Wnt proteins are crucial for development/homeostasis by controlling cell fate including apoptosis (Moon RT et al. 1997). Three humanWnt13 isoforms were identified: the secreted Wnt13A, mitochondrial Wnt13B, and nuclear Wnt13C forms; and nuclear Wnt13 had an increased sensitivity to LPS/TNF-induced apoptosis in primary endothelial cells (EC); both Wnt13B and C mRNA contain two start codons (AUG+1 and +74), but the same protein encoded from AUG+74 by Wnt13C was expressed lower than Wnt13B (Struewing IT et al.2006). We hypothesize that during EC apoptosis, the nuclear Wnt13C expression is regulated translationally; nuclear Wnt13 favors apoptosis through regulating the activity/expression of apoptosis-related factors; Wnt13 isoforms may have differential effects on EC apoptosis and apoptosis-related factors. 1. The protein levels, but not the mRNA levels of Wnt13C were induced by apoptosis-inducers. And the Myc-tag insertion at the AUG+1 in Wnt13C mRNA inhibited its expression, indicating the RNA sequences/structures are critical. Therefore, nuclear Wnt13C is regulated during apoptosis at translational levels. 2. Nuclear Wnt13 increased caspase-3/7 expression with/without LPS, followed by an increase in LPS-induced caspase-3/7 cleavage; and nuclear Wnt13 upregulated the pro-apoptotic Bcl-2 family member Bim expression, suggesting that nuclear Wnt13 increased caspase activation through upregulating caspase and Bim expression. Wnt13 isoforms increased EC apoptosis with different strengths: nuclear > mitochondrial > secreted forms. 3. Both caspase-3 and Bim are FOXO target genes; and nuclear Wnt13 increased the nuclear localization of FOXOs, suggesting increased FOXO activity. Nuclear Wnt13 also upregulated SOD2, another FOXO target gene related to oxidative stress-resistance. Nuclear Wnt13 did not increase FOXO activity at the SOD2 promoter, but increased the SOD2-intron 2 element luciferase activity upon LPS, where a novel putative FOXO site was found, implying intron 2 may be responsible for enhanced SOD2 transcription by nuclear Wnt13. Altogether, our results pinpoint the interplay between the expression and functions of Wnt13 forms during EC apoptosis, forming a positive cycle further facilitating the apoptotic program completion, which is important for EC homeostasis.

