Deoxyhypusine synthase (DHS) catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF5A). In human cells, two eIF5A isoforms are present, eIF5A-1 and eIF5A-2, and DHS catalyzes the hypusination of both. Since both eIF5As are substrates for DHS, the biological functions of DHS are likely to be exerted through the various post-translational forms of these two eIF5As. The lysine form of eIF5A-1 has been associated with apoptosis, while the hypusinated form of eIF5A-1 has been associated with cell viability and proliferation. eIF5A-2 has been found to be over-expressed in certain cancers and has been proposed to function as an oncogene. Dhs is also over-expressed in certain human cancers and is a metastatic signature gene. The purpose of the present study was to investigate the role of DHS in cancer cell invasiveness, cell proliferation, and apoptosis using RNA interference. The main finding of the study is that DHS siRNA treatment decreases invasiveness of PC3 cells in vitro. Both DHS 0 siRNA treatment and DHS 1/b siRNA treatment significantly reduced cell invasiveness of PC3 cells as measured by the Matrigel invasion assay. Potential confounding variables, such as differences in cell proliferation or differences in apoptosis in response to DHS siRNA treatment, were assessed using the XTT cell proliferation assay and the Annexin V/Pi apoptosis assay, and they were found not to have an effect. In the absence of serum, DHS siRNA treatment did not result in significant decrease in cell proliferation compared to the control siRNA treatment. Furthermore, DHS siRNA treatment did not induce apoptosis in PC3 cells under the present experimental conditions. In conclusion, depletion of DHS with RNAi reduces invasiveness, but does not induce apoptosis in PC3 cells. The significance of the research is that the anti-invasiveness effect of DHS depletion in metastatic cancer cells is shown for the first time in the present study. Thus, DHS depletion may be useful to combat cancer in conjunction with L-eIF5A-1 over-expression.

Deoxyhypusine synthase (DHS) catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF5A). In human cells, two eIF5A isoforms are present, eIF5A-1 and eIF5A-2, and DHS catalyzes the hypusination of both. Since both eIF5As are substrates for DHS, the biological functions of DHS are likely to be exerted through the various post-translational forms of these two eIF5As. The lysine form of eIF5A-1 has been associated with apoptosis, while the hypusinated form of eIF5A-1 has been associated with cell viability and proliferation. eIF5A-2 has been found to be over-expressed in certain cancers and has been proposed to function as an oncogene. Dhs is also over-expressed in certain human cancers and is a metastatic signature gene. The purpose of the present study was to investigate the role of DHS in cancer cell invasiveness, cell proliferation, and apoptosis using RNA interference. The main finding of the study is that DHS siRNA treatment decreases invasiveness of PC3 cells in vitro. Both DHS 0 siRNA treatment and DHS 1/b siRNA treatment significantly reduced cell invasiveness of PC3 cells as measured by the Matrigel invasion assay. Potential confounding variables, such as differences in cell proliferation or differences in apoptosis in response to DHS siRNA treatment, were assessed using the XTT cell proliferation assay and the Annexin V/Pi apoptosis assay, and they were found not to have an effect. In the absence of serum, DHS siRNA treatment did not result in significant decrease in cell proliferation compared to the control siRNA treatment. Furthermore, DHS siRNA treatment did not induce apoptosis in PC3 cells under the present experimental conditions. In conclusion, depletion of DHS with RNAi reduces invasiveness, but does not induce apoptosis in PC3 cells. The significance of the research is that the anti-invasiveness effect of DHS depletion in metastatic cancer cells is shown for the first time in the present study. Thus, DHS depletion may be useful to combat cancer in conjunction with L-eIF5A-1 over-expression.