Messenger RNA transcripts of αs2- and к-casein are translated at 25% of the efficiency of αs1- and β-casein transcripts; however, the molecular mechanisms governing the difference are unknown. We hypothesized that the bovine casein translational efficiency is influenced by characteristics of the untranslated regions (UTRs) and coding regions. The main objective of this study was to identify molecular mechanisms that explain differential translational regulation between bovine β- and αs2-casein by assessing the role of each putative translational regulatory factor found throughout full-length sequences in both in cellular and cell-free translation systems. This dissertation begins with the cloning and initial characterization of bovine β- and αs2-casein. Transcript analysis indicates that the two genes share similar characteristics of nucleotide sequence around the coding region and secondary structure. It is confirmed that αs2-casein mRNA has a lower translational efficiency compared to that of β-casein in a cell-free system. The latter portion of this thesis investigates further the UTRs and codon usage effect on difference in translational efficiency between β- and αs2-casein. Overall, our data suggest that β-casein 3’ UTR and αs2-casein 5’ UTR exert stimulatory effects on translation yet their effectiveness depends on the upstream and downstream sequences with which they are associated. Replacement of the UTRs of αs2-casein mRNA with those of β-casein did not stimulate translation. A stronger effect on translational efficiency was found in the coding region of αs2-casein which displays unfavourable codons at the 3’ terminus. Deletion of a 28-codon fragment from the 3’ terminus of the αs2-casein coding region increased translation to a par with β-casein. We suggest that the last 28 codons of αs2-casein is the main regulatory sequence that attenuates its expression and is responsible for the different translational expression of β- and αs2-casein mRNAs. Identification of regulatory factors that are responsible for translation efficiency improves our understanding of the molecular mechanisms of control of milk protein prodiction in secretory cells of the bovine mammary glands. / NSERC canada

Orientador: Cleslei Fernando Zanelli / Resumo: O fator de início de tradução 5A (eIF5A) é altamente conservado em arqueas e eucariotos e essencial para a viabilidade celular. eIF5A sofre uma modificação pós-traducional exclusiva e essencial para sua função, em que um resíduo específico de lisina é convertido em uma hipusina. Apesar eIF5A já ter sido relacionado com o início da tradução, uma quantidade crescente de estudos recentes têm estabelecido sua função na etapa de elongação da tradução, mais especificamente na elongação de sequências que são capazes de induzir um stalling (atraso ou parada) do ribossomo. Entretanto, existem ainda poucos trabalhos realizados com perfil proteômico na ausência de função de eIF5A, de maneira que atualmente pouco se sabe sobre as proteínas que têm sua tradução dependente de eIF5A. Desta forma, o presente projeto visa a busca de proteínas que têm sua tradução dependente de eIF5A através da comparação de perfil proteômico entre linhagens selvagens e mutantes de eIF5A em Saccharomyces cerevisiae. Para isto, utilizamos neste trabalho uma estratégia de perfil proteômico celular in vivo por fluorescência de GFP utilizando uma coleção de 4.156 linhagens, cada uma contendo uma ORF diferente em fusão com GFP no C-terminal, e uma proteína RFP (variante E2Crimson) constitutivamente produzida como normalizador, tanto no background selvagem (HYP2) como mutante para eIF5A (hyp2-3). Esta tese apresenta a análise dos dados de GFP/RFP e a validação desta análise utilizando-se western blot. Os resultados ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The translation factor 5A (eIF5A) is conserved and essential for cell viability. This is the only protein known to contain the amino acid residue hypusine, essential for eIF5A function, generated by a post-translational modification. Although it was initially suggested a function for eIF5A in the translation initiation, eIF5A has been demonstrated to have a role in translation elongation. More recent studies have established that eIF5A is necessary for the elongation of specific sequences, which are able to induce a ribosome stalling. Still, there are few studies with proteomic profile in the absence of eIF5A function and the proteins which syntheses are dependent on eIF5A are not well known. Thus, the present study aims to search for the