Le virus de l’hépatite C (VHC) dépend de facteurs cellulaires pour accomplir son cycle viral et persister dans l’hôte. L’une des stratégies de notre laboratoire consiste à étudier de manière approfondie le réseau d’interactions virus-hôte, afin d’identifier de nouvelles cibles thérapeutiques cellulaires et de développer des antiviraux plus efficaces pour vaincre la résistance virale. Durant ma thèse j’ai étudié deux facteurs cellulaires importants pour le VHC. Le premier est la protéine ribosomale RACK1. Nous avons montré que cette protéine est spécifiquement requise pour la traduction IRES-dépendante du VHC, et non pour la traduction coiffe-dépendante. Le deuxième facteur est une protéine de surface des gouttelettes lipidiques appelée TIP47. Nous avons montré que cette protéine est importante à la fois pour l’assemblage et pour l’export des particules virales. L’ensemble de ces travaux montre que de nouvelles cibles thérapeutiques pourraient être envisagées pour lutter contre le VHC. / The hepatitis C virus (HCV) relies on cellular factors to complete its life cycle and persist in its host. One of the strategies employed by our laboratory is the in-depth study of the network of virus-host interactions to identify new therapeutic cellular targets and develop more effective antivirals to overcome viral resistance.During my PhD, I studied two cellular factors involved in the HCV life cycle. The first factor is the ribosomal protein RACK1. We have shown that this protein is specifically required for the HCV IRES-mediated translation but not for the cap-mediated translation. The second factor is the lipid droplets binding protein TIP47. We have shown that this protein is important for both assembly and export of viral particles. This work shows that new therapeutic targets could be considered in the fight against HCV.

La glycyl-ARNt synthétase humaine (GRS) est une enzyme clé dans la traduction des protéines dans le cytosol et la mitochondrie. Chez l’Homme, des mutations de la GRS conduisent à la neuropathie périphérique Charcot-Marie-Tooth (CMT). Bien que l’activité de la GRS soit ubiquitaire, les mutations associées à la CMT n’affectent que les nerfs périphériques, suggérant un rôle supplémentaire de la GRS dans les neurones. Pour comprendre ce rôle, nous avons d’abord élucidé le mécanisme particulièrement complexe qui contrôle l’expression de la GRS mitochondriale et cytosolique à partir du même gène. Nous avons identifié deux ARNm : un codant pour les deux enzymes ; et un autre plus long qui contient une IRES fonctionnelle et un uORF. Cet ARNm complexe, ne génère que la GRS cytosolique et montre que son expression et localisation sont étroitement contrôlées. De plus, nous avons montré une distribution particulière de la GRS dans des neurones, qui est un premier indice sur un rôle non canonique. / Human Glycyl-tRNA synthetase (GRS) is a housekeeping enzyme with a key role in protein synthesis, both in the cytosol and the mitochondria. In human, mutations in GRS cause the Charcot-Marie-Tooth (CMT) peripheral neuropathy. Though GRS activity is required in all cells, the CMT-associated mutations affect only the peripheral nervous system, suggesting an additional non canonical role.To understand how GRS is involved in CMT pathology, we first elucidated the original post-transcriptional regulatory mechanism that controls the expression of both the mitochondrial and the cytosolic GRS from a single gene. We identified two mRNA isoforms: one coding for both enzymes; and a longer one containing a functional IRES and an uORF encoding only the cytosolic GRS, evidence that expression and localization of human GRS are tightly controlled. Furthermore, we found a particular Ca2+ dependant distribution of GRS in neurons, giving us a first clue about a potential non-canonical role in neurons.