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est responsable de la pandémie du SIDA (syndrome de l’immunodéficience acquise). Des souches virales résistantes aux antirétroviraux actuellement utilisés apparaissent rapidement. Il est donc important d’identifier de nouvelles cibles dans le cycle de réplication du VIH-1 pour développer de nouveaux agents contre ce virus. La traduction des protéines de structure et des enzymes du VIH-1 est une étape essentielle du cycle de réplication virale. Ces protéines sont exprimées à partir de l’ARN messager (ARNm) pleine-longueur (ARNmPL) à la fin du cycle de réplication. L’ARNmPL du VIH-1 peut utiliser un mode d’initiation de la traduction coiffe-dépendant, comme la majorité des ARNm cellulaires, mais peut aussi utiliser un mode d’initiation alternatif, car sa région 5’ non-traduite (5’UTR) contient un site interne d’entrée du ribosome (IRES), ce qui lui permet d’initier la traduction suivant un mode IRES-dépendant. L’initiation IRES-dépendante permet à l’ARNmPL d’être traduit quand l’initiation coiffe-dépendante est inhibée. L’activité de l’IRES de la région 5’UTR de l’ARNmPL du VIH-1 (IRES5’UTR) est faible dans des conditions physiologiques, mais est stimulée lorsque la cellule est arrêtée à la transition G2/M du cycle cellulaire, un arrêt qu’induit l’infection par le VIH-1. Une grande portion de l’IRES5’UTR, que nous nommons IRES5’UTRc, est présente dans tous les ARNm viraux et a une activité semblable à celle de l’ IRES5’UTR, ce qui indique que le mode IRES-dépendant peut être utilisé par tous les messagers du VIH-1. Lors de mes études doctorales, j’ai caractérisé le fonctionnement de l’IRES5’UTR du VIH-1. J’ai transfecté des cellules lymphocytaires Jurkat T, dérivées des cibles naturelles du VIH-1, avec un vecteur dual-luciférase contenant les séquences codantes des luciférases de la Renilla (Rluc) et de la luciole (Fluc) séparées par la région 5’UTR de l’ARNmPL du VIH-1. La traduction de la Rluc est coiffe-dépendante alors que celle de la Fluc dépend de l’IRES5’UTR. J’ai d’abord effectué une analyse mutationnelle et j’ai identifié trois régions qui stimulent l’activité de l’IRES5’UTR et une tige-boucle qui réprime l’activité de cet IRES, que j’ai nommée IRENE (IRES negative element). J’ai montré que l’effet répresseur d’IRENE est aboli lorsque les cellules sont soumises à un stress oxydatif, un type de stress induit lors d’une infection par le VIH-1. Nous proposons que IRENE maintiendrait l’IRES5’UTR dans une conformation peu active dans des conditions physiologiques. On sait que les IRES sont activés par divers facteurs cellulaires, appelés ITAF (IRES trans-acting factors). Nous proposons que l’IRES5’UTR adopterait une conformation active suite à la liaison d’un ITAF exprimé ou relocalisé lors d’un stress oxydatif. Ces travaux ont fait l’objet d’une publication (Gendron et al., 2011, Nucleic Acids Research, 39, 902-912). J’ai ensuite étudié l’effet de la protéine virale Tat sur l’activité de l’IRES5’UTR. En plus de son rôle essentiel dans la transactivation de la transcription des ARNm viraux, Tat stimule leur traduction coiffe-dépendante, en empêchant l’inhibition d’un facteur d’initiation canonique, eIF2, induite par la protéine kinase modulée par l’ARN double-brin (PKR) et en déroulant la structure TAR présente à l’extrémité 5’ de tous les ARNm du VIH-1. Elle affecte aussi l’expression de plusieurs gènes cellulaires. J’ai montré que les isoformes Tat86 et Tat72, mais non Tat101, stimulent l’activité de l’IRES5’UTR. Cet effet est indépendant de PKR et de TAR, mais dépendrait de la conformation de Tat. Nous proposons que Tat activerait un facteur de transcription cellulaire qui déclenche l’expression d’un ITAF de l’IRES5’UTR ou encore qu’elle activerait directement un tel ITAF. J’ai de plus montré que PKR stimule l’activité de l’IRES5’UTR, ce qui est surprenant puisque PKR est une protéine antivirale. Cet effet est indépendant de l’inhibition d’eIF2 par PKR et pourrait résulter de l’activation d’un ITAF. Sachant qu’une portion active de l’IRES5’UTR, IRES5’UTRc, est présente dans tous les ARNm viraux, notre hypothèse est que la stimulation de cet IRES par PKR permettait de traduire l’ARNm de Tat au début du cycle de réplication, ce qui permettrait ensuite la traduction coiffe-dépendante des ARNm du VIH-1, qui est stimulée par Tat. Ces travaux font l’objet d’un manuscrit (Gendron et al., soumis à RNA). Mes résultats, couplés aux données de la littérature, me conduisent à la conclusion que, à la fin du cycle de réplication du VIH-1, l’activité de l’IRES5’UTR est stimulée par le stress oxydatif, l’arrêt en G2/M et la présence de quantités élevées de Tat, alors que la traduction coiffe-dépendante est compromise. L’initiation IRES-dépendante serait alors indispensable pour que le VIH-1 traduise l’ARNmPL. L’IRES5’UTR constituerait donc une cible très intéressante pour développer des agents anti-VIH. / The human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS (acquired immunodeficiency syndrome). Viral