proteins which syntheses are dependent on eIF5A by proteomic profile comparison between wild-type strains and eIF5A mutants in Saccharomyces cerevisiae. We present a proteomic profile for GFP fluorescence using a 4156 collection of strains, each one containing a different ORF fused to the C-terminal GFP and a protein RFP constitutively produced as normalizing, both in the wild and eIF5A mutant background. This thesis presents GFP / RFP data analysis and data validation using western blot. Our data using an in vivo proteome profile of the ORFs-GFP collection in a hyp2-3 mutant background demonstrating that yeast eIF5A shows several mitochondrial proteins downregulated in the eIF5A mutant. To confirm eIF5A involvement with mitochondrial functi... (Complete abstract click electronic access below) / Doutor

Current approaches aimed at improving the efficacy of radiation as a cancer treatment modality involve the development and application of molecularly targeted radiosensitizers, a strategy that requires a thorough understanding of the fundamental processes comprising the cellular radioresponse. Recent data indicating that radiation modifies gene expression primarily through translational control rather than transcriptional events suggests that mRNA translation contributes to cell survival after irradiation. The overall goal of this project is to determine whether the regulatory/rate-limiting components of the translational machinery provide targets for tumor cell radiosensitization. The majority of translation in mammalian cells occurs in a cap-dependent manner and is highly dependent on eIF4E. As such, we investigated a regulatory role for eIF4E in cellular radiosensitivity. eIF4E knockdown enhanced the radiosensitivity of tumor but not normal cells. eIF4E knockdown inhibited the dispersal of radiation-induced γH2AX foci. Furthermore, radiation was found to increase the binding of >1000 unique mRNAs to eIF4E, many involved in DNA replication, recombination, and repair. S6 kinase 1 (S6K1), also an important regulatory component of the translational machinery, enhances the translation of specific mRNA subpopulations, independent from eIF4E, and mediates ribosome biogenesis. The role of S6K1 in determing cell survival after radiation was determined in several tumor cell lines and one normal cell line. S6K1 knockdown enhanced the radiosensitivity of all 3 tumor lines. In contrast S6K1 knockdown had no effect on the cellular radiosensitivity of the one normal line tested. The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. Importantly, it plays a major role in regulating eIF4E availability as well as S6K1 activity. The radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the ATP competitive mTOR inhibitor PP242 were compared. Based on immunoblot analyses, whereas rapamycin only partially inhibited mTORC1 activity and had no effect on mTORC2, PP242 inhibited the activity of both mTOR containing complexes. In the two tumor cell lines evaluated, PP242 treatment 1h before irradiation increased radiosensitivity, whereas rapamycin had no effect. PP242 had no effect on the cellular radiosensitivity of a normal lung fibroblast line. PP242 exposure did not influence the initial level of γH2AX foci after irradiation, but did significantly delay the dispersal of radiation-induced γH2AX foci. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiation-induced tumor growth delay. A next generation analog of PP242, INK128, is currently undergoing analysis in clinical trials. Given our data showing ATP-competitive mTOR inhibition is a strategy for tumor radiosensitization as well as the fact that radiotherapy is a primary treatment modality for locally advanced pancreatic ductal adenocarcinoma, the effects of INK128 on pancreatic cancer radiosensitivity were determined. In three pancreatic cancer cell lines addition of INK128 immediately after radiation resulted in radiosensitization. Consistent with the effects of PP242 on other cell lines, INK128 exposure did not influence the initial level of γH2AX foci after irradiation, but did significantly delay the dispersal of radiation-induced γH2AX foci. Furthermore, in pancreatic tumor xenografts INK128 inhibits mTOR activity as well as cap-complex formation in a time-dependent manner. Lastly, INK128 treatment significantly prolonged the radiation-induced tumor growth delay of pancreatic tumor xenografts. In summary, the data provided in this thesis have begun to characterize the role of the translational machinery in determining the cellular response to radiation.

