The N-7 methylguanosine cap structure is conserved from yeast to man. It is essential for cell proliferation as it influences several steps in eukaryotic gene expression including transcription, pre-mRNA processing, RNA export and translation. The N-7 methylguanosine cap is added co-transcriptionally to RNA pol II transcripts. In mammals, two enzymes catalyse the synthesis of the N7-methylguanosince cap. RNGTT adds an inverted guanosine group to the first transcribed nucleotide and RNMT methylates the guanosine cap at the N7-position. RNMT consists of a catalytic domain and an N-terminal domain that is absent in lower eukaryotes. Experiments presented in this thesis revealed that the N-terminus mediates RNMT recruitment to transcription start sites. Furthermore, it was found that the RNMT N-terminal domain is phosphorylated at Threonine-77 (T77) by CDK1/Cyclin B in a cell cycle-dependent manner during G2/M-phase. RNMT T77 phosphorylation activates cap methyltransferase activity in vitro. Furthermore, it negatively regulates the interaction of RNMT with KPNA2 (Importin-a), which was found to inhibit RNMT activity in vitro. RNMT T77 phosphorylation is required for normal cell proliferation suggesting an important biological function. Initial experiments indicated that RNMT T77 phosphorylation functions to regulate gene expression in a gene-specific manner. Future work is focused on establishing an experimental system to perform a genome-wide study in order to elucidate which transcripts are affected by RNMT T77 phosphorylation. To summarise, this study for the first time revealed that the RNA cap methyltransferase activity is regulated in a cell-cycle dependent manner.

While actin was discovered in the nucleus over 50 years ago, research lagged for decades due to strong skepticism. The revitalization of research into nuclear actin occurred after it was found that cellular stresses both induce the nuclear localization and alter the structure of nuclear actin. These studies provided the first hints that actin has a nuclear function. Subsequently, it was established that the nuclear import and export of actin is highly regulated. While the structures of nuclear actin remain unclear, it can function as monomers, polymers, and even rods. Furthermore, even within a given structure, distinct pools of nuclear actin that can be differentially labeled have been identified. Numerous mechanistic studies have uncovered an array of functions for nuclear actin. It regulates the activity of RNA polymerases, as well as specific transcription factors. Actin also modulates the activity of several chromatin remodeling complexes and histone deacetylases, to ultimately impinge on transcriptional programing and DNA damage repair. Further, nuclear actin mediates chromatin movement and organization. It has roles in meiosis and mitosis, and these functions may be functionally conserved from ancient bacterial actin homologs. The structure and integrity of the nuclear envelope and sub-nuclear compartments are also regulated by nuclear actin. Furthermore, nuclear actin contributes to human diseases like cancer, neurodegeneration, and myopathies. The work presented in this thesis aims to describe the nuclear localization and functions of actin during Drosophila oogenesis. Drosophila oogenesis, i.e. follicle development, provides a developmental system with which to study nuclear actin. Follicles are composed of roughly 1000 somatic follicle cells and 16 germline cells, including 15 nurse or support cells and a single oocyte. Follicles progress through a series of 14 morphological stages, from the germanium to Stage 14 (S14). Ovary staining using the anti-actin C4 antibody reveals one pool of nuclear actin during early oogenesis (germarium through S9), including in the germline and somatic stem cells, a subset of mitotic follicles cells, and a subset of nurse cells during S5-S9. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged actin results in nuclear actin rod formation. These findings indicate that nuclear actin is tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of C4 nuclear actin, but does not alter the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate C4 nuclear actin. Evidence indicates that Fascin positively regulates C4 nuclear actin through Cofilin. Indeed, loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high C4 nuclear actin levels. Thus, through Cofilin, Fascin positively regulates C4 nuclear actin. These studies identified Fascin as a novel means of nuclear actin regulation. Having established Drosophila oogenesis as an in vivo, developmental system to study nuclear actin, I sought to identify the functions of nuclear actin. To uncover the functions of nuclear actin, I manipulate nuclear actin levels by blocking its nuclear import (Importin 9) and export (Exportin 6). Knockdown of Importin 9, results in female sterility and defects within the germarium, supporting a role for nuclear actin in stemness. Additionally, reduced Importin 9 levels cause chromatin organization defects. Loss or knockdown of Exportin 6 causes reduced female fertility, abnormal nucleolar morphology, alterations in the nuclear envelope, and aberrant heterochromatin status. These data suggest several functions for nuclear actin in the ovary: nuclear actin is essential for stem cell differentiation, proper chromatin organization and dispersal, nucleolar structure and likely function, nuclear envelope morphology, heterochromatin status and likely gene expression. Ultimately, nuclear actin is absolutely required for the highly conserved process of follicle development. These studies provide insight into the regulation and function of nuclear actin in Drosophila oogenesis. The data presented here indicate that nuclear actin is critical for chromatin organization, nucleolar morphology, nuclear envelope shape, and heterochromatin status and suggest that nuclear actin ultimately impacts transcription, a process essential for all cells. Considering the high level of sequence and functional conservation of actin, studies in Drosophila oogenesis will provide insight into the conserved functions of nuclear actin in follicle development across higher organisms. The study of nuclear actin in the many cell types of the Drosophila ovary provide insight into the functions of nuclear actin for all cell types across evolution. Further, aberrant nuclear actin regulation has been implicated in several disease states. The studies in Drosophila provide insight into the regulation of nuclear actin and how misregulation contributes to disease states. Together, the data presented in this thesis advance our understanding of the nuclear localization and functions of actin.

Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a green fluorescent protein (GFP):N fusion in plant cells. Deletion analysis and alanine-scanning mutagenesis identified two amino acid motifs, 419QKR421 and 432KR433, that were shown to be essential for nuclear import and interaction with importin-α. Additional bimolecular fluorescence complementation showed that the PYDV-N-NLS mutants cannot be ferried into the nucleus via interaction with PYDV-P or-M. In contrast, interaction with N-NLS mutants appeared to retard the nuclear import of PYDV-P. Taken together, it was determined that PYDV-N contains the bipartite NLS 419QKRANEEAPPAAQKR433. Similarly, alanine-scanning mutagenesis was performed to determine the regions responsible for the nuclear import of PYDV-M and -P. A non-canonical NLS was identified in PYDV-P, consisting of three regions in the N-terminus of the protein required for nuclear import. PYDV-P does not interact with any Nicotiana benthamiana importins, but was found to interact with importin-α7 and -α9 of the non-host plant Arabidopsis thaliana. Two amino acids of PYDV-M, 225KR226, were found to be critical for nuclear import and interaction with importin-α. In addition, site-directed mutagenesis identified that amino acids 223LL224 of PYDV-M, which are adjacent to the two amino acids identified as responsible for nuclear import, are critical for inducing invaginations of the inner nuclear membrane. Bimolecular fluorescence complementation (BiFC) was then used to identify any differences in localization and interaction caused by the mutations introduced to PYDV-P and -M. The PYDV-P and -M proteins were still able to interact with other PYDV proteins, although the localization of the interaction differs between mutants.