strains that are resistant to antiretroviral agents used for the treatment of HIV-1 infected patients rapidly emerge. It is thus important to study the viral replication cycle in order to discover new targets for the development of novel agents against HIV-1. Translation of structural proteins and viral enzymes is a key step of the viral replication cycle. These proteins are translated from the HIV-1 full-length mRNA during late stages of the replication. This mRNA can be translated by a cap-dependent mode which is used by the majority of cellular mRNAs. However, since its 5’ untranslated region (5’UTR) contains an internal ribosome entry site (IRES) that we call IRES5’UTR, it can also be translated by an IRES-dependent mode. The IRES-dependent mode enables the full-length mRNA to be translated when the cap-dependent mode is impaired. The activity of the IRES5’UTR is weak in physiological conditions, but it is stimulated when the cell cycle is arrested at the G2/M transition, an arrest induced by HIV-1 infection. A large portion of this IRES, which we name IRES5’UTRc, is present in all HIV-1 mRNAs and its activity is similar to the activity of the complete IRES, which indicates that the IRES-dependent mode can be used by all HIV-1 mRNAs. During my doctoral studies, I investigated how the HIV-1 IRES5’UTR functions. I transfected Jurkat T cells, a lymphocytic cell line derived from the natural target cells of HIV-1, with a dual-luciferase reporter containing the coding sequences of the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) separated by the complete 5’UTR of the HIV-1 full-length mRNA. Translation of Rluc is cap-dependent while translation of Fluc depends on HIV-1 IRES5’UTR. First, I performed a mutational analysis and I discovered three regions that stimulate the activity of IRES5’UTR and a stem-loop that represses its activity, which we named IRENE (IRES negative element). I showed that the repression induced by IRENE is relieved when cells are exposed to oxidative stress, a type of stress caused by HIV-1 infection. We propose that IRENE maintains the IRES5’UTR in a weakly active conformation in physiological conditions. It is known that IRESes are activated by cellular factors, called ITAFs (IRES trans-acting factors). We propose that the IRES5’UTR adopts an active conformation triggered by the binding of an ITAF that is expressed or relocalized during oxidative stress. These results generated a publication (Gendron et al. Nucleic Acids Research, 2011, 39, 902-912). I then decided to study the effect of the viral protein Tat on the IRES5’UTR activity. In addition to its essential role in the transcription of HIV-1 mRNAs, Tat stimulates the cap-dependent translation of HIV-1 mRNAs by interfering with the inhibition of a canonical initiation factor, eIF2, induced by the protein kinase modulated by double-stranded RNA (PKR) and by unwinding the TAR structure present at the 5’end of all HIV-1 mRNAs. Tat also affects the expression of several cellular genes. I showed that the Tat86 and Tat72 isoforms, but not Tat101, stimulate the activity of the IRES5’UTR. This effect is independent of PKR and TAR, but appears to be dependent upon the conformation of Tat. We suggest that Tat could activate a transcription factor that controls the expression of an ITAF of the IRES5’UTR or else that Tat could directly activate such an ITAF. I also showed that PKR stimulates the IRES5’UTR activity, which is surprising since PKR is an antiviral protein. This effect is independent of the inhibition of eIF2 by PKR and could result from the activation of an ITAF. Knowing that IRES5’UTRc, an active portion of IRES5’UTR is present in all HIV-1 RNAs, our hypothesis is that the stimulation of the IRES activity by PKR would allow Tat mRNA to be translated in the beginning of the replication cycle. This would subsequently allow the cap-dependent translation of HIV-1 mRNAs to proceed, which is stimulated by Tat. These results generated a manuscript that is submitted for publication to RNA. Altogether, my results, coupled to data from literature, lead me to conclude that, in the late phases of the replication cycle, the activity of the HIV-1 IRES5’UTR is stimulated by oxidative stress, by the cell cycle arrest in G2/M and by the presence of high amounts of Tat, while cap-dependent translation is impaired. The IRES5’UTR would thus be critical to translate the HIV-1 full-length mRNA. Consequently, the IRES5’UTR would constitute a very interesting target for the development of novel anti-HIV agents.