<p>mRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.</p> / Dissertation

The inner membrane of mitochondria harbors the complexes of the respiratory chain and the ATP synthase, which perform the key metabolic process oxidative phosphorylation. These complexes are composed of subunits from two different genetic origins: the majority of constituents is synthesized on cytosolic ribosomes and imported into mitochondria, but a handful of proteins, which represent core catalytic subunits, are encoded in the organellar DNA and translated on mitochondrial ribosomes. Using yeast as a model organism, I investigated the mitochondrial ribosomal tunnel exit, the region of the ribosome where the nascent chain emerges and that in cytosolic ribosomes serves as a platform to bind biogenesis factors that help the newly synthesized protein to mature. This study provided insights into the structural composition of this important site of mitochondrial ribosomes and revealed the positioning of Cbp3 at the tunnel exit region, a chaperone required specifically for the assembly of the bc1 complex. In my further work I found that Cbp3 structurally and functionally forms a tight complex with Cbp6 and that this complex exhibits fundamental roles in the biogenesis of cytochrome b, the mitochondrially encoded subunit of the bc1 complex. Bound to the ribosome, Cbp3-Cbp6 stimulates translation of the cytochrome b mRNA (COB mRNA). Cbp3-Cbp6 then binds the fully synthesized cytochrome b, thereby stabilizing and guiding it further through bc1 complex assembly. The next steps involve the recruitment of the assembly factor Cbp4 to the Cbp3-Cbp6/cytochrome b complex and presumably acquisition of two redox active heme b cofactors. During further assembly Cbp3-Cbp6 is released from cytochrome b, can again bind to the ribosome and activate further rounds of COB mRNA translation. The dual role of Cbp3-Cbp6 in both translation and assembly allows the complex to act as a regulatory switch to modulate the level of cytochrome b synthesis in response to the bc1 complex assembly process. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Vif (Viral Infectivity Factor) est une protéine auxiliaire qui augmente le «fitness» viral dans l’hôte infecté. Vif est essentielle à la formation de particules virales infectieuses dans les cellules dites «non-permissives», alors que des virus ΔVif se répliquent efficacement dans des lignées cellulaires T dites « permissives ». Les cellules non-permissives expriment les facteurs de restriction APOBEC3G (APOlipoprotein B mRNA-Editing enzyme Catalytic polypeptide 3G ou A3G) et A3F, deux cytidine désaminases dont l’action hypermutatrice est létale pour le virus. Vif réduit de façon considérable le taux d’expression des protéines A3G/3F par deux mécanismes principaux : (1) en recrutant une E3 ubiquitine ligase, Vif induit la dégradation d’A3G par le protéasome et (2) en se fixant sur l’ARNm, Vif régulant négativement la traduction d’A3G par un mécanisme dépendent de la région 5'-UTR. L'objectif de mon projet a été de déterminer le rôle et le mécanisme de la régulation traductionnelle du facteur de restriction A3G par la protéine Vif du VIH-1 ex vivo. En parallèle, nous nous sommes intéressés à déterminer les domaines de la protéine Vif impliqués dans l’activité chaperonne d'ARN. Par l’analyse des « westerns blots » issus de co-transfections de différents vecteurs d’expression d’A3G en présence ou absence de Vif et d’un dominant négative de la Cullin 5, nous avons démontré ex vivo que Vif requiert les tiges boucles 2 et 3 de la région 5’UTR (de façon simultanée) pour inhiber la traduction d’A3G. La régulation traductionnelle d’A3G par Vif cause 50% de la réduction total d’A3G en présence de Vif. Ensuite, nous avons observé qu’une une petite uORF (Upstream Open Reading Frame), contenue entre ces deux tiges-boucles est nécessaire pour l’inhibition d’A3G par Vif. Les uORFs, présents dans 50 % des gènes eucaryotes, interviennent principalement dans des mécanismes de régulation traductionnelle. Ainsi, nous avons observé in vitro et ex vivo que l’uORF régule négativement la traduction de l’ORF majeure d’A3G. Par l’analyse de l’expression de différents mutants de l’uORF, nous avons démontré ex vivo que 60% des complexes d’initiation de la traduction synthétisent A3G par « leaky scanning » et 40 % sont recrutés dans la traduction de l’uORF, en inhibant ainsi l’expression d’A3G. A partir de ces résultats nous proposons un modèle de l’inhibition d’A3G par Vif. Dans ce modèle, Vif pourrais inhiber l’étape de terminaison de la traduction de l’uORF en causant un « stalling » des ribosomes en empêchant ainsi à des nouveaux complexes d’initiation d’attendre l’ORF majeur. Nous avons aussi déterminé l’impact de la régulation traductionnelle d’A3G par Vif sur l’incorporation d’A3G dans les particules virales et sur l’infectivité virale. Nous avons alors observé que l’inhibition traductionnel d’A3G par Vif réduit l’incorporation d’A3G dans les particules virales avec un profil de diminution d’A3G similaire à celui observé dans les cellules. En utilisant des cellules indicatrices TZM-bl, nous avons ensuite observé que l’inhibition traductionnelle d’A3G par Vif augmente l’infectivité virale de 50%. Finalement, nous avons déterminé les domaines de la protéine Vif impliqués dans l’activité chaperonnes d'ARN. En utilisant des essaies de dimérisation des fragments d’ARN du VIH-1, nous avons pu mettre en évidence que le domaine C-terminal de la protéine Vif était impliqué dans cette activité. Ces résultats nous ont permis de mieux comprendre ce phénomène de restriction cellulaire et pourraient être importants dans le développement de nouvelles stratégies d’inhibition de la réplication virale qui ciblerait spécifiquement l’interaction de Vif avec l’ARNm d’A3G. / The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting cellular cytosine deaminases APOBEC3G (A3G) and A3F by different mechanisms and thus preventing its incorporation into viral particles. The Vif-induced degradation of A3G through the proteasome pathway has been extensively studied, but little is known about the translational repression of A3G mRNA by Vif. After cellular co-transfection of A3G mRNA constructs mutated in their untranslated regions (UTRs) in presence or absence of Vif, and in conditions where the proteasome-induced degradation of A3G was inhibited, we show that the 5’-UTR of A3G mRNA is crucial for the translational inhibition by Vif. The core binding factor, CBF-, required to stabilize the Vif/A3G complex is dispensable for this specific repression. According to our previous secondary structural model of the 5’-UTR, the two distal stem-loop structures are sufficient for a complete translational inhibition of A3G. We show that residue K26 of Vif is critical for A3G neutralization, both for its proteasome-induced degradation and translation inhibition of itsmRNA. Interestingly, we observe a strict correlation between the cellular reduction of A3G through translation inhibition and the quantity of A3G incorporated into viral particles. Both mechanisms account for about 50% decrease of A3G in cell. Thus, we showed for the first time that A3G mRNA translational inhibition by Vif is a 5’-UTR mRNA-dependent mechanism, and that any of these two mechanisms, degradation or translation, is sufficient to restore viral infectivity. Regulating the translation of A3G could thus be considered as a new target to restore a functional expression of A3G and viral restriction.