Human immunodeficiency virus type 1 (HIV-1) infection causes general loss of immune response in humans. Presently, an estimated 34 million (31.4-35.9 million) people worldwide are HIV-1 positive and many more are being newly infected. In the absence of a definitive cure, anti-HIV-1 drug therapy helps to manage the infection by suppressing virus replication. However, extensive drug resistance against most of existing drugs demands alternative anti-HIV-1 strategies. The proper knowledge about HIV-1 replication is essential to guide the development of new anti-HIV-1 strategies. The research presented in this thesis aims to understand the role of HIV-1 Integrase (IN) and cellular co-factors interactions in the early stage virus replication. In the cytoplasm, HIV-1 cDNA exists as a high molecular weight nucleoprotein complex called pre-integration complex (PIC). The cDNA enters the nucleus as a part of PIC by active nuclear import and integrates into the host genome. HIV-1 Integrase (IN) protein has been recognized as a primary viral factor for HIV-1 nuclear import, but the key contributing cellular factor(s) is unknown. We have examined the requirement of different Importinα (Impα) isoforms for HIV-1 replication and identified the requirement of Impα3 for HIV-1 replication in HeLa cells, C8166T cells, and human macrophages. Further investigations showed the specific requirement of Impα3 for HIV-1 nuclear import. By analyzing the Impα3 interaction with HIV-1 proteins, we detected the IN interaction with Impα3 and C-terminal domain (CTD) of IN was essential for Impα3 interaction. These data led to the conclusion that Impα3 is required for HIV-1 nuclear import and interacts with IN. The IN-CTD consists of conserved basic amino acid rich motifs (211KELQKQITK, 236KGPAKLLWK, and 262RRKAK) that closely resemble the consensus classical nuclear localization signal (NLS) for Impα interaction. By substitution mutation and interaction analysis, 211KELQKQITK and 262RRKAK motifs in IN were identified as required for Impα3 interaction, IN nuclear localization, and HIV-1 nuclear import. Together, these data were useful in explaining the molecular mechanism of IN and Impα3 interaction and its requirement for HIV-1 nuclear import. Retrograde transportation of macromolecules in the cytoplasm is one of the prerequisites for their nuclear import. Although an earlier study implicated the dynein complex in retrograde transport of HIV-1, cellular and viral factors that are involved in this process are unknown. In this study, we have elucidated the HIV-1 IN interaction with the dynein light chain 1 (DYNLL1) in 293T cells, in vitro, and in HIV-1 infected cells. DYNLL1 is one of the adapter proteins that mediate the cargo recruitment to dynein complex. However, our data suggested that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not for nuclear import. Surprisingly, DYNLL1 interaction of IN was dispensable for HIV-1 recruitment to dynein complex. These data led to the conclusion that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not required for HIV-1 recruitment to the dynein complex or for retrograde transport. In summary, this study advances our knowledge on the role of IN and cellular factors interactions in different early steps of HIV-1 replication and offers potential contributions in the development of future anti-HIV-1 strategies.

La protéine Rev du VIH-1 est une petite protéine qui permet l'export nucléaire des transcrits viraux partiellement ou non épissés. L'import nucléaire de Rev est réalisé par Importin β (Impβ), un facteur de transport cellulaire qui reconnaît le signal de localisation nucléaire (NLS) riche en arginine présent sur Rev. Le but de cette thèse est de déterminer la structure tridimensionnelle du complexe formé par Impβ et Rev. Après des essais de cristallogenèse infructueux, nous avons sondé la structure du complexe Impβ/Rev en utilisant différentes approches biochimiques, biophysiques et computationnelles. Les résultats suggèrent un nombre limité de modèles tridimensionnels du complexe Impβ/Rev. Ces travaux augmentent notre compréhension de la façon dont Impβ reconnaît Rev et devraient dans l'avenir, faciliter la détermination de la structure de ce complexe à haute résolution. / HIV-1 Rev is a small protein which mediates the nuclear export of unspliced and partially spliced viral transcripts. The nuclear import of Rev is mediated by the host cell protein Importin β (Impβ), a transport factor that recognizes the arginine- rich nuclear localization signal (NLS) present on Rev. The goal of this project is to determine the three-dimensional structure of the complex formed by Impβ and Rev. Because attempts to crystallize this complex were unsuccessful, we sought structural information using a range of biochemical, biophysical and computational approaches. The results suggest a limited number of hypothetical configurations for the Impβ/Rev complex, which can be specifically verified with additional experiments. The work described in this thesis increases our understanding of how Impβ recognizes Rev and should facilitate future efforts at determining the high-resolution structure of this complex.

No description available.

No description available.

No / Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107–Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.