MAGALHÃES, Vladimir Gonçalves. Prospecção bioquímica e molecular de fatores possivelmente envolvidos na defesa de feijão-de-corda [Vigna unguiculata (L.) Walp] ao vírus do mosaico severo do caupi (CPSMV). 2011. 109 f. : Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2011. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-15T14:36:03Z No. of bitstreams: 1 2011_dis_vgmagalhaes.pdf: 4392892 bytes, checksum: 571bbf928e7a06498a9e935694d2ab1d (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:26:15Z (GMT) No. of bitstreams: 1 2011_dis_vgmagalhaes.pdf: 4392892 bytes, checksum: 571bbf928e7a06498a9e935694d2ab1d (MD5) / Made available in DSpace on 2016-08-02T20:26:15Z (GMT). No. of bitstreams: 1 2011_dis_vgmagalhaes.pdf: 4392892 bytes, checksum: 571bbf928e7a06498a9e935694d2ab1d (MD5) Previous issue date: 2011 / Cowpea [(Vigna unguiculata (L.) Walp.)] has a major socioeconomic importance in Northeastern Brazil. However, its production is low due abiotic and biotic factors. Amongst the biotic factors the cowpea severe mosaic virus (CPSMV, Secoviridae family) has a great importance because it causes the most prevalent and serious virus disease that affects this crop in the country. Although there are resistant cultivars to CPSMV, the defense mechanisms involved is not understood. For this reason, a comparative study was conducted between resistant and susceptible cultivars, using two experimental approaches. In the first one, the biochemical approach, possible differences of enzyme activities related to oxidative stress (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase) and pathogenesis (β-1,3-glucanase and chitinase), in addition of phenyl amonium lyase, and H2O2 generation in the leaves of the cultivars Pitíuba (susceptible) and Macaibo (resistant) were analyzed. The secondary leaves were harvested at 6, 12, 24, 48, 72 h after treatment with CPSMV or carborundum (controls) and these above parameters were measures in the protein extracts obtained. It was shown that, in general, the response amongst the cultivars did not differ significantly, suggesting that the defense mechanisms of cowpea are different from the classic response of defense observed for several plant species. In the second approach, molecular, the nucleotide sequences of the genes that code for the translation initiation factors (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G and nCBP) and the primary strucuture of the correspondent putative proteins were analyzed in order to search patterns of polymorphis between the studied cowpea cultivars that could be related to a constitutive defense conferred by recessive genes. After sequence analysis, it was found that eIF4E showed polymorphisms between cultivars, and, in at least two positions (68 and 108), there were differences between susceptible and resistant cultivars (Arg68/Pro68; Val108 or Pro108/Ala108). The molecular modeling revealed that differences in amino acid are located in two external loops close to the cap (m7G) binding domain, well reported in cases of recessive resistance within the Potyviridae family. Through immunodetection studies with the leaf extracts and the protein fractions obtained after the affinity chromatography on a Sepharose-7-metil-guanosina column, it was found that the amino acid mutations found did not impair the ability of eIF4E to bind to M7G in vitro. However, as it was observed two variants for eIF4E comparing the resistant and susceptible cultivars to CPSMV, at spatially neighboring regions, it could not be ruled out the hypothesis that this constitutive/recessive resistant trait is correlated with these mutations detected, which could impair, consequently, the in vivo interaction of eIF4E with the viral VPg. / O feijão-de-corda [Vigna unguiculata (L.) Walp.] tem grande importância socioeconômica no Nordeste brasileiro. Entretanto, sua produção é baixa devida a diversos fatores abióticos e bióticos. Dentre os fatôres bióticos, o vírus do mosaico severo do caupi (CPSMV, família Secoviridae) apresenta grande destaque, por causar a virose que mais acomete essa cultura no país. Embora existam cultivares resistentes ao vírus, não se sabe quais os mecanismos de defesa envolvidos. Por