<p>The activation and maintenance of plant immune responses require a significant amount of energy because they are accompanied by massive transcriptional reprogramming. Spurious activation of plant defense machinery can lead to autoimmune diseases and growth inhibition. So it is important for plants to tightly regulate the immune system to ensure the balance between growth and defense. However, neither the molecular mechanisms nor the design principles of how plants reach this balance are understood. </p><p>In this dissertation work, I showed how transcriptional and translational control of plant immune system can help avoid the constant immune surveillance and elicit a proper level of defense responses when necessary. These fine tunings of the immune system ensure the balance between growth and defense. </p><p>My research on the transcriptional regulation of plant defense responses led to the surprising discovery that even without pathogen, plant can 'anticipate' potential infection according to a circadian schedule under conditions that favor the initiation of infection. Functional analysis of 22 novel immune components unveiled their transient expression at dawn, when the infection is most likely to happen. This pulse expression pattern was shown to be regulated by the central circadian oscillator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) since these 22 genes are no longer induced in the cca1 mutant. Moreover, the temporal control of the transcription level of these 22 immune genes by CCA1 also fine tunes their expression pattern according to the perceptions of different pathogenic signals. At the basal defense level, the expression of these genes can be transiently induced upon perceptions of critical infection stages of the pathogen. When an elevated level of defense response is needed, the high expression levels of these genes are maintained to confer a stronger immunity against pathogen. Since this stronger form of defense may also cause the suicidal death of the plant cells, the interplay between the circadian clock and defense allows a better decision on the proper level of the immunity to minimize the sacrificial death. The circadian clock is also known to regulate the growth-related cellular functions extensively. So the circadian clock can help to balance the energy used in growth and defense through transcriptional regulation on both sides.</p><p>Besides the integrated control by the circadian clock, the translational control on a key transcription factor involved in the growth-to-defense transition can also maintain the balance between growth and defense.TBF1 is a major transcription factor that can initiate the growth-to-defense transition through transcriptional repression of growth-associated cellular functions and induction of defense-related machinery. Bioinformatics studies identified 2 upstream open reading frames (uORFs) encoding multiple phenylalanine at 5' of the translation initiation codon of TBF1. Under normal conditions, these 2 uORFs can repress the translation of TBF1 to prevent accidental activation. However, pathogen infection may cause rapid and transient depletion of phenylalanine, a well-known precursor for cell wall components and the SAR signal SA. This depletion signal can be reflected by the increase of uncharged tRNAPhe, which subsequently leads to the phosphorylation of eIF2á and the release of uORFs' repression on TBF1. These findings provided the molecular details of how uORF-based translational control can couple transcriptional reprogramming with metabolic status to coordinately trigger the growth-to-defense transition. </p><p>In summary, my dissertation work has identified previously unrecognized regulatory mechanisms by which plant immune responses are balanced with growth. These new findings will further investigations into these novel interfaces between plants and pathogens. Future studies will definitely further improve our understandings of the plant-microbe interactions.</p> / Dissertation

