila melanogaster ha permesso di identificare una possibile interazione delle due ribonucleoproteine SQUID e HRB87F con CRYPTOCROME. Nella regolazione molecolare dell’orologio circadiano, si sono evoluti diversi eventi di regolazione post trascrizionale che aggiustano e consolidano l’espressione ritmica dei geni orologio e delle corrispettive proteine, secondo un meccanismo a feedback negativo. Per tale motivo lo scopo di questo lavoro è stato studiare e analizzare il possibile coinvolgimento delle hnRNPs nel controllo post-trascrizionale dell’orologio circadiano in Drosophila. In una fase iniziale, per confermare i risultati ottenuti in precedenza, sono stati condotti esperimenti di Co-Immunoprecipitazione e western-blot in mosche transgeniche in grado di esprimere una versione di CRY associata all’epitopo HA (HACRY) sotto il controllo del driver timGal4. L’anticorpo anti-SQUID ha identificato una proteina nel campione co-immunoprecipitato, confermando l’interazione, mentre il legame con HRB87F è stato osservato mediante l’utilizzo del sistema del doppio ibrido di lievito. In seguito è stato valutato il possibile coinvolgimento dell’orologio circadiano nell’espressione delle hnRNPs nell’intera testa del moscerino. I livelli di espressione del mRNA di Hrb87F mostrano un trend oscillatorio in LD (luce-buio) e DD (buio costante), sia nel wt che nel mutante per0, mentre la proteina oscilla in LD e DD nelle mosche wt, ma non nelle per0, suggerendo un possibile ruolo dell’orologio circadiano nel controllo traduzionale o post-traduzionale della proteina. L’analisi dell’espressione del gene Squid ha dimostrato, invece, che né mRNA né proteina vengono espressi in maniera ritmica, né in LD né DD. Successivamente, è stato studiato l’intervento delle due ribonucleoproteine nella generazione della ritmicità circadiana, analizzando l’attività locomotoria di mosche mutanti per entrambi i geni. Tutti i mutanti hanno riportato un’alterazione della ritmicità circadiana, con una perdita dell’anticipazione dell’attività mattutina in LD e bassi livelli di ritmicità in DD a 29°C, 23°C, 18°C e 15°C. I risultati ottenuti suggeriscono che Squid e Hrb87F possano partecipare alla generazione di un fenotipo ritmico nell’attività locomotoria. Questa analisi è stata completata studiando, inoltre, l’attività locomotoria del mutante squid in condizione di luce costante (LL), poiché esibiva un fenotipo associabile a quello in cui è presente un’alterazione del meccanismo di sincronizzazione della luce; si è visto che, le mosche mutanti presentano un risposta in LL paragonabile al wt, sottolineando un corretto funzionamento del meccanismo di sincronizzazione della luce. Abbiamo analizzato i profili di espressione dei geni period e timeless e delle rispettive varianti dovute a splicing alternativo legato alla temperatura. I risultati ottenuti mostrano che l’espressione è alterata nel mutante Squid, rispetto al controllo wt, sia in LD sia in DD ad ogni temperatura considerata, evidenziando che è presente un elevata attività di splicing. In fine, in collaborazione con la Dott. Milena Damulewicz (Università di Jagiellonian di Kracovia - Polonia), è stata condotta un’analisi dell’espressione di PERIOD nei neuroni orologio in mosche mutanti Squid e wt, mediante immunocitochimica a 18°C e 23°C in LD e DD. Si è visto che sia a 23°C sia a 18°C, l’oscillazione di PER nei neuroni orologio l-LNv, viene persa sia in LD sia in DD, mentre nei neuroni s-LNv sia a 23°C sia a 18°C si assiste a un ritardo nell’accumulo della proteina seguito da una cinetica di degradazione più lenta rispetto ai controlli. L’analisi delle proiezioni del neuropeptide PDF ha evidenziato una profonda disorganizzate nel mutante squid. Nel complesso, tutti questi risultati suggeriscono un coinvolgimento di HRB87F e SQUID nella generazione e nel mantenimento della ritmicità circadiana in Drosophila melanogaster.
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Okamoto, Yusuke. "FANCD2 protects genome stability by recruiting RNA processing enzymes to resolve R‐loops during mild replication stress". Kyoto University, 2019. http://hdl.handle.net/2433/242379.
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Garneau, Daniel. "Modulation de l'épissage alternatif de Bcl-x par les protéines hnRNP F et H in vitro". Mémoire, Université de Sherbrooke, 2004. http://savoirs.usherbrooke.ca/handle/11143/3390.
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L'épissage alternatif est un mécanisme permettant d'augmenter la diversité protéique dans une cellule. Dans certains cas, les isoformes ainsi créés peuvent avoir des activités complètement différentes. Une telle situation implique le gène apoptotique Bcl-x qui produit des isoformes ayant des activités antagonistes dans l'apoptose. Ces isoformes sont formés par l'utilisation de sites d'épissage 5' alternatifs. L'isoforme Bcl-xL possède une activité anti-apoptotique alors que Bcl-xS possède une activité pro-apoptotique. Mes recherches ont permis de trouver deux régions exoniques responsables de la régulation de l'épissage alternatif de ce gène. Une première région B2, située en aval du site d'épissage de Bcl-xS , active la formation de l'isoforme Bcl-xS alors qu'une deuxième région B3, située en amont du site d'épissage de Bcl-xL, active la formation de l'isoforme Bcl-xL. Plus précisément, une sous-région B2G est principalement responsable de l'activité de la région B2 et possède une séquence constituée de trois séries de G pouvant recruter les protéines hnRNP F et hnRNP H. De plus, les deux dernières séries de G sont importantes pour l'activité de l'élément B2G ainsi que pour la liaison des protéines à cette séquence. Nous avons aussi montré que l'ajout de hnRNP F ou H dans une réaction d'épissage in vitro permet de moduler l'épissage de Bcl-x en favorisant la formation de l'isoforme Bcl-xS. Cette modulation n'est cependant pas obtenue lorsque que l'élément B2G est muté. Mon étude montre aussi que, in vitro, l'ajout des protéines hnRNP Al et SRp30c stimule la production de l'isoforme Bcl-xL. Cet effet se ferait via un autre élément que B2G puisque ni hnRNP A1, ni SRp30c ne lient cette séquence.