essa razão, foi elaborado um estudo comparativo entre cultivares resistentes e susceptíveis, utilizando duas abordagens experimentais. Na abordagem bioquímica, possíveis diferenças de atividades de enzimas relacionadas ao estresse oxidativo (dismutase do superóxido, peroxidase do ascorbato, peroxidase) e à patogênese (β-1,3-glucanase e quitinase), além da fenilamônia liase, e teores de H2O2 foram estudadas nos cultivares Pitiúba (susceptível) e Macaibo (resistente). Após tratamento com o CPSMV ou com apenas carborundum (plantas controles), foram realizadas coletas nos tempos de 6, 12, 24, 48 e 72 h, tendo sido realizadas as análises bioquímicas nos extratos protéicos obtidos das folhas secundárias. Foi verificado que, de maneira geral, a resposta entre os cultivares não diferiram significamente, sugerindo que os mecanismos de defesa de feijão-de-corda sejam diferentes da resposta clássica de defesa. Na segunda abordagem, molecular, as sequências nucleotídicas dos genes codificantes para os fatores de iniciação de tradução (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G e nCBP) e as sequências primárias putativas das proteínas correspondentes foram analisados, no intuito de se averiguar a existência de padrões de polimorfismos entre cultivares resistentes e susceptíveis, que pudessem estar relacionados à defesa constitutiva conferida por genes recessivos. Após análise das sequências, foi observado que eIF4E apresentava polimorfismos entre os cultivares, sendo que, em pelo menos duas posições nas sequências primárias putativas do fator (68 e 108), existiram diferenças entre cultivares susceptíveis e resistentes (Arg68/Pro68; Val108 ou Pro108/Ala108). A modelagem molecular revelou que as diferenças em aminoácidos situam-se em dois loops externos, próximos ao domínio de ligação ao capacete (m7G), bastante relatados em casos de resistência recessiva para a família Potyviridae. Através de estudos de imunodectecção posterior ao passo cromatográfico em coluna de afinidade, foi observado que as mudanças de aminoácidos não comprometiam, a capacidade de eIF4E em se ligar ao m7G in vitro. Entretanto, como foram observadas duas variantes para eIF4E, entre cultivares resistentes e susceptíveis ao CPSMV, em regiões próximas espacialmente, não se pode descartar a hipótese de que a resistência recessiva constitutiva esteja associada com essas mutações detectadas nessas sequências, que iriam modificar, consequentemente, a interação da VPg viral com eIF4E in vivo.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O feijÃo-de-corda [Vigna unguiculata (L.) Walp.] tem grande importÃncia socioeconÃmica no Nordeste brasileiro. Entretanto, sua produÃÃo à baixa devida a diversos fatores abiÃticos e biÃticos. Dentre os fatÃres biÃticos, o vÃrus do mosaico severo do caupi (CPSMV, famÃlia Secoviridae) apresenta grande destaque, por causar a virose que mais acomete essa cultura no paÃs. Embora existam cultivares resistentes ao vÃrus, nÃo se sabe quais os mecanismos de defesa envolvidos. Por essa razÃo, foi elaborado um estudo comparativo entre cultivares resistentes e susceptÃveis, utilizando duas abordagens experimentais. Na abordagem bioquÃmica, possÃveis diferenÃas de atividades de enzimas relacionadas ao estresse oxidativo (dismutase do superÃxido, peroxidase do ascorbato, peroxidase) e à patogÃnese (β-1,3-glucanase e quitinase), alÃm da fenilamÃnia liase, e teores de H2O2 foram estudadas nos cultivares PitiÃba (susceptÃvel) e Macaibo (resistente). ApÃs tratamento com o CPSMV ou com apenas carborundum (plantas controles), foram realizadas coletas nos tempos de 6, 12, 24, 48 e 72 h, tendo sido realizadas as anÃlises bioquÃmicas nos extratos protÃicos obtidos das folhas secundÃrias. Foi verificado que, de maneira geral, a resposta entre os cultivares nÃo diferiram significamente, sugerindo que os mecanismos de defesa de feijÃo-de-corda sejam diferentes da resposta clÃssica de defesa. Na segunda abordagem, molecular, as sequÃncias nucleotÃdicas dos genes codificantes para os fatores de iniciaÃÃo de traduÃÃo (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G e nCBP) e as sequÃncias primÃrias putativas das proteÃnas correspondentes foram analisados, no intuito