<p>Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.</p><p> Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.</p> / Dissertation

The tumour suppressor p53 actually exists as 9 protein isoforms. Among them, D133p53a, b and g result from the use of an alternative promoter and lack the N-terminal transactivation domain. In addition to its multiple functions maintaining cell integrity, p53 is also able to block angiogenesis, a process strongly contributing in tumour development. Here I have examined the role of p5 isoforms in the regulation of angiogenesis and tumor progression. I also focused my work on FGF-2 regulation by p53. In a first part, full length p53 (p53) and/or D133p53 isoforms were selectively knocked-down with siRNAs in human glioblastoma cells U87. Conditioned medium produced by tumour cells knocked- down for D133p53 inhibited endothelial cell - EC - migration and tubulogenesis. Furthermore, in the chicken chorioallantoïd membrane CAM, D133p53 knockdown gave rise to smaller tumours devoid of vessels, whereas, in mice, it strongly inhibited tumour growth. Interestingly, the double knockdown of p53 and D133p53 also slowed town tumour growth in mice. Taqman Low Density Array revealed distinct gene expression profiles of pro and anti-angiogenic factors regulation following D133p53 and/or p53 knockdown. In particular, D133p53 knockdown resulted in specific down-regulation of Angiogenin and hepatocyte growth factor, whereas the main angiogenic factors FGF-2 and VEGF-A were not significantly affected. Secondly we investigated the regulation of FGF-2 by p53 and its isoforms D133p53 in a human osteosarcoma cell line U2OS, at translational, transcriptional and secretion levels. It resulted in a sophisticated mode of regulation mediated by a transient IRES-dependent translation inhibition of FGF-2. Our data reveal D133p53 isoforms as activators of angiogenesis and tumour progression, through a specific modulation of the angiogenic balance. These isoforms exhibit dominant-negative effect towards p53 but also intrinsic activities, while underlining the importance of considering D133p53 expression in cancers, as well as the potential antitumoural interest of drugs targeting this p53 isoform.

Can sustainability work in the real world? John Robinson is determined to prove it with his forcoming "greener than green" living laboratory.

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.

HCV genome is a positive sense single-stranded RNA containing a single open reading frame (ORF) flanked by untranslated regions (UTRs), 5’UTR and 3’UTR.Initiation of HCV RNA translation is mediated by internal ribosome entry site (IRES) present in 5’ UTR and this process is independent of cap-structure and requires only a small subset of canonical initiation factors. Hence, HCV IRES-mediated translation initiation mechanism is quite different from canonical cellular mRNA translation initiation. The IRES is organized into highly structured domains, namely domain II, III and IV. High affinity interactions between structured RNA elements present in the IRES and 40S ribosomal proteins mediate 40S recruitment to HCV IRES. However, details of the RNA elements and region of ribosomal proteins involved in these interactions are poorly understood. In recent days, RNA-based molecules like siRNAs, antisense RNAs and RNA decoys have become promising candidates for antiviral molecules. So designing short RNA molecules that target unique HCV translation initiation mechanism might help in developing novel anti-HCV molecules. HCV 3’UTR and antisense-5’ UTRs serve as sites for replication initiation to synthesize negative and positive strand and this process is catalyzed by NS5B protein (RNA-dependent RNA polymerase). Hence, host proteins binding to both 3’UTR and antisense-5’UTR might play important role in HCV replication. This puts the study of HCV RNA–host protein interactions and its role in viral translation and replication in perspective. Studying the HCV IRES-ribosomal protein S5 interactions and its role in HCV IRES function Previous studies from our laboratory have demonstrated that binding of La protein to GCAC close to initiator AUG enhances ribosomal protein S5 (RPS5) binding with HCV IRES and stimulates HCV translation. However in-detail study on HCV IRES–RPS5 interactions and its implication on HCV translation initiation were lacking. In present study computational modelling suggested that domain II and IV interact majorly with the beta hairpin structure and C-terminal helix of RPS5. Filter-binding and UV cross-linking studies with peptides derived from predicated RNA-binding region of RPS5 and mutational studies with RPS5 demonstrated that beta hairpin structure present in RPS5 is critical for IRES–RPS5 interaction. In parallel, we have studied RNA elements involved in the IRES–RPS5 interactions using deletions and substitution mutations, which we had generated on the basis of the computational model. Direct and competition UV cross-linking experiments performed with these IRES mutants and 40S subunits as a source of RPS5 suggested that structure and sequence of both domain II and IV play crucial role in IRES–RPS5 interactions. We further investigated the effect of these mutations on IRES activity by in vitro translation assay and found that all the mutants that were compromised in binding to RPS5 showed reduced IRES activity. Moreover, ribosome assembly experiments on HCV IRES demonstrated that mutations affecting IRES–RPS5 interactions result in reduction of 80S peak and slight increase of 48S