Chez les eucaryotes, les ARNm cellulaires subissent une traduction dépendante de la coiffe qui nécessite des facteurs appelés eIFs pour produire des protéines, mais les ARNm Hox sont traduits dans un mécanisme non canonique en raison de deux régulateurs d'ARN dans l'élément 5'UTR appelé Internal Ribosome Entry Site (IRES) qui recrute le ribosome sans le besoin de coiffe, et un élément inhibiteur de traduction (TIE) qui empêche la translation dépendant de coiffe. L'objectif de ma thèse est de déchiffrer le mécanisme de deux éléments TIE a3 et a11 dans les ARNm Hox. Pour cela, nous avons utilisé le système de traduction sans cellules RRL. Notre modèle pour TIE a3 suggère qu'il inhibe la traduction en uORF qui se traduit par un ARNm Hox a3 UTR 5'UTR de pleine longueur et produit un peptide de 9 KDa avec l'implication de eIF2D, un facteur d'initiation non canonique indépendant du GTP. Pour TIE a11, il séquestre le ribosome 80S sur une combinaison de codons start-stop à 19 nucléotides en amont d'une structure de boucle de tige riche en GC qui bloque le 80S au codon stop. / In eukaryotes, cellular mRNAs undergo cap-dependent translation which requires factors called eIFs to produce proteins.However, Hox mRNAs are translated in a non-canonical mechanism due to two RNA regulons in the 5’UTR called Internal Ribosome Entry Site (IRES) element which recruits the ribosome without the need of a cap, and a Translation Inhibitory Element (TIE) which inhibits cap-dependent translation. The objective of my PhD is to decipher the mechanism of two TIE elements a3 and a11 in Hox mRNAs. For that, we used RRL cell-free translation system. Our model for TIE a3 suggests that it inhibits translation to a uORF which translates through full length 5’UTR Hox a3 mRNA and produces a peptide of 9 KDa with the involvement of eIF2D, a non-canonical GTP-independent initiation factor. For TIE a11, it sequesters 80S ribosome on a start-stop codon combination at 19 nucleotides upstream of a GC-rich stem loop structure which blocks the 80S at the stop codon.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphorylation of eukaryotic initiation factor 2 (eIF2) is an important mechanism regulating global and gene-specific translation during different environmental stresses. Repressed global translation by eIF2 phosphorylation allows for cells to conserve resources and elicit a program of gene expression to alleviate stress-induced injury. Central to this gene expression program is eIF2 phosphorylation induction of preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in stress remediation, a pathway referred to as the Integrated Stress Response (ISR). We investigated whether there are additional transcription factors whose translational expression is regulated by eIF2 kinases. We found that the expression of the transcriptional regulator ATF5 is enhanced in response to many different stresses, including endoplasmic reticulum stress, arsenite exposure, and proteasome inhibition, by a mechanism requiring eIF2 phosphorylation. ATF5 is regulated by translational control as illustrated by the preferential association of ATF5 mRNA with large polyribosomes in response to stress. ATF5 translational control involves two upstream open reading frames (uORFs) located in the 5′-leader of the ATF5 mRNA, a feature shared with ATF4. Mutational analyses of the 5′-leader of ATF5 mRNA fused to a luciferase reporter suggests that the 5′-proximal uORF1 is positive-acting, allowing scanning ribosomes to reinitiate translation of a downstream ORF. During non-stressed conditions, when eIF2 phosphorylation is low, ribosomes reinitiate translation at the next ORF, the inhibitory uORF2. Phosphorylation of eIF2 during stress delays translation reinitiation, allowing scanning ribosomes to bypass uORF2, and instead translate the ATF5 coding region. In addition to translational control, ATF5 mRNA and protein levels are significantly reduced in mouse embryo fibroblasts deleted for ATF4, or its target gene, the transcriptional factor CHOP. This suggests that ISR transcriptional mechanisms also contribute to ATF5 expression. To address the function of ATF5 in the ISR, we employed a shRNA knock-down strategy and our analysis suggests that ATF5 promotes apoptosis under stress conditions via caspase-dependent mechanisms. Given the well-characterized role of CHOP in the promotion of apoptosis, this study suggests that there is an ATF4-CHOP-ATF5 signaling axis in the ISR that can determine cell survival during different environmental stresses.

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.