de se averiguar a existÃncia de padrÃes de polimorfismos entre cultivares resistentes e susceptÃveis, que pudessem estar relacionados à defesa constitutiva conferida por genes recessivos. ApÃs anÃlise das sequÃncias, foi observado que eIF4E apresentava polimorfismos entre os cultivares, sendo que, em pelo menos duas posiÃÃes nas sequÃncias primÃrias putativas do fator (68 e 108), existiram diferenÃas entre cultivares susceptÃveis e resistentes (Arg68/Pro68; Val108 ou Pro108/Ala108). A modelagem molecular revelou que as diferenÃas em aminoÃcidos situam-se em dois loops externos, prÃximos ao domÃnio de ligaÃÃo ao capacete (m7G), bastante relatados em casos de resistÃncia recessiva para a famÃlia Potyviridae. AtravÃs de estudos de imunodectecÃÃo posterior ao passo cromatogrÃfico em coluna de afinidade, foi observado que as mudanÃas de aminoÃcidos nÃo comprometiam, a capacidade de eIF4E em se ligar ao m7G in vitro. Entretanto, como foram observadas duas variantes para eIF4E, entre cultivares resistentes e susceptÃveis ao CPSMV, em regiÃes prÃximas espacialmente, nÃo se pode descartar a hipÃtese de que a resistÃncia recessiva constitutiva esteja associada com essas mutaÃÃes detectadas nessas sequÃncias, que iriam modificar, consequentemente, a interaÃÃo da VPg viral com eIF4E in vivo. / Cowpea [(Vigna unguiculata (L.) Walp.)] has a major socioeconomic importance in Northeastern Brazil. However, its production is low due abiotic and biotic factors. Amongst the biotic factors the cowpea severe mosaic virus (CPSMV, Secoviridae family) has a great importance because it causes the most prevalent and serious virus disease that affects this crop in the country. Although there are resistant cultivars to CPSMV, the defense mechanisms involved is not understood. For this reason, a comparative study was conducted between resistant and susceptible cultivars, using two experimental approaches. In the first one, the biochemical approach, possible differences of enzyme activities related to oxidative stress (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase) and pathogenesis (β-1,3-glucanase and chitinase), in addition of phenyl amonium lyase, and H2O2 generation in the leaves of the cultivars PitÃuba (susceptible) and Macaibo (resistant) were analyzed. The secondary leaves were harvested at 6, 12, 24, 48, 72 h after treatment with CPSMV or carborundum (controls) and these above parameters were measures in the protein extracts obtained. It was shown that, in general, the response amongst the cultivars did not differ significantly, suggesting that the defense mechanisms of cowpea are different from the classic response of defense observed for several plant species. In the second approach, molecular, the nucleotide sequences of the genes that code for the translation initiation factors (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G and nCBP) and the primary strucuture of the correspondent putative proteins were analyzed in order to search patterns of polymorphis between the studied cowpea cultivars that could be related to a constitutive defense conferred by recessive genes. After sequence analysis, it was found that eIF4E showed polymorphisms between cultivars, and, in at least two positions (68 and 108), there were differences between susceptible and resistant cultivars (Arg68/Pro68; Val108 or Pro108/Ala108). The molecular modeling revealed that differences in amino acid are located in two external loops close to the cap (m7G) binding domain, well reported in cases of recessive resistance within the Potyviridae family. Through immunodetection studies with the leaf extracts and the protein fractions obtained after the affinity chromatography on a Sepharose-7-metil-guanosina column, it was found that the amino acid mutations found did not impair the ability of eIF4E to bind to M7G in vitro. However, as it was observed two variants for eIF4E comparing the resistant and susceptible cultivars to CPSMV, at spatially neighboring regions, it could not be ruled out the hypothesis that this constitutive/recessive resistant trait is correlated with these mutations detected, which could impair, consequently, the in vivo interaction of eIF4E with the viral VPg.

