Proper mRNA nuclear export is essential for harmonious growth and maintenance of a cell. An effective weapon influenza virus employs to hijack a host cell is its ability to inhibit such export. Exactly how influenza virus achieves this inhibition is not fully known. Here, we demonstrate that upon infection, influenza virus degrades two nucleopore proteins (Nup98 and Nup96), which play a key role in mRNA nuclear export. Also, a main virulence factor of influenza virus (non-structural protein 1, NS1) binds directly to NXF1 and E1B-AP5, two key constituents of the mRNA export pathway (NXF1/NXT pathway) responsible for exporting bulk (~70%) mRNA from the nucleus. By increasing the expression levels of members of the NXF1/NXT pathway, we were able to reverse NS1-mediated inhibition of gene expression. On the other hand, by decreasing the levels of members of the NXF1/NXT pathway, we demonstrated that host cells become more sensitive to influenza virus infection and produce more viral particles. These results demonstrate undiscovered influenza-mediated host interactions that may be used to medicinally inhibit influenza virus. To this end, high-throughput screens were designed to identify small molecule antagonists of both NS1-mediated inhibition of gene expression and influenza virus-mediated cell death. Seventy-one compounds were identified, and the most potent molecule (named compound #8) was examined further. We found that compound #8 releases influenza virus-mediated mRNA nuclear export blockage and decreased viral replication and viral gene expression. Thus, the bulk mRNA nuclear export machinery is vital to antiviral response, and compound #8 enhances its ability to fight the cytopathic effects of NS1 and influenza virus. In conclusion, our data demonstrate that the mRNA export machinery is disrupted by influenza virus, and that this machinery also facilitates an antiviral function. We have also shown that these two events can be manipulated chemically to attenuate the negative effect of the virus and enhance the positive antiviral effect of the mRNA export machinery, thereby providing a powerful, new strategy against the ever-present, global threat of influenza virus.

Yes / Tumour suppressor proteins, such as p53, BRCA1, and ABC, play key roles in preventing the development of a malignant phenotype, but those that function as transcriptional regulators need to enter the nucleus in order to function. The export of proteins between the nucleus and cytoplasm is complex. It occurs through nuclear pores and exported proteins need a nuclear export signal (NES) to bind to nuclear exportin proteins, including CRM1 (Chromosomal Region Maintenance protein 1), and the energy for this process is provided by the RanGTP/RanGDP gradient. Due to the loss of DNA repair and cell cycle checkpoints, drug resistance is a major problem in cancer treatment, and often an initially successful treatment will fail due to the development of resistance. An important mechanism underlying resistance is nuclear export, and a number of strategies that can prevent nuclear export may reverse resistance. Examples include inhibitors of CRM1, antibodies to the nuclear export signal, and alteration of nuclear pore structure. Each of these are considered in this review.

No description available.

Tubulin is the basic building block of microtubules which ensure intracellular transport, morphological changes, the connection of proteins of metabolic pathways, the segregation of chromosomes during mitosis and many other essential processes in plant cells. The localization of tubulin regarding nucleus is strictly controlled and during the interphase, tubulin is usually not present in the nucleus. As we previously showed, one of the exceptions of the presence of tubulin in the nucleus is the cold stress condition. Also, we discovered several plant-specific nuclear export sequences (NES) in tubulin molecules: one in α-tubulin and two in β-tubulin. In this work I found that double mutation of both functional β-tubulin NESes cause slight accumulation of the protein in the nuclei. However, the phenotype of the transgenic plants expressing β-tubulin with mutations is probably not affected by the presence of the mutated protein. The effect of the expression of the mutated β-tubulins was observed in Nicotiana tabacum BY-2 cells as well. The expression of β-tubulin carrying a single NES3 mutation or double mutation of both NESes bring changes of the division activity of the cells, while the NES2 mutation does not have any effect. Furthermore, it was discovered that the 0řC cold tr eatment does not cause...