Maize is an important food crop in most parts of the world including the United States. The plants growing in the field are constantly challenged with various biotic stresses like insect herbivores and fungal pathogens. The physical wounds produced on the growing crops by the insects render the plants more vulnerable to the fungal pathogens. Hence developing both insect and fungal resistant maize varieties is crucial to benefit more from the harvest. Several studies have been in advance in this direction and as a consequence insect, in particular lepidopteran larve resistant maize genotype Mp708 and Aspergillus flavus resistant genotype Mp313E were developed. This study particularly focuses on understanding the functional involvement of the major phytohormones in the signal transduction and expression of the unique defense protein, Maize insect resistance 1-cysteine protease (Mir1-CP) shown to accumulate in response to herbivory by lepidopteran larvae, Spodoptera frugiperda (Fall armyworm, FAW) as a defense mechanism. Using a pharmacological approach involving exogenous hormone and hormone inhibitor treatments and analyzing the expression and accumulation of Mir1-CP protein and mir1 transcript by immunoblot and qRT-PCR analysis respectively, both JA and ET were found to be involved in mediating Mir1-CP accumulation with JA acting upstream of ET. Results also indicate that Mir1-CP accumulation involves both transcriptional and post-transcriptional (or post-translational) regulations. A different part of the study involved in understanding and evaluating the performance of Aspergillus flavus on the resistant and susceptible maize genotypes during infection. As of part of this study I also analyzed and compared the defense response offered by the resistant maize genotype, Mp313E and the susceptible genotype, Va35 by looking at the expression levels of the various defense related genes. The potency of the resistant maize genotype in sustaining the fungal infection in the field was of particular focus. Resistant maize genotype Mp313E was found to potentially oppose A.flavus proliferation and colonization and also delay aflatoxin biosynthesis unlike Va35. The up regulation of the maize defense genes during the early time points of infection, in Mp313E, indicate the potential role of these genes in conferring resistance against fungal pathogens.

No description available.

No description available.