La traduction est une étape clef de l’expression des gènes, et mon travail a consisté à étudier l’implication de la protéine ribosomique S1 d’Escherichia coli dans l’initiation de la traduction des ARNm structurés. Mes résultats montrent que 1) S1 est requise pour la formation du complexe d’initiation des ARNm portant une séquence SD faible et/ou des structures stables, 2) elle est dotée d’une activité chaperonne, débobinant les ARNm afin de les placer dans le canal de décodage ; et 3) le ribosome favorise son action. Par la suite, j’ai montré un rôle inattendu de S1 dans la régulation post-transcriptionnelle médiée par les ARNnc. En effet, la dégradation rapide de l’ARNm sodB, induite par l’ARNnc RyhB en absence de fer, est perdue dans une souche dont l’extrémité C-terminale de S1 a été supprimée, montrant ainsi un lien fonctionnel entre S1 et le dégradosome. Ainsi, S1 exerce de multiples fonctions qui se placent au carrefour de la traduction et de la régulation de l’expression des gènes / The translation is a key step for the gene expression, and the aim of my PhD was to analyze the involvment of Escherichia coli ribosomal protein S1 in the translation initiation of structured mRNAs.My results show that 1) S1 is required for the establishment of the active translation initiation complex involving mRNAs with a weak SD sequence and/or stable structures, 2) S1 has a RNA chaperone activity, unwinding the mRNA in order to accommodate it in the decoding channel, and 3) the ribosome promotes its activity.In the second part of my thesis, I unexpectedly showed that S1 is involved in the ncRNAmediated regulation. Indeed, the fast degradation of sodB mRNA, induced by RyhB ncRNA under iron depletion, is impaired in a strain depleted of the C-terminal part of S1 protein, thus highlighting a functional link between S1 and the degradosome.All in one, my results show that S1 is endowed with multiple functions, at the cross-road between translation and regulation of gene expression.

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.

Eukaryotic translation initiation is a complex and highly regulated process involving the ribosome, mRNA and proteins called eukaryotic initiation factors (eIFs). The overall aim of translation initiation is to position the ribosome at the initiation codon of the mRNA. eIF2, in its GTP-bound conformation, binds the initiator tRNA (Met-tRNAiMet) and delivers it to the 40S ribosomal subunit. When the anticodon of the tRNA is bound to the initiation codon, the GTP on eIF2 is hydrolysed to GDP. The guanine nucleotide exchange factor (GEF) eIF2B regenerates eIF2-GTP. eIF2 and eIF2B are multisubunit/multidomain protein complexes. Because information regarding the interface between each complex is limited, particularly the interface on the eIF2γ subunit, which binds the guanine-nucleotides and Met-tRNAiMet, interactions between the minimal GEF domain of eIF2Bε, εGEF, and eIF2 were mapped using mutagenesis and an in vitro cysteine cross-linking approach, with the cross-linker Mts-Atf-Biotin. Site-directed mutagenesis (SDM) was used to mutate five N-terminal and five C-terminal surface-exposed εGEF residues to cysteines. The mutant alleles were analysed in Saccharomyces cerevisiae and it was found that the gcd6-R574C allele was lethal and the gcd6-T572C was Gcd-. Further gcd6-R574 mutant alleles were also found to be lethal in yeast but expressed in vivo.εGEF-R574C has dramatically reduced GEF activity in vitro and binding assays showed that this mutant has significantly reduced affinity for eIF2. The εGEF-T572C and εGEF-S576C mutants also have severe and minor eIF2-binding defects respectively, while the C-terminal εGEF-Cys mutants have slightly reduced affinity for eIF2. The N-terminal εGEF-Cys mutants cross-link specifically to eIF2γ, while the C-terminal εGEF-Cys mutants interact predominantly with eIF2β. From the data obtained in this study, we propose a new model for eIF2B-mediated guanine-nucleotide exchange that reduces the importance of eIF2β and suggests εGEF resembles other GEFs in binding primarily to its G protein partner eIF2γ.