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and major regulator of the DNA damage response (DDR). One critical ATM target is protein phosphatase 2A (PP2A) known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylation of many proteins during the recovery from the DDR while returning the cell to normalcy. Interestingly, ATM and PP2A are known to form an auto-regulatory yin-yang kinase-phosphatase relationship. Herein, we show that the phosphorylation of the PP2A-Aα structural subunit at S401 by ATM results in nuclear export, which regulates the DDR at multiple levels and affects genomic stability and cell growth. We generated PP2A-Aα conditional knockout mouse embryonic fibroblasts expressing PP2A-Aα-WT, S401A (cannot be phosphorylated), or S401D phosphomimetic) transgenes by floxing out the endogenous PP2A-Aα alleles with Cre. The S401D mutant cells displayed increased ERK and AKT signaling, resulting in an enhanced growth rate. Phosphorylation of PP2A-Aα at S401 caused the dissociation of ATM with the holoenzyme, an effect that could be recapitulated with S401D. Additionally, the S401A and S401D mutants exhibited significantly more chromosomal aberrations and underwent increased mitotic catastrophe after radiation. Both the S401A and the S401D cells showed impaired DSB repair (Non-homologous end joining and Homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Time-lapse video and cellular localization experiments showed that the PP2A-Aα subunit was exported to the cytoplasm after radiation possibly by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects seen. In conclusion, our study demonstrates using a genetically defined system that ATM phosphorylation of a single, critical amino acid S401 is essential for regulating DDR. To study how the interplay between ATM and PP2A affects DDR in the brain, we are in the process of generating a brain specific PP2A-Aα conditional knockout mouse. Loss of many DDR related proteins like ATM and PP2A can lead to severe neuropathological effects. This model will be helpful in dissecting the PP2A-Aα/ATM regulatory circuit in the brain in response to DDR.

Thesis advisor: Junona Moroianu / In this study we investigated the hydrophobic interactions between HPV11 E7 and the FG regions of Nup62N through transfection assays with EGFP-11E7 fusion plasmids in HeLa cells and binding assays with GST-Nup62N immobilized on Glutathione-Sepharose beads. We found that EGFP-11cE7 binds to Nup62N. This suggests a possible mechanism for the nuclear import of HPV11 E7 through direct hydrophobic interactions between its carboxy-terminus and the FG region of Nup62. The interaction between HPV11 E7 and CRM1 nuclear export receptor was also examined using similar methods. Binding between these proteins suggest that nuclear export of 11E7 is mediated by CRM1 binding to its leucine-rich nuclear export signal (NES). / Thesis (BS) — Boston College, 2013. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

Thesis advisor: Junona Moroianu / Some non melanoma skin cancers (NMSC) have been associated with human papillomavirus (HPV) pathogenesis, like epidermodysplasia verruciformis (EV) and squamous cell carcinoma (SCC). EV is a genetically inherited skin disease that develops when the individuals are infected with cutaneous HPV types belonging to the β-genus, especially types 5 and 8. Transgenic mouse lineages expressing all early genes of cutaneous HPV8 develop papillomas, dysplasias and SCC after UV irradiation and this correlates with enhanced HPV8 oncogenes expression. We have previously discovered that the nuclear localization of mucosal HPV16 E7 and HPV11 E7 proteins is mediated by their zinc-binding domain via a Ran-dependent pathway and independent of nuclear import receptors and that a patch of hydrophobic residues within the zinc-binding domain of HPV16 E7 and HPV11 E7 proteins is responsible for their nuclear import via hydrophobic interactions with FG nucleoporins. Here we investigated the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein using confocal microscopy to analyze the intracellular localization of EGFP-8E7, its subdomains and its mutants after transient transfections. We also investigated the nuclear import ability of GST-8E7, its subdomains and mutants using in vitro nuclear import assays in digitonin-permeabilized HeLa cells. In addition, we performed isolation assays