peak. Since the 40S subunit had been previously reported to bind with HCV 3’UTR, we explored the possible interaction of RPS5 with HCV 3’UTR. From direct and competition UV cross-linking assays, we found that RPS5 does not bind to 3’UTR and the interaction is unique to IRES (5’UTR). Interestingly, partial silencing of RPS5 preferentially inhibited HCV translation with marginal effect on cap-dependent translation. Recently, reduction in 40S subunit abundance was reported to preferentially inhibit HCV translation. So, we investigated the abundance of free 40S subunit upon silencing RPS5 and results showed reduction in free 40S subunit level. So, we hypothesize that silencing of RPS5 reduces free 40S abundance to inhibit HCV translation. Taken together, results identified specific RNA elements present in HCV IRES that are critical for IRES–RPS5 interactions and demonstrated the role of these interactions in HCV translation initiation. Targeting ribosome assembly on HCV IRES using short RNAs Stem-loops (SL) IIIe and IIIf of HCV IRES are known to play an important role in stable IRES–40S complex formation. However interaction of these stem-loops with 40S subunit in isolation, independent of other regions of HCV IRES, was not studied. In this study, using electrophoretic mobility shift assay (EMSA) and sucrose gradient centrifugation experiments, we demonstrate that short RNA containing both SLIIIe and SLIIIf together (SLRef RNA) binds to 40S subunit, while short RNAs containing either of the stem-loops (SLRe RNA and SLRf RNA) lose their ability to interact with 40S subunit. Further, SLRef RNA inhibited ribosome assembly on the IRES, whereas SLRe and SLRf RNA failed to inhibit the same. Since SLRef RNA is derived from IRES, we investigated the interaction SLRef RNA with IRES–trans-acting factors (ITAFs). UV cross-linking of radio-labelled HCV IRES with cytoplasmic extract (S10) in presence of unlabelled short RNAs suggested possible interactions of La and RPS5 proteins with SLRef RNA. Studies with recombinant La protein and RPS5 further confirmed their interaction with SLRef RNA. Ex vivo experiments with HCV bicistronic RNA suggested that SLRef RNA specifically inhibits HCV translation. In addition to that SLRef RNA inhibited the HCV RNA synthesis in JFH1 HCV cell culture system. Moreover, specific delivery of pSUPER construct expressing SLRef RNA (pSUPERSLRef) to mice liver along with HCV bicistronic construct using Sendai virosomes demonstrated specific inhibition of HCV IRES activity by SLRef RNA in mice hepotocytes. In summary, short RNA derived from HCV IRES was shown to bind with La protein and RPS5 to inhibit ribosome assembly on HCV IRES. Further, targeted delivery of SLRef RNA into mice liver using Sendai virosome resulted in inhibition of HCV RNA translation in mice hepatocytes. Characterizing the interaction of host proteins with antisense-5’UTR and 3’UTR and its significance in HCV replication Antisense-5’UTR and 3’UTR of HCV RNA are the sites of replication initiation. Hence, host proteins binding to both of these RNA sequences are potential candidates for regulation of HCV replication. In this study, we have investigated host proteins binding with antisense-5’UTR and 3’UTRof HCV RNA by performing UV cross-linking experiments with cytoplasmic extract of Huh7 cells, and found that a protein of ~42kDa protein interacts with both antisense-5’UTR and 3’UTR. Based on earlier report, we predicted that the ~42kDa protein could be hnRNPC1/C2. Results of UV cross-linking followed by immuno pull-down (UV-IP assay) and UV cross-linking experiments with recombinant hnRNPC1 protein confirmed that hnRNPC1 indeed binds to antisense-5’UTR and 3’UTR. Further, filter-binding experiments demonstrated that hnRNPC1 protein binds to 3’UTR with higher affinity compared to antisense-5’UTR. Subsequently, we investigated the regions within 3’UTR and antisense-5’UTR that interact with hnRNPC1protein. Results demonstrated that poly-(U/UC) region of 3’UTR and region containing stem-loops SL-IIIa’, SL-IIIb’, SL-IIIcdef’ and SL-IV’ in antisense-5’UTR were mostly involved in the interaction. Interestingly, studies with confocal microscopy suggested that hnRNPC1/C2 re-localizes from nucleus to cytoplasm upon JFH1 infection, which might in turn influence HCV replication. To investigate the role of hnRNPC1/C2 in HCV replication, partial silencing of hnRNPC1/C2 was performed in HCV cell culture system (JFH1) and results demonstrated that hnRNPC1/C2 is critical for HCV RNA synthesis. However experiments with HCV bicistronic RNA suggested that hnRNPC1/C2 does not play significant role in HCV translation. Taken together, results suggested that hnRNPC1/C2 re-localizes from nucleus to cytoplasm upon JFH1 infection and binds to HCV 3’UTR and antisense- 5’UTR to regulate HCV replication. In summary, this thesis provides novel insights into the interaction of host proteins with HCV RNA and its significance in HCV translation and replication. Inhibition of the ribosome assembly and consequent reduction in HCV translation with mutations interfering with IRES–RPS5 interaction, reported in the present study, unfolds the novel role of this interaction in HCV translation. Further, results obtained in the present study with a small RNA SLRef, derived from HCV IRES, provide proof of concept for using short RNAs to specifically inhibit HCV translation. In addition, studies of interaction of hnRNPC1/C2 with HCV RNA and its re-localization upon HCV infection sheds light on the significance of host–virus interaction in viral RNA replication.