La protéine Vif du VIH-1 contrecarre le facteur de restriction APOBEC3G (A3G) en diminuant son niveau d'expression dans les cellules infectées. Ceci est mis en œuvre entre autres par l'inhibition de sa traduction, un mécanisme encore peu compris. La première partie de ma thèse contribue à la caractérisation d'une petite ORF (uORF) qui se situe dans la 5'-UTR de l'ARNm d'A3G et d'A3F en amont de leurs ORF respectives. Cette uORF s'est révélée cruciale pour la régulation de la traduction d'A3G en présence et absence de Vif. Dans la deuxième partie de cette thèse, différents protocoles ont été mis en œuvre pour identifier les protéines associées avec l'ARNm d'A3G, qui pourraient jouer un rôle dans le mécanisme d'inhibition traductionnelle d'A3G par Vif. Ainsi, plusieurs protéines ont été identifiées dont la présence sur l'ARNm d'A3G semble modulée par Vif. / The HIV-1 Vif protein counteracts the restriction factor APOBEC3G (A3G) by downregulating its expression level in infected cells. This is achieved in different ways, one of which is translational inhibition, a mechanism that is still poorly understood. The first part of my thesis contributes to the characterization of a small upstream ORF (uORF), that is found in the 5'-UTR of A3G and A3F mRNAs. This uORF has been found to be crucial for regulation of A3G translation and is necessary to allow Vif-mediated translational inhibition. In the second part of this thesis, different protocols have been set up in order to identify A3G mRNA-associated cellular proteins which might play a role in the mechanism of Vif-mediated translational inhibition. Several proteins, whose presence on A3G mRNA seems to be modulated by Vif have been identified.

Translation control is crucial during virus-host interaction, in which the host relies on the translation machinery to mount an antiviral response or induce the inflammation response to reduce virus spread, while the virus aims to take control of this system to thwart the host defense while producing viral progeny. The field of oncolytic virus (OV) therapy relies on replicating, engineered viruses that preferentially infect tumor cells to induce direct oncolysis or promote an antitumor immune response. Despite the importance of translation control in virus-host interaction, not much has been described on the interaction at the translation level between OV and cancer cells. I propose that this knowledge gap could reveal significant improvements in OV efficacy in treating cancer. In my first study, I set out to characterize the translatome of an infection-resistant breast cancer cell line infected by three clinically advanced OVs to identify residual antiviral activity in cancer cells regulated by translation control. I found the inositol phosphatase Inpp5e to be a novel antiviral gene that is translationally induced during infection via a transcript variant shift. Mechanistically, I showed that the majority of Inpp5e transcripts in uninfected cells contain a long 5’ UTR that harbor four translationally inhibitory upstream reading frames (uORF). Yet, OV infection induced the expression of a shorter 5’ UTR with a spliced intron that removes three uORFs, derepressing the translation of Inpp5e mRNA. CRISPR-Cas9 knockout of Inpp5e also enhanced the infectivity of oncolytic HSV1 and VSV. My study suggests the existence of a class of translationally regulated antiviral genes in cancer cells. In my second study, I sought to adapt the translation of transgenes to the unique translation condition imposed by the infecting virus via the incorporation of a viral 5’UTR. I identified HSV1 5’UTRs by locating the transcription start site of most HSV1 genes using RNA-seq data, then determined the 5’UTR of US11 as a potent translation enhancer during HSV1 infection. Incorporation of this 5’UTR into the transgene expression cassette inserted into the HSV1 genome enhanced transgene expression significantly at the translation level. In my third study, I set out to explore the mechanism of miR-223 mediated inflammation inhibition. miR-223 is a protective miRNA in the context of atherogenesis via suppressing inflammatory signaling. Using transcriptome and translatome profiling (RNA-seq and Ribo-seq), I found that the inhibitory effect of miR-223 on inflammation occurs primarily at the translation level. Overall, my work highlights the importance of translation control in OV-cancer cells interaction, as well as in inflammation-related diseases.

No description available.