to study the direct interaction between HPV8 E7 and two FG nucleoporins, Nup62 and Nup153 or the nuclear export receptor, CRM1. We found that the nuclear import of cutaneous HPV8 E7 is mediated by a nuclear localization signal (NLS) located within its zinc-binding domain. Furthermore, we determined that the hydrophobic residues within the 65LRLFV69 patch are responsible for the nuclear import and nuclear localization of HPV8 E7 via direct hydrophobic interactions with FG nucleoporins, Nup62 and Nup153, whereas the positively charged arginine 66 plays no significant role in the function of the NLS. In addition, we examined the nuclear export mechanism of cutaneous HPV8 E7 protein and showed that it has a leucine-rich nuclear export signal (NES) in its C-terminal domain that is recognized by the CRM1 nuclear export receptor. These studies are essential for understanding the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Junona Moroianu / Human papillomaviruses (HPVs) have been estimated to be the most common sexually transmitted infection in the United States. In addition to condyloma accuminata, infection of the squamous basal epithelium by high-risk HPVs, notably type 16 (HPV16), has been shown to be the primary etiological agent in the majority of cervical carcinomas. The E7 major transforming protein of HPV16, along with E6, has been linked to tumorigenesis and malignancy. While the E7 protein itself possesses no enzymatic activity, its ability to bind a number of nuclear and cytoplasmic targets subverts a variety of cellular regulatory complexes and facilitates viral replication. Previous studies in the Moroianu Lab have shown the HPV16 E7 oncoprotein to translocate across the nuclear pore complex (NPC) in a facilitated manner dependent on a non-canonical, c-terminal, nuclear localization signal (cNLS) for import, and a consensus leucine-rich nuclear export sequence (NES) for export (28). While the leucine-rich NES has been characterized, a full examination of the cNLS has yet to be performed. Here we present evidence that the karyopherin independent nuclear import mediated by the cNLS of 16E7 is dependent on its c-terminal Zn binding domain. Furthermore, we demonstrate that nuclear import is mediated by the direct interaction of a small patch of hydrophobic residues, 65LRLCV69, with the FG domain of the central FG-nucleoporin Nup62. In addition, we examined a potential regulatory mechanism of 16E7 nucleocytoplasmic translocation. Previous work has shown that a serine conserved in the high-risk HPVs at position 71 is phosphorylated by an unknown kinase. Here we present evidence that while phosphorylation of S71 is not required for either 16E7 nuclear localization or nuclear export in HeLa cells, mimicking phosphorylation of the S71 residue results in a statistically significant shift in the distribution of localization phenotypes of the resultant cell population toward a larger percentage exhibiting more nuclear localization. These data suggest that nucleocytoplasmic transport of 16E7 is, at least in part, a regulated process. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Junona Moroianu / The E7 protein of low risk HPV11 has been shown to interact with multiple proteins, including pRb, in both the cytoplasm and the nucleus. High risk HPV16 E7 and low risk HPV11 E7 share a novel nuclear import pathway independent of karyopherins but dependent on the GTPase Ran (Angeline, et al., 2003; Knapp, et al., 2009; Piccioli, et al., 2010). We continued to analyze the nucleocytoplasmic transport of HPV11 E7 in vivo through transfection assays in HeLa cells with EGFP-HPV11 E7 wild type and mutant fusion constructs. We found that nuclear localization of HPV11 E7 is mediated by a nuclear localization signal located in the C-terminal domain which contains a unique zinc-binding domain. Mutations of cysteine residues that interfered with zinc-binding clearly disrupted the nuclear localization of the EGFP-11cE7 and EGFP-11E7 mutants. These data suggest that the integrity of the zinc-binding domain is essential for the nuclear localization of HPV11 E7. In addition, we discovered that HPV11 E7 has a leucine-rich C-terminal nuclear export signal (NES) (76IRQLQDLLL84) mediating the nuclear export of HPV11 E7 in a CRM1-dependent manner. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology Honors Program. / Discipline: Biology.