Scrub typhus is a potentially fatal infection that threatens one billion persons in the Asia-Pacific region and is caused by the obligate intracellular bacterium, Orientia tsutsugamushi. How this organism facilitates its intracellular survival and pathogenesis is poorly understood. Intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) into the host cell to modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank libraries as well as Type 1 and Type 4 secretion systems (T1SS and T4SS), which are expressed during infection. In silico analyses of the Anks’ C-termini revealed that they possess characteristics of T1SS secretion signals. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks. In addition to infecting endothelial cells, O. tsutsugamushi infects professional phagocytes. To better understand why these innate immune cells are unable to eliminate O. tsutsugamushi, we addressed the activity of host NF-κB proinflammatory transcription factor. Screening of O. tsutsugamushi infected cells at an MOI of 1 revealed inhibition of NF-κB nuclear accumulation as early as 8 hours in HeLa and bone-marrow derived macrophage cells. When stimulating infected cells with TNF-α, IκBα degradation still occurs, however NF-κB dependent gene transcription remains downregulated. Immunofluorescence microscopic analysis of TNF-α treated cells ectopically expressing all O. tsutsugamushi Anks revealed that two nuclear trafficking Anks, Ank1 and Ank6, result in a significant decrease in NF-κB nuclear accumulation. Additionally, these Anks also significantly inhibited NF-κB dependent gene transcription. Co-immunoprecipitation experiments revealed that both Anks interact with importin-β1, exportin-1, and the p65 NF-κB subunit. Treating cells with importazole significantly reduces the nuclear accumulation of Ank1 and Ank6. Finally, treating infected cells or cells ectopically expressing Ank1 or Ank6 with leptomycin B resulted in restoration of NF-κB nuclear accumulation. With these data, we propose that O. tsutsugamushi secretes Ank1 and Ank6 to initially interact with importin-β1, which permits their nuclear entry where they then interact with NF-κB and subsequently exportin-1 to prevent NF-κB nuclear accumulation.

Le virus de la grippe est un virus à ARN négatif appartenant à la famille des Orthomyxoviridae qui se compose de 7 membres dont les virus influenza A, B, C et D. Le génome viral comprend 7 à 8 particules ribonucléoprotéiques (RNP) au sein desquelles l’ARN viral (ARNv) est recouvert de multiples copies de nucléoprotéines (NP) et est associé à l’ARN polymérase virale via ses extrémités 3’ et 5’. Au cours de ce travail de thèse, nous nous sommes tout d’abord focalisés sur l’étude biochimique de NP A et NP B et avons pu mettre en évidence des comportements différents en ce qui concerne leurs propriétés d’oligomérisation en présence ou en absence d’ARN et en fonction de la concentration en sel. Pour la première fois nous avons pu observer une structure similaire aux RNP mais reconstituée uniquement à partir de NP A et d’un ARN de 12 nucléotides. Nous avons pu formuler l’hypothèse que 12 nucléotides de l’ARN serait fixés à la NP avec une forte affinité tandis que le reste de l’ARN fixerait la NP avec une affinité beaucoup plus faible. En parallèle nous avons résolu la structure cristallographique de la nucléoprotéine de la grippe de type D et réaliser la caractérisation de son interaction avec l’importine-α7 humaine. Enfin nous avons étudié la fixation de l’ARN sur NP D et mis en évidence l’importance de l’extrémité C-terminale dans le processus de fixation à l’ARN. Ces informations ont permis de formuler de nouvelles hypothèses quant au fonctionnement du virus de la grippe et permettre d’inscrire ce projet de thèse dans une dynamique globale de lutte contre ce virus. / Influenza virus is a negative RNA virus belongs to the Orthomyxoviridae family which consists of 7 members including influenza viruses A, B, C and D. The viral genome comprises 7 to 8 ribonucleoprotein particles (RNP) in which the viral RNA (vRNA) is coated with multiple copies of nucleoproteins (NP) and is associated with the viral RNA polymerase by its 3 'and 5' ends. In this thesis, we first focused on the biochemical study of NP A and NP B and we demonstrate that there are different behaviors with regard to their oligomerization properties in the presence or absence of RNA and as a function of the salt concentration. For the first time we were able to observe a structure very similar to RNP which was reconstituted only from NP A and a 12 nucleotide RNA. Thus, we formulate the hypothesis that 12 nucleotides of the RNA would bind NP with a very strong affinity while the rest of the RNA would bind NP with a lower affinity. In parallel, we solved the crystallographic structure of the nucleoprotein of influenza D virus and we characterized its interaction with human importin-α7. Finally, we studied the binding of RNA on NP D and we demonstrated the importance of the C-terminal end in the RNA binding process. This thesis project made it possible to formulate new hypotheses concerning the functioning of the influenza virus and to include this thesis project in a global dynamic of combating the influenza virus.