Evolutionär konservierte, kleine offene Leserahmen (upstream open reading frames, uORFs) sind translational aktive Kontrollelemente, die bevorzugt in Boten-Ribonukleinsäuren von Schlüsselgenen zur Regulation von Zellwachstum, Proliferation und Differenzierung vorkommen. In dieser Arbeit wurden Mäuse analysiert, die defizient für das uORF Initiationscodon des Transkriptionsfaktors CCAAT/enhancer binding protein beta (C/EBPbeta-Delta-uORF) sind. Proteinanalysen verschiedener Gewebe zeigten, dass C/EBPbeta-Delta-uORF Mäuse im Gegensatz zu Wildtyptieren nicht in der Lage sind, die kurze, auto-antagonistische C/EBPbeta LIP Isoform zu induzieren. Die verminderte LIP Expression verursachte eine gestörte Differenzierung knochenabbauender Osteoklasten und ging mit einer Zunahme von mineralisiertem Knochengewebe in C/EBPbeta-Delta-uORF Mäusen einher. Nach partieller Hepatektomie führte der Verlust der uORF-vermittelten Induktion von LIP in regenerierenden C/EBPbeta-Delta-uORF Lebern zu einer Überaktivierung C/EBPbeta-regulierter Akute Phase Gene. Im Vergleich zum Wildtyp wiesen Hepatozyten von C/EBPbeta-Delta-uORF Tieren einen verzögerten und abgeschwächten Wiedereintritt in die S-Phase des Zellzyklus auf. Genomweite Genexpressionsanalysen zeigten, dass die verminderte S-Phase Aktivität in regenerierenden C/EBPbeta-Delta-uORF Lebern mit einer persistierenden Repression von Zellzyklusgenen korrelierte, wobei insbesondere die verminderte Expression zahlreicher E2F-regulierter Gene auffällig wurde. Chromatinimmunpräzipitations- und Reportergenexperimente führten zur Entwicklung eines mechanistischen Modells, das eine isoformspezifische C/EBPbeta-Koregulation E2F-kontrollierter Zellzyklusgene vorschlägt. Die Analyse der C/EBPbeta-Delta-uORF Mäuse belegt erstmals die Funktionalität der uORF-gesteuerten translationalen Kontrolle im Säugetier und weist auf eine entscheidende Bedeutung dieses Kontrollmechanismus bei zahlreichen physiologischen und pathopysiologischen Prozessen hin. / Evolutionary conserved small upstream open reading frames (uORFs) are translational control elements predominantly prevalent in the 5'' mRNA regions of key regulatory genes of growth, proliferation, and differentiation. This thesis comprises the evaluation of mice deficient for the uORF initiation codon of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta-Delta-uORF). Protein analysis of various tissues demonstrated that C/EBPbeta-Delta-uORF mice, in contrast to wildtype control animals (C/EBPbeta-WT), fail to induce translation of the truncated, auto-antagonistic C/EBPbeta LIP isoform. The reduced expression of LIP was associated with impaired differentiation of bone resorbing osteclasts and resulted in an increased bone volume of C/EBPbeta-Delta-uORF mice. After partial hepatectomy the loss of uORF-mediated LIP induction resulted in super activation of acute phase response genes in regenerating livers. Furthermore, C/EBPbeta-Delta-uORF hepatocytes showed a delayed and blunted re-entry into the cell cycle after partial hepatectomy as compared to C/EBPbeta-WT animals. Genome-wide transcript expression analyses revealed that the reduced S-phase activity in regenerating C/EBPbeta-Delta-uORF livers correlated with a persistent repression of cell cycle regulatory genes and showed a remarkable underrepresentation of genes regulated by the E2F family of transcription factors. Chromatinimmunoprecipitations and luciferase reporter gene assays allowed the development of a mechanistic model that suggests C/EBPbeta isoform-specific co-regulation of E2F-controled cell cycle genes. The analysis of C/EBPbeta-Delta-uORF mice validates the functionality of uORF-mediated translational control in vertebrates and suggests a comprehensive role of uORF regulation in physiology and the etiology of disease.