Bovine adenovirus (BAdV)-3 is a non-enveloped icosahedral DNA virus, which replicates in the nucleus of infected cells, and is being developed as a vector for vaccination for humans and animals. The genome of BAdV-3 is organized into early, intermediate and late genes and it has thirty three predicted open reading frames (Reddy et al., 1998). The late region of BAdV-3 is divided into seven families (L1-L7) (Reddy et al., 1998). One of the proteins expressed in the L-6 region encodes a protein called pVIII, which is a minor capsid protein connecting the core with the inner surface of the capsid. The objective of the current study was to characterize pVIII protein of BAdV-3 and to examine its role in the life cycle of BAdV-3. Anti-pVIII serum detected a protein of 24 kDa at 12-48 hr post infection and an additional protein of 8 kDa at 24-48 hr post infection. While a 24 kDa protein is detected in empty capsids, only the C-terminal cleaved protein of 8 kDa is detected in the mature virion suggesting that amino acids 147-216 of conserved C- terminus of BAdV-3 pVIII are incorporated in mature virions. The pVIII protein predominantly localizes to the nucleus of BAdV-3 infected cells utilizing the classical importin α /β dependent nuclear import pathway. Analysis of mutant pVIII demonstrated that amino acids 57-72 of the conserved N-terminus bind to importin α-3 with high affinity and are required for the nuclear localization. Detection of hexon associated with both, precursor (24 kDa) and cleaved (8 kDa) form of pVIII suggests that the C-terminus of pVIII interacts with Hexon. Based on yeast II hybrid screening assay, we identified the cellular protein DDX3 as an interacting protein partner of pVIII. Earlier, targeting of DDX3 by few viral proteins has defined its role in mRNA transport (Yedavalli et al., 2004) and induction of interferon production (Schroder et al., 2008; Wang et al., 2009). Here, we provide evidence regarding the involvement of DDX3 in cap dependent cellular mRNA translation and show that targeting of DDX3 by the adenovirus pVIII protein abolishes cap-dependent mRNA translation function of DDX3 in virus infected cells. Adenovirus late protein pVIII interacts with DDX3 in transfected and bovine adenovirus (BAdV-3) infected cells. pVIII inhibited capped mRNA translation in-vitro and in-vivo by limiting the amount of DDX3 and eIF3. Diminished amount of DDX3 and eIFs including eIF3, eIF4E and PABP were present in cap binding complex in BAdV-3 infected or pVIII transfected cells with no trace of pVIII in the cap binding complex. The total amount of eIFs appeared similar in uninfected or BAdV-3 infected cells. The co-immunoprecipitation experiments indicated the absence of direct interaction between pVIII and eIF3, eIF4E or PABP. These data indicate that interaction of pVIII with DDX3 depletes eIF3, eIF4E and PABP from the cap-binding complex. We conclude that DDX3 promotes cap-dependent cellular mRNA translation and BAdV-3 pVIII inhibits translation of capped cellular mRNA by excluding functional cap-binding complex from the capped cellular mRNA. BAdV-3 infection of DDX3 positive cells significantly inhibits cellular protein synthesis at late times post-infection. Interestingly, knockdown of DDX3 resulted in significant reduction in virus yield and expression of BAdV-3 late proteins at late times post-infection. Our results suggest that selective translation of BAdV-3 late mRNAs observed at late time post-infection of DDX3 positive cells is abrogated in DDX3 knock down cells. Moreover, the reduction in the extent of protein synthesis is evidenced by less functional 80S and polysomes in pVIII expressing plasmid transfected cells. Alternatively, DDX3 and pVIII binds to BAdV-3 tripartite leader (TPL) and the translation of mRNAs containing TPL at their 5’ ends is enhanced in the presence of pVIII and DDX3 proteins. From this observation, we concluded that pVIII and DDX-3 might promote the translation of late viral mRNAs by interacting with TPL.