RESUME<p><p><p>Le proto-oncogène c-myc code pour un facteur de transcription qui est impliqué dans de multiples processus cellulaires tels que la prolifération, la différenciation et l’apoptose. Une dérégulation de son expression suite à des altérations génétiques (mutation, translocation, amplification) est retrouvée dans plusieurs tumeurs telles que le lymphome de Burkitt, des plasmacytomes murins ainsi que des tumeurs non-lymphoïdes.<p>c-myc est un gène dont l’expression est régulée à différents niveaux. Chez l’homme, le gène c-myc est transcrit à partir de quatre promoteurs alternatifs appelés respectivement P0, P1, P2 et P3. P1 et P2 sont les deux promoteurs les plus utilisés. Ensemble, ils permettent de former 90% des transcrits c-myc dans des cellules normales. <p>Les promoteurs P0, P1 et P2 permettent la transcription de trois ARNms qui comportent deux codons d’initiation de la traduction (un CUG et un AUG). L’utilisation alternative de ces deux codons d’initiation est à l’origine de la synthèse de deux protéines (c-Myc 1 et c-Myc 2) ayant à la fois des fonctions identiques et distinctes. <p> La grande taille des parties 5’ non-traduites ainsi que la présence dans celles-ci de phases ouvertes de lecture sont des éléments défavorables à la traduction de l’ORF codant pour les protéines Myc par un mécanisme classique d’initiation de la traduction. Notre laboratoire avait précisément montré que les protéines c-Myc sont synthétisées par un processus d’initiation interne de la traduction. Les ARNms dont l’initiation de la traduction s’effectue par entrée interne des ribosomes présentent une structure spécifique appelée IRES (Internal Ribosome Entry Site). Cette structure permet la fixation du ribosome directement à proximité du codon d’initiation. Dans le cas des ARNms c-myc, on retrouve une IRES se situant en amont des codons CUG et AUG qui permet la synthèse des protéines c-Myc1 et 2 respectivement. Un tel mécanisme permet la synthèse des protéines c-Myc dans des conditions où toute traduction dépendante de la coiffe est inhibée (mitose, apoptose).<p><p>Au cours de mon travail, tout d’abord j’ai montré qu’une séquence de 40 nt dans les transcrits P2 permet à elle seule une initiation interne efficace de la traduction. Nous avons déterminé aussi que cette séquence, appelée B4, est active dans quatre types cellulaires différents avec une efficacité variable et qu’elle active la traduction indépendamment de l’ORF placée en aval. D’autre part, il a été déterminé que la séquence B4 recrute le complexe de préinitiation 43S, qui ensuite scanne le messager jusqu’aux codons initiateurs comme c’est le cas de l’IRES du rhinovirus. <p>Une analyse plus détaillée de la séquence B4 a permis d’identifier trois plus petites séquences de plus ou moins 14 nt (Ti1, Boucle, Ti2), qui indépendamment l’une de l’autre permettent une entrée interne des ribosomes. Il a été déterminé que la présence du motif A-N6-AC dans la séquence de Ti2 était importante pour l’activité IRES de celle-ci. Cependant, ce même motif également présent dans la séquence Ti1 n’est pas essentiel à l’activité IRES de Ti1. <p>Par la suite, nous avons démontré que l’IRES de c-myc nécessite pour son activité un évènement nucléaire. Nous avons donc entrepris la recherche de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc. Dans un premier temps, nous avons exclu le rôle de certaines protéines connues pour activer d’autres IRES dont le mécanisme de recrutement du complexe de préinitiation est similaire. Ainsi, nous avons montré, par des expériences de complémentation d’un RRL, que les protéines PTB et unr connues pour activer l’IRES du rhinovirus ne contribuent pas à l’activité de l’IRES de c-myc. De plus, la complémentation de RRL avec des extraits S10 ou nucléaires de cellules HeLa n’a pas permis d’identifier des protéines impliquées dans l’activité IRES de c-myc.<p>D’autre part, des méthodes alternatives d’interaction d’ARN et de protéine comme le triple hybride ou la chromatographie d’affinité d’ARN n’a pas permis dans un premier temps de détecter une interaction entre un facteur non canonique et l’IRES de c-myc. Dès lors, l’existence de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc reste à déterminer.