La traduction est considérée comme une étape clé de l'expression des gènes permettant à la cellule de s'adapter aux variations de son environnement en réponse aux signaux internes ou externes. Des études bioinformatiques ont montrés que la moitié des ARN messagers chez l'homme portent, en amont de leur phase codante, des éléments régulateurs appelés uORF. Le laboratoire a montré qu'un défaut de terminaison de la traduction par déplétion du facteur de terminaison eRF3 modifie l'expression de gènes dont l'ARNm contient des uORF comme le gène ATF4. Cette modification se fait soit par un mécanisme de réinitiation après traduction de l'uORF soit par une augmentation de la stabilité de l'ARNm résultant d'un défaut de sa dégradation par la voie du "Nonsense-mediated mRNA Decay" (NMD). A travers leur association dans le même complexe et leur implication dans la terminaison de la traduction et la NMD, eRF3 et Upf1 contribuent à la régulation fine de l'expression des gènes. Cependant, on ne sait pas dans quelle mesure ces deux facteurs affectent la traduction et la stabilité des ARNm. Nous avons évalué la traduction par ribosome profiling et le taux de transcrits par RNA-seq dans les cellules humaines déplétées en eRF3 ou en Upf1. Ces analyses nous ont permis de dresser une carte des uORF traduites dans le transcriptome des cellules humaines HCT116. Nous avons également observé que peu de gènes cibles sont communs entre la déplétion en eRF3 ou en Upf1. Nos résultats appuient fortement l'hypothèse qu'il y a au moins deux classes de transcrits portant des uORF, l'une dont la régulation implique la terminaison de la traduction et l'autre dont la régulation implique la NMD. / Regulation of gene expression at the translational level is increasingly being recognized as a key mechanism by which cells can rapidly change their gene expression pattern in response to internal or external stimuli. Bioinformatic studies revealed that half of human transcripts present at least one expression regulatory element uORF in the 5’ leader sequence preceding the main ORF. We have previously shown that translation termination disruption caused by eRF3a depletion induces upregulation of the transcriptional activator ATF4 and its targeted genes partly by a translational control at uORFs, and partly in relation to a defect in Nonsense-mediated mRNA Decay activation, increasing ATF4 mRNA stability. Through their physical association and their involvement in translation termination and NMD, eRF3 and Upf1 are regulating the protein and mRNA levels of a significant number of genes and thus contribute to the fine-tuning of their expression. It is not known yet, in what extent both of these factors affect translational control and what is the subset of genes that are regulated by these factors. In this study, we evaluated translation by ribosome profiling and mRNA level by RNA-seq in human cells subjected to either eRF3a or Upf1 depletion. These analyses allowed us to draw a transcriptome-wide map of uORFs and obtained a list of functional uORFs in our reference HCT116 transcriptome. We also observe that only a small fraction of these are common targets for both eRF3a and Upf1. Our results provide strong support for the notion that different classes of transcripts bearing uORFs are regulated either by translational processes involving translation termination or by NMD.

Multidrug resistance protein 2 (MRP2) is the second member the C subfamily in the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) efflux transporters. MRP2 is a critical player for generation of bile acidindependent bile flow and biliary excretion of glutathione, glucuronate and sulfate conjugates of endo- and xenobiotics. Dysfunctional expression of MRP2 is associated with Dubin-Johnson Syndrome. Pathological and physiological states or xenobiotics change the MRP2 expression level. Under some conditions, expression of the human MRP2 and rat Mrp2 proteins are regulated at the translation level. There are several transcription initiation sites in MRP2/Mrp2 gene. The 5’ untranslated regions (5’UTRs) of MRP2/Mrp2 contains multiple translation start codons. The focus of this study, therefore, was investigation of the translational regulatory mechanisms mediated by the upstream open reading frames (uORF) of MRP2/Mrp2. Using in vitro translation assays and transient cotransfection assays in HepG2 cells, we showed that the rat uORF1 starting at position -109 (relative to the ATG of Mrp2) and the human uORF2 starting at position -105 (relative to the ATG of MRP2) are two major cis-acting inhibitors of translation among the rat and human multiple uORFs, respectively. Translational regulation mediated by the uORFs in the rat Mrp2 mRNA is a combined effect of the leaky scanning model and the reinitiation model, and also results from interaction of the multiple uORFs. In addition, by Ribonuclease Protection Assays (RPA), we detected multiple transcription initiation sites of MRP2/Mrp2 gene in tissues. We also found that the relative abundance of the rat Mrp2 mRNA isoforms with different 5’UTRs differed in the rat liver, kidney, jejunum, ileum, placenta, and lung. This is the first study on the translational regulatory mechanisms of the MRP2/Mrp2 gene.