Gene expression at the level of mRNA translation is critical for cells to respond to external signals; it allows changes in protein synthesis without triggering transcription of a new set of genes. Control of mRNA translation and stability is important in several cellular processes including cell growth and differentiation. Thus regulation of the cellular machinery involved in mRNA translation is crucial. Poly (A) binding protein (PABP1), eukaryotic elongation factor 1A (eEF1A) and ribosomal protein S6 (RPS6) are important members of the cellular mRNA translation machinery, the mRNAs that encode these proteins belong to the terminal oligo pyrimidine tract (TOP) containing family. Translation of the TOP mRNAs is regulated by growth signals and usually codes for several proteins involved in mRNA translation. Our laboratory has previously reported up regulation of PABP1 mRNA translation during recovery from heat shock. It was also shown that the terminal oligopyrmidine tract (TOP) cis-element of PABP1 mRNA is responsible for the preferential increase of PABP1 mRNA translation; however the mechanism for achieving this is unknown. In the studies reported here, we showed that translation of eEF1A and RPS6 expression was similarly enhanced during recovery from heat shock. Analyses of samples of in vivo cross linked RNA– protein complexes, immunoprecipitated by ZNF9 antibody, for the presence of specific mRNAs showed that the cellular nucleic acid binding protein ZNF9 binds not only to TOP mRNAs but also mRNA that lack the TOP element such as to β-actin mRNA. To elucidate the mechanism of activation of TOP mRNA translation, as a candidate trans acting factor, siRNA was used to deplete the cellular level of ZNF9 from heat shocked HeLa cells to examine its potential role in stimulation of TOP mRNA translation during recovery from heat shock. Results show that the knock down of ZNF9 disallowed the preferred stimulation of PABP1, eEF1A and RPS6 expression during recovery from heat shock. There was no detectable effect on the constitutive expression of either β-actin or PABP1, eEF1A and RPS6 in exponentially growing HeLa cells. These results suggest that binding of ZNF9 to TOP mRNAs per se does not inhibit translation, but more likely it acts as a general facilitator of mRNA translation. It is possible that modification of the interaction between ZNF9 with other unknown protein factors is responsible for its preferred effect on all three TOP mRNAs studied here. Additionally, results also suggest that a different TOP sequences amongst the observed TOP mRNAs responds similarly to ZNF9.

Adaptation to variations in oxygen concentration is a conserved mechanism in all metazoans as this process is central for the maintenance of cell and tissue homeostasis. Two major and highly conserved processes contribute to hypoxia-induced gene reprogramming. The first one relies on the transcriptional activation of gene expression by the Hypoxia Inducible Factors (HIFs) leading to the upregulation of a large panel of genes. The second one corresponds to a strong modification of the translation program. While the mechanisms underlying HIF-dependent transcriptional activation have been well characterized, the ones governing translation reprogramming are only partially understood. 
To uncover how mRNA translation takes place at low oxygen tension, we used Drosophila as our research model since both Drosophila flies and S2 cells are highly resistant to low O2. We first demonstrate that several genes are efficiently translated under hypoxia in Drosophila S2 cells. By a gene reporter-based approach, we demonstrate that Ldh mRNA 3’UTR is sufficient to promote reporter mRNA association to polysomes in hypoxia. A deletion analysis of Ldh 3’UTR leads to the identification of a ACAAA-rich sequence important for polysomal association and translation in hypoxia.Cap-binding factors play a key role in controlling translational initiation. We have shown that the cap-binding translation initiation factor eIF4EHP (4EHP) plays a dual role on translation under hypoxic conditions. Despite having a general repressive function on global translation under normoxic and hypoxic conditions, we demonstrated that 4EHP also positively controls the translation of specific mRNAs under hypoxia. Inactivation of 4ehp reduces LDH protein synthesis and impairs reporter mRNA translation in hypoxia. Deletion of 4ehp inhibites the translation of several candidate genes harboring a ACAAA motif in 3’UTR under hypoxia, suggesting that 4EHP is required for hypoxic translation of mRNAs carrying ACAAA-rich motifs. Most interestingly, we observed that 4EHP is strongly enriched in polysomal fractions in hypoxia, further supporting a role of this initiation factor in hypoxic translation. The reduction of 4ehp expression also impairs Drosophila development under hypoxic conditions. Taken together, our results indicate that specific mRNAs can bypass the