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Apoptosis is the mechanism by which complex multicellular organisms induce the programmed death of damaged cells, thus maintaining tissue homeostasis. One of the main hallmarks of cancer, apoptosis is tightly regulated by pro- and anti-apoptotic factors whose equilibrium will decide of the fate of the cell. Among these factors, the cellular inhibitor of apoptosis cIAP1 is a key regulator of nuclear factor-κB dependent signaling and of caspase-8 mediated apoptosis. cIAP1 expression is controlled primarily at the translational level through an internal ribosome entry site (IRES) that facilitates the recruitment of the ribosome to the translation initiation start independently of the 5’ cap. We have previously identified four putative IRES trans-acting factors (ITAFs) that bind specifically to the cIAP1 IRES, namely NF45, NF90, IGF2BP1 and RH1. My research project characterised NF45 as an ITAF that positively regulates the IRES-mediated translation of cIAP1 and of the Xlinked inhibitor of apoptosis, XIAP. This regulation is important for maintaining Survivin and Cyclin E protein levels and insuring proper cell division. Furthermore, I showed that IGF2BP1 is another ITAF that is overexpressed in rhabdomyosarcoma cancer (RMS) and positively regulates cIAP1 translation, thus leading to apoptotic resistance in these cells. Importantly, the use of Smac mimetics, chemical compounds that cause cIAP1 proteasomal degradation, induces TNFα-mediated apoptosis of RMS cells and leads to growth inhibition of RMS xenograft tumors as well as significantly improved survival. Finally, I show that certain modulators of innate immunity synergize with Smac mimetics to improve the killing of RMS cancer cells. Hence, cIAP1 translation regulation by NF45 and IGF2BP1 is highly important for maintaining proper functioning of the cell and dysregulation of these ITAFs can lead to carcinogenesis.

The conserved secondary structural RNA motifs of EncephaloMyoCarditis Virus (EMCV) have been well characterised biochemically and shown to play an important role in translation initiation by a novel cap-independent mechanism called Internal Ribosomal Entry Site (IRES). However, the three dimensional structure and interactions of these conserved motifs are not known, and hence the mechanism is not fully understood. The NMR results described in this thesis have provided, for the first time, new structural knowledge on the conformation of these motifs, their affinity for Mg2+ and their intermolecular interactions. RNA motifs selected from two separate domains (I and J) of the IRES structure were investigated using a range of 2D and 3D NMR techniques. The apical ‘hammerhead’ region of the I domain contains a highly conserved 16mer RNA which hosts a stable and mutationally sensitive G547CGA550 tetraloop. Sequence specific assignments were carried out on this motif, along with its Mg2+ complex, and a large number of NMR experimental constraints were generated for the RNA structure determination. Similarly, high resolution NMR structures of a distal 17mer RNA, which has been predicted to be a potential receptor for the GCGA tetraloop, and its Mg2+ complex were also produced. Thus, we were able to demonstrate that Mg2+ stabilises the RNA tertiary structure via non-specific interactions. Since the largest changes were induced at the tetraloop motif, we propose that Mg2+ stabilises the 16mer into an optimum conformation which is essential for IRES function. The determination of the structures of the above motifs led us to investigate the 16mer-17mer binary (1:1) complex at 1 GHz, in the presence of Mg2+. Significant changes were observed in the 1H and 31P chemical shift, NOE intensity and line width, clearly demonstrating RNA-RNA interactions taking place between the two components. The most interesting result to emerge was the distinct absence of NOEs from G547{NH} of the stable tetraloop, thus highlighting an important structural role for this functionally critical residue. Since no previous work has shown a clear interaction between the two RNAs, the results obtained in this project provide the first direct experimental evidence for intramolecular interactions in the I domain of EMCV IRES.Finally, we show how isotopically labelled RNAs can be successfully used as an aid in NMR assignment, analysis and structure determination. The J domain of EMCV IRES binds to eIF4GII protein and is essential for translation initiation. A suite of 3D NMR techniques were carried out on a highly enriched and uniformly 13C, 15N-labelled 39mer RNA. Several key features of the RNA, which may be involved in protein recognition, were identified. Further, a selectively 19F-labelled 16mer RNA from the I domain, was also studied to show how fluorine NMR can be used to probe RNA structure, dynamics and interactions. The RNA motifs of the EMCV IRES were shown to exhibit high stabilities, which are brought about by the complex folding of the various secondary structural elements involving RNA- Mg2+, RNA-RNA and RNA-protein tertiary interactions. It is these vital interactions that enable the IRES to recruit the ribosome in the translation initiation step of protein synthesis, and have laid a strong foundation for further NMR investigation of the whole IRES.