The ZNF9 gene on chromosome 3 encodes the cellular nucleic acid binding protein (CNBP), a ubiquitously expressed, 177 amino acid (≈19.5kDa) protein that is highly conserved among vertebrates. The function of the protein is largely unknown, however an expansion in the first intron of the protein results in myotonic dystrophy type 2 (DM2), a multisystemic disease featuring cardiac arrhythmia, muscle wasting, cataracts, and a range of neuropathologies. Remarkably, we recently discovered that CNBP is involved in regulating the activity of β-secretase, the enzyme that produces the first cleavage event in the generation of the amyloid-β peptide (Aβ). The progressive fibrillization and deposition of Aβ is widely believed to be the primary causal factor in the development of Alzheimer’s disease (AD), and AD-like pathology in individuals with Down syndrome (DS). DS provides a unique model for evaluating how these factors change in the aged brain as compared to young brain, and how such changes affect the proportion of DS patients with AD. In the AD brain, both BACE1 and BACE2 increased from an early stage of disease; in DS brains, BACE1 significantly decreased (p<0.04) with age, whereas BACE2 was unchanged, even though the gene for BACE2 is located within the DS obligate region of chromosome 21. BACE1 and BACE2 activity levels were highly correlated in this series (r2 = 0.95), indicating that there may be a higher degree of shared regulation than previously believed. This implicates regulators of BACE as potentially critical for the development of AD, and our data suggests that CNBP may be one such regulator. In AD, CNBP increases early in the disease process, a change that does not occur in the normal aging process or in DS. CNBP and BACE protein levels were correlated in these cases (p<0.001), while there was no relationship between CNBP and age, or CNBP and Aβ, in either the human or mouse brain, indicating that CNBP does not increase as a consequence of normal aging. Thirty day overexpression of CNBP following adeno-associated viral delivery in murine gastrocnemius muscle resulted in an increase in BACE1 protein (p<0.01) and a consequential increase in Aβ production (p<0.01). Other experiments indicated that CNBP overexpression did not affect the half-life of BACE1 mRNA or protein, but resulted in an increase in BACE1 translation. These data indicate that CNBP is an important regulator of β-secretase, and may play an important role in the onset and progression of AD.

Du fondamental à la clinique, cette thèse a été construite autour d’un seul mot clefs : KCC2, grâce à plusieurs projets collaboratifs. Ce co-transporteur d’ion est une molécule à plusieurs facettes dont la multiplicité des rôles et ses implications dans diverses pathologies font d’elle un élément clef de l’organisme vivant. En plus des études phylogénétiques et thérapeutiques effectuées au cours de cette période, mon principal travail a été de déterminer le rôle physio-pathologiques in vitro et in vivo d’un mécanisme de régulation post-traductionnelle de KCC2. Nous avons dans un premier temps montré que la déphosphorylation des Thréonines (Thr) 906 et 1007 in vitro était un puissant activateur de la protéine. En effet, nous avons montré que l’état de phosphorylation des sites dicté par la voie Wnk-Spak/OSR1 était impliqué dans le niveau d’expression en surface de la protéine. Par la suite, nous avons pu révéler que les souris porteuses d’une mutation phospho-mimétique Glu906 et Glu1007 “(KCC2E/+)” sur un allèle de la protéine, présentaient un décalage de l’émergence de la force inhibitrice GABAergique, une altération de la balance excitation/inhibition, ainsi qu’une augmentation de la susceptibilité à générer des crises. De plus, ces même souris développent des troubles de la communication chez le jeune ainsi qu’un défaut de sociabilité chez l’adulte, deux symptômes clefs des TSA. Ces résultats suggèrent que la régulation post-traductionnelle est un mécanisme physio-pathologique clef de la protéine. / From basic to clinical aspects, this thesis comprises different collaborative projects focusing on KCC2. KCC2 is a complex protein with multiple roles and implications in various pathologies that makes this molecule a key element of living organisms. In addition to the phylogenetic and therapeutic studies performed during this period, my main work has been to determine the in vitro and in vivo physio-pathological role of a KCC2 post-translational regulatory mechanism. We first showed in vitro that dephosphorylation of Threonines (Thr) 906 and 1007 was a potent activator of the protein. We have shown that phosphorylation state by the Wnk-Spak/OSR1 pathway of these two residues is implicated in the level surface expression of KCC2. Subsequently we have revealed that mice carrying in one allele a phospho-mimetic mutations Glu906 and Glu1007 “(KCC2E/+)”, preventing the developmental dephosphorylation at these sites, exhibited a delayed onset of fast synaptic GABA inhibition, a decreased ratio of spontaneous GABA- to glutamate-driven post-synaptic responses, and a significantly reduced flurothyl-induced seizure threshold. Furthermore, KCC2E/+ pups and adult mice, respectively, exhibited impaired communication and sociability, classic ASD phenotypes. These results suggest that post-translational regulation is a key physio-pathological mechanism of KCC2.

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence for a model in which Crc and Hfq co-target multiple RNA motifs to bring about a two-tiered regulation to subvert catabolism of phenols in the face of preferred substrates – one at the level of the regulator DmpR and another at the level of translation of the catabolic enzymes.