Le processus d'excision-épissage des RNAs pré-messagers (pré-mRNAs) est une étape essentielle dans l'expression de la majorité des gènes eucaryotiques. Lépissage se déroule dans le noyau au sein dun complexe macromoléculaire appelé spliceosome, ou particule dépissage, qui sassemble sur des sites précis le long des pré-mRNAs. Il consiste en cinq petites particules nucléaires ribonucléoprotéiques dénommées snRNPs (small nuclear Ribonucleoproteins) constituées de snRNAs (small nuclear RNAs) riches en uridines (U1, U2, U4/U6 et U5) et denviron 150 protéines associées (Patel et Steitz, 2003; Jurica et al., 2004). Lépissage requiert également de nombreuses protéines non constitutives des snRNPs appelées de manière générique facteurs essentiels dépissage. Parmi ceux-ci, les protéines SR constituent une famille de facteurs dépissage conservés chez les Eucaryotes (Barta et al., 2008; Long et Caceres, 2009). Ces protéines possèdent toutes un ou deux domaines de liaison au RNA appelé RRM (RNA Recognition Motif) en N-terminal et un domaine riche en dipeptides sérine et arginine (SR ou RS) en C-terminal. Elles jouent un rôle crucial dans lépissage constitutif et alternatif et ce, par un jeu complexe d'interactions protéine-protéine et protéine-RNA (Bourgeois et al., 2004; Reddy, 2007). Le mécanisme dépissage alternatif permet de produire différents mRNAs à partir dun seul pré-mRNA et de ce fait peut amener à la synthèse de plusieurs isoformes protéiques. Chez Arabidopsis de ~20 à 40% des pré-mRNAs sont épissés alternativement (Campbell et al., 2006; Wang et Brendel, 2006; Severing et al., 2009; Filichkin et al., 2010). Le séquençage complet du génome dArabidopsis a révélé que ~80% des régions codantes des gènes nucléaires sont interrompues par des introns (Iida et al., 2004) et a également permis lidentification de 19 protéines SR (Kalyna et Barta, 2004). Ces protéines sont classées en 7 sous-familles, certaines ayant leur homologue chez lhomme, dautres étant spécifiques aux végétaux (Kalyna et Barta, 2004). Le nombre de protéines SR est plus élevé chez Arabidopsis comparé à lhomme, chez qui on en dénombre seulement 11. Cela soulignerait des différences entre ces deux règnes au niveau des mécanismes dépissage et des facteurs impliqués dans ce processus. Les travaux antérieurs réalisés par immunofluorescence (Docquier et al., 2004) et par fusion traductionnelle avec la GFP (Green Fluorescent Protein) ont permis de montrer que les protéines SR dArabidopsis se localisent dans le noyau et présentent une organisation nucléaire en speckles (ou Splicing Factors Compartments, SFCs) et ce dans différents types cellulaires (Ali et al., 2003; Docquier et al., 2004; Fang et al., 2004; Tillemans et al., 2005). Les speckles sont considérés comme des sites de stockage et/ou dassemblage des complexes dépissage (Lamond et Spector, 2003). La phosphorylation des protéines SR joue un rôle important dans la régulation de leur localisation et de leurs fonctions. Une hyper- ou hypophosphorylation réduit leur activité générale suggérant que leur niveau de phosphorylation est strictement régulé in vivo (Misteli et Spector, 1996; Lai et al., 2003; Lin et al., 2005). Ainsi, la relocalisation des protéines SR au sein des speckles est activement dépendante de leur état de phosphorylation (Misteli, 2000; Docquier et al., 2004; Huang et Steitz, 2005; Tillemans et al., 2005). Le flux des facteurs nucléaires au sein de ces compartiments et leurs interactions transitoires et rapides soulignent une organisation spatiale et temporelle très dynamique (Eils et al., 2000; Phair et Misteli, 2000; Dundr et Misteli, 2001). Le but de cette thèse de doctorat est détablir un profil dexpression précis de RSZp22 au cours du développement de la plante, de caractériser les propriétés nucléocytoplasmiques de RSZp22 et enfin définir les rôles des domaines de liaison au RNA dans sa dynamique. RSZp22 est un homologue de la protéine 9G8 humaine. Cette protéine