translational blockade imposed by hypoxic conditions. This process is controlled by mRNA 3’UTR “CA” rich element and is positively regulated by the translation initiation factor 4EHP. / L'adaptation aux variations de la concentration en oxygène est un mécanisme conservé chez tous les métazoaires car ce processus est central pour le maintien de l'homéostasie cellulaire et tissulaire. Deux processus hautement conservés contribuent à la reprogrammation génétique induite par l'hypoxie. Le premier repose sur l'activation transcriptionnelle de l'expression génique par les facteurs inductibles de l'hypoxie (HIF) conduisant à l’induction d'un large panel de gènes. Le second correspond à une forte modification du programme traductionnel. Alors que les mécanismes sous-jacents à l'activation transcriptionnelle dépendante de HIF ont été bien caractérisés, ceux qui régissent la reprogrammation de la traduction ne sont que partiellement compris.Pour découvrir comment la traduction de l'ARNm se déroule à faible tension d'oxygène, nous avons utilisé la drosophile comme modèle d’étude, car les mouches Drosophila melanogaster et les cellules S2 issue de cet organisme sont très résistantes à de faibles teneurs en O2. Nous avons tout d’abord démontré que plusieurs gènes sont efficacement traduits en hypoxie dans les cellules S2 de drosophile. Par une approche basée sur l’utilisation de gènes rapporteurs, nous avons démontré que la région 3’ Non traduite (3’UTR) de l’ARNm Ldh est suffisante pour promouvoir l’association de l’ARNm du rapporteur aux polysomes en conditions hypoxiques. Une analyse par délétion de la région 3’UTR de l’ARNm Ldh a conduit à l’identification d’une séquence riche en ACAAA importante pour l’association polysomale et la traduction en hypoxie. La reconnaissance de la coiffe joue un rôle clé dans le contrôle de l'initiation de la traduction Nous avons montré que le facteur d'initiation de la traduction eIF4EHP (4EHP), qui se lie à la coiffe, joue un double rôle sur la traduction dans des conditions hypoxiques. Bien qu'il ait une fonction répressive sur la traduction générale dans des conditions normoxiques et hypoxiques, nous avons démontré que 4EHP contrôle aussi positivement la traduction d'ARNm spécifiques dans des conditions hypoxiques. L'inactivation de 4ehp réduit la synthèse de la protéine LDH et altère la traduction de l'ARNm rapporteur contenant la partir 3’UTR du messager Ldh en hypoxie. La délétion de 4ehp peut atténuer la traduction de plusieurs gènes candidats contenant un motif ACAAA dans leur région 3’UTR 3' en hypoxie, ce qui suggère que 4EHP est nécessaire pour la traduction hypoxique des ARNm portant des motifs riches en ACAAA. De façon intéressante, nous avons observé que 4EHP est fortement enrichi dans les fractions polysomales en hypoxie, ce qui confirme le rôle de ce facteur d'initiation dans la traduction en hypoxie. La réduction de l'expression de 4ehp altère également le développement de la Drosophile dans des conditions hypoxiques. Ensemble, nos résultats indiquent que des ARNm spécifiques peuvent contourner le blocage traductionnel imposé par les conditions hypoxiques. Ce processus est contrôlé par l'élément riche en "CA" situé dans la partie 3'UTR de l’ARNm et est régulé positivement par le facteur d'initiation de la traduction 4EHP. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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La qualité germinative (vigueur) des semences est un caractère agronomique majeur. Elle correspond à la capacité d'un lot de semences à germer de façon rapide et homogène dans une large gamme de conditions environnementales. Cette qualité germinative est notamment contrôlée par une interaction antagoniste entre deux phytohormones, l'acide abscissique (ABA) qui induit et maintient la dormance et les gibbérellines (GAs) qui stimulent la germination et la croissance de la plantule. La dormance, qui correspond à un blocage physiologique de la germination, est un paramètre non souhaitable d'un point de vue agronomique. Par conséquent la compréhension de la régulation hormonale sur la qualité des semences représente un intérêt fort pour la communauté scientifique mais aussi pour les acteurs de la filière "semences". De nombreuses études ont démontré l'existence d’une importante régulation de la synthèse des protéines au cours de l’imbibition des graines. Cette régulation traductionnelle contribuerait à la mise en place des programmes métaboliques différents en fonction de l’état physiologique des semences pour maintenir un état de dormance ou initier le processus de germination.Le travail réalisé dans le cadre de cette thèse s’est concentré à apporter des éléments nouveaux sur le rôle de l’ABA dans la détermination de la qualité physiologique des semences au cours du développement de la graine et au cours de la germination. L’impact de l’ABA a été particulièrement décortiqué à l’aide de mutant d’Arabidopsis thaliana présentant des