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est un virus à simple brin positif qui appartient au genre Lentivirus dans la famille retroviridae et qui constitue l’agent étiologique du SIDA pandémique.Pendant le cycle réplicatif du VIH-1, la traduction de protéines virales dépend exclusivement de la machinerie traductionnelle cellulaire. Pour cette raison, nous avons cherché à comprendre le rôle de quelques facteurs cellulaires qui pourraient contrôler la traduction du VIH-1 à différents nivaux. Nous avons centré nos recherches sur la traduction de l’ARN génomique (ARNg) du virus qui sert en même temps de génome pour être encapsidé et comme ARN messager pour la traduction des protéines virales Gag et Gag-Pol. 1) Le rôle de l’hélicase d’ARN DDX3 dans la traduction du VIH-1. L’ARNg du VIH-1 possède une région 5’ non traduite très structurée, raison pour laquelle nous avons spéculé sur un possible rôle de DDX3 dans la traduction du VIH-1. Nous avons utilisé une combinaison de techniques in vitro et ex vivo afin de pouvoir démontrer que DDX3 était capable de lier et faire des complexes avec l’ARN de la région 5’ non traduite pour promouvoir l’initiation de la traduction. Nous avons aussi pu démontrer que DDX3 formait des complexes avec les facteurs d’initiation de la traduction PABP, eIF4G et eIF4E. 2) Le changement programmé du cadre de lecture (PRF) dans l’ARN génomique du VIH-1. La traduction de la polyprotéine Gag-Pol du VIH-1 nécessite un décalage de phase de 1 nucléotide en arrière. Ce mécanisme permet la synthèse des protéines Gag et Gag-Pol avec des ratios de 95 et 5% respectivement à partir du même ARN. Cette proportion doit être conservée pour assurer la réplication du virus. Nous avons utilisé un système de double gène rapporteurs et un système de réplication complète du provirus pour montrer que la protéine associé aux granules de stress TIAR pouvait contrôler la réplication viral en régulant la proportion de ribosome qui assurent / Human Immunodeficiency virus type 1 (HIV-1) is a positive strand RNA virus belonging to the lentivirus genus of the retroviridae family and it is the etiological agent of the pandemic AIDS, which is a major health concern worldwide. Throughout HIV-1 replication cycle, the production of viral proteins depends exclusively on the cellular translational machinery. This is the reason why we have explored the role of some cellular factors that could control HIV-1 translation at different stages. We have focused our studies on the translation of the full length genomic RNA (gRNA), which serves both as genome for viral encapsidation and as a messenger for translation of Gag and Gag-Pol viral polyproteins.1) The role of the RNA helicase DDX3 in HIV-1 translation Initiation The fact that HIV-1 possesses a highly structured 5’ untranslated region (5’UTR) prompted us to speculate that DDX3 may be involved in HIV-1 translation. We used a combination of in vitro and ex-vivo approaches to show that DDX3 was able to bind and form complexes with the 5’-UTR of HIV-1 to assist translation initiation. We also demonstrated that DDX3 can form a complex with initiation factors such as PABP, eIF4G and eIF4E. 2) Programmed Ribosomal Frameshift (PRF) in the genomic RNA of HIV-1Translation of HIV-1 Gag-Pol polyprotein requires a -1 PRF. This mechanism allows the synthesis of Gag and Gag-Pol polyproteins, using the same mRNA template, at ratios of 95 and 5% respectively. Keeping the -1PRF ratio is important as any change leads to reduction in virus infectivity.By means of a dual reporter construct and full provirus replication system we were able to demonstrate that the stress granules associated protein TIAR, controls HIV-1 infectious progeny by regulating the ratio of the HIV-1 PRF.

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