SR possède un domaine Zn-knuckle à motif CCHC localisé entre un domaine RRM unique et le domaine RS. Le domaine RRM reconnait les séquences activatrices dépissage présentes sur les exons (ESEs Exonic Splicing Enhancers) alors que le domaine RS est impliqué dans les interactions protéine-protéine et protéine-RNA (Shen et al., 2004). Le rôle du Zn-knuckle, qui se retrouve dans deux sous-familles de protéines SR dArabidopsis (RSZ et RS2Z), nest pas encore bien caractérisé et les spécificités dinteractions des domaines RRM et Zn-knuckle nont pas encore été étudiées. Parmi les protéines SR étudiées, RSZp22 semble être la seule à se localiser et se concentrer au sein du nucléole selon les conditions physiologiques dans lesquelles la cellule se trouve (Tillemans et al., 2005). Les travaux réalisés récemment au laboratoire montrent que RSZp22 fusionnée à la GFP et surexprimée transitoirement dans des cellules foliaires est une protéine hautement dynamique. Sa mobilité dépend du niveau de phosphorylation et de la concentration en ATP de la cellule. Le développement puis lutilisation des approches de FRAP (Fluorescence Recovery After Photobleaching) et de FLIP (Fluorescence Loss In Photobleaching) ont permis de suggérer que RSZp22 est une protéine SR dynamique et quelle peut transiter entre le noyau et le cytoplasme. RSZp22 ferait ainsi partie des protéines SR dites navettes comme, entre autres, son homologue humain 9G8 (Tillemans et al., 2006). Basés sur la délétion de domaines entiers de la protéine, nos travaux montrent également que le domaine RS est impliqué dans lorganisation en speckles des protéines SR dArabidopsis au sein du nucléoplasme et quil jouerait le rôle de signal de localisation nucléaire (NLS Nuclear Localisation Signal). Cette étude suggère aussi que le domaine RRM nest pas indispensable pour lorganisation en speckles de RSZp22 et que le domaine Zn-knuckle interviendrait dans lexportation de la protéine (Tillemans et al., 2006). Les études antérieures de la dynamique des protéines SR d'Arabidopsis et en particulier de RSZp22 ont été réalisées après surexpression ectopique -et quelquefois hétérologue- des facteurs d'épissage. Dans cette étude, nous avons exprimé la protéine RSZp22-GFP sous le contrôle du promoteur endogène RSZp22 après transformation stable dArabidopsis. Parallèlement, une analyse de RT-PCR quantitative et lutilisation du gène rapporteur -glucuronidase (GUS), nous ont permis d'établir un profil dexpression précis de RSZp22 et de complémenter -et valider- la localisation tissulaire de la protéine de fusion. Nous avons ensuite étudié en plantes transgéniques, la dynamique de la protéine RSZp22-GFP dans des types cellulaires spécifiques. Par analyses de FLIP cytoplasmique (dénommé FLIP-shuttling), nous avons démontré que RSZp22 est bien une protéine SR navette nucléocytoplasmique et établi une cinétique d'exportation. Nous avons également confirmé que son exportation vers le cytoplasme est partiellement dépendante de la voie du récepteur CRM1/XPO1. Notre travail a ainsi permis de mettre en évidence les avantages complémentaires des techniques de surexpression en transformation transitoire et de lexpression stable et spécifique pour létude de la dynamique des protéines nucléaires. Enfin, nous avons montré par mutagenèse dirigée que les motifs RNP1 et Zn-knuckle ne sont pas nécessaires pour la localisation nucléaire de la protéine RSZp22 ni pour sa concentration en speckles. Ces motifs de liaison au RNA sont cependant impliqués dans lexportation de RSZp22 par la voie CRM1/XPO1. De plus, les expériences de FRET (Fluorescence Resonance Energy Transfert) indiquent que ces motifs interviennent dans les interactions moléculaires impliquant RSZp22. Ainsi, ce travail a permis de caractériser la dynamique nucléocytoplasmique de RSZp22 dans des tissus spécifiques dArabidopsis et de mettre en évidence limportance de son interaction avec le mRNA pour son exportation par la voie CRM1/XPO1.