Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

Les mitochondries jouent un rôle majeur dans le métabolisme de l'ATP des cellules eucaryotes. Le maintien de l'ADN mitochondrial (ADNmt) est fondamental pour la production d'énergie chez les organismes aérobie stricte. De grandes délétions de ADNmt sont à l'origine d'anomalies mitochondriales entrainant des maladies chez l'homme. Plusieurs gènes nucléaires impliqués dans le métabolisme de l’ADNmt ont été identifiés et caractérisés chez l'homme. Cependant, l’ensemble des facteurs et leurs activités requis pour le maintien de l'ADNmt reste largement inconnu. L'identification de ces facteurs et la détermination de leurs activités dans des systèmes modèles simples peuvent contribuer à l’étude du maintien de l'ADNmt et à la compréhension des mécanismes induisant des délétions de l’ADNmt chez l'homme. Le champignon filamenteux Podospora anserina est un modèle d'étude du maintien de l’ADNmt. Chez P. anserina, l’accumulation de délétions région-spécifiques de l’ADNmt (Δmt) est corrélée à la présence de la mutation AS1-4 dans le gène nucléaire codant la protéine cytosolique ribosomale S15. L'altération de la protéine S15 pourrait modifier la traduction de transcrits codant des protéines impliquées dans le maintien de l'ADNmt et indirectement causer l'accumulation des Δmt. Par une approche globale (translatome), nous avons analysé l’ensemble des transcrits associés aux ribosomes AS1-4 en cours de traduction. A partir des données obtenues, deux gènes candidats, PaIML2 et PaYHM2 potentiellement impliqués dans le maintien de l'ADNmt, ont été identifiés et étudiés. L'analyse fonctionnelle a été principalement développée pour PaYHM2. La protéine PaYHM2 partage 68% d’identité avec la protéine mitochondriale bi-fonctionnelle Yhm2 de levure, impliquée dans le transport de métabolites dans la mitochondrie et possèdant un domaine de liaison à l'ADN. J'ai démontré que le gène PaYHM2 est essentiel pour P. anserina, un organisme aérobie stricte et que la protéine PaYHM2 est mitochondriale. Par mutagénèse, j'ai montré que c'est la fonction de transport qui est essentielle à la survie du champignon et non pas la putative capacité à se lier à l'ADN. Les résultats obtenus suggèrent également que PaYHM2 participe au métabolisme de l'acétyl-CoA chez P. anserina. / Mitochondria play main role as adenosine triphosphate (ATP)-energy factories of the eukaryotic cells. To ensure energy production, mitochondrial DNA (mtDNA) maintenance is essential for all obligate-aerobe eukaryotic organisms. Large-scale mtDNA deletions are major causes of mitochondrial dysfunction in human diseases. Several nuclear genes implicated in mtDNA metabolism were identified and characterized in human. Nuclear-encoded factors and their activities required for mtDNA maintenance are, however largely unknown. Identification of these factors and discovery of their activities in simple model systems can contribute to the comprehension of mtDNA maintenance and of the mechanisms leading to mtDNA deletions in human. The filamentous fungus Podospora anserina is a useful model system for studying mtDNA maintenance. An S15 cytosolic ribosomal protein mutant in P. anserina, named AS1-4 mutant, shows a positive correlation with the accumulation of specific large mtDNA deletion (Δmt) at the time of death. Alteration of S15 protein might modify translation of transcripts encoding proteins related to mtDNA maintenance and indirectly cause Δmt accumulation. Polysome profiling (called translatome), a global approach giving genome-wide informations about modified transcripts on translation, was performed on AS1-4 mutant. From the data of this translatome, two candidate genes potentially related to mitochondrial DNA maintenance, the PaIML2 gene and PaYHM2 gene has been identified and functionally analyzed. The function of the PaYHM2 gene has been especially characterized in this project. This gene encodes a protein sharing 68% of identity with yeast Yhm2, a bi-functional protein as a mitochondrial carrier and as a protein with DNA-binding activity. I demonstrated that the PaYHM2 gene is essential for P. anserina, an obligate-aerobe organism and that the PaYHM2 protein localizes to mitochondria. Through mutagenesis approach, I showed that the transport function decides the essentiality of mitochondrial carrier PaYHM2 while the putative DNA binding activity of PaYHM2 protein is important for P. anserina. Furthermore, I found that the function of PaYHM2 probably participates in the cytosolic acetyl-CoA metabolism