teneurs en ABA endogènes contrastées. Par une approche multi-omique combinant des analyses transcriptomiques, protéomiques et métabolomiques, nous avons étudié les mécanismes moléculaires et biochimiques associées avec la mise en place de la qualité physiologique des semences en relation avec l’ABA. Nos résultats ont montré qu’au-delà du contenu en ABA, l’origine tissulaire de cette hormone dans les graines gouverne de nombreux réarrangements métaboliques qui participent au déterminisme de la profondeur de dormance et de la vigueur germinative. Il apparaît un lien entre l’ABA et l’activité traductionnelle, étroitement associé au métabolisme énergétique et à l’homéostasie RedOx.L’effet de l’ABA sur l’activité traductionnelle a été suivi par une adaptation des méthodes SILAC (stable-isotope labelled amino acids in cell culture) aux grains d’Arabidopsis. Cette technique a été utilisée pour décrire la dynamique du protéome dépendante du contenu en ABA des graines au cours de leur imbibition. Nos résultats montrent que cette approche originale permet d’enrichir les connaissances sur la biologie fondamentale des semences. En effet, nous avons montré que l’ABA est un régulateur clé de la synthèse protéique dans les graines et est un contributeur majeur dans la mise en place des différents programmes traductionnels. Cette approche a montré que l’ABA exerce un contrôle sur la traduction de plus de 400 ARNm au cours de l’imbibition des graines et ouvre de nouvelles pistes pour la compréhension de la régulation de la synthèse protéique chez les semences et chez les plantes. Ces données générées offrent un nouveau regard sur le processus germinatif et de sa régulation par l’ABA.Sur la base des données existantes au laboratoire et celles générées au cours de cette thèse, nous avons également développé une utilisation de bio-marqueurs pour l’évaluation de la qualité des semences et nous avons mis au point des traitements de semences innovants. Ces technologies ont été développé en accord avec les attentes des industriels de la filière « semences ». La récente obtention d’un financement pour ce projet de recherche appliquée démontre la complémentarité des recherches effectuées au sein du laboratoire avec les besoin des industriels de la filière « semences ». / Germination vigor is a main concern in agriculture. High seed vigor is defined as the capacity of a seed lot to germinate rapidly, uniformly and in a wide range of environmental conditions. Seed quality is controlled by a dynamic balance between two antagonistic hormones, abscisic acid (ABA), which induces and maintains dormancy and gibberellins (GAs), which stimulate seed germination and seedling establishment. Seed dormancy corresponds to a block to the completion of germination and is an undesirable characteristic from an agronomic point of view. Thus, investigation of seed quality toward a better understanding of hormonal regulation is of fundamental concern for scientific community and seed industry.Recent studies have highlighted the intensive regulation of protein synthesis during seed germination. Translational regulation would govern the implementation of different metabolic programs during seed imbibition in order to maintain seed dormancy or to initiate the germination process. In this thesis, we explore the role of ABA in the control of germination quality during seed development and seed germination, using Arabidopsis thaliana mutant displaying contrasted ABA content.By combined “omic” approaches, we have highlighted the impact of ABA level on metabolic rearrangements during seed maturation. Our results showed that ABA origin in the seeds governs many metabolic rearrangements controlling dormancy depth and germination vigor. In addition, the present work suggests an intimate linkage between translational activity and ABA content, in association with energetic pathways and redox homeostasis.The impact of ABA on proteome turnover during seed germination was studied by adapting a metabolic labeling of neosynthesized proteins based on SILAC methods (stable isotope labelled amino acids in cell culture) to Arabidopsis seeds. Our results suggest that ABA is a key regulator of protein synthesis and modulates metabolic changes during seed imbibition. Indeed, this novel approach has highlighted that ABA controls the translation of more than 400 mRNAs during seed imbibition. This work provides an original perspective on the contribution of ABA and mRNA translation in seed germination and provides a valuable basis for further investigation of translational regulation in seeds and in plants.Based on existing data and those generated during this thesis, we also developed innovative seed treatments and new biomarkers for seed quality assessment. Recent funding for a maturation program dedicated to improve these biotechnologies demonstrates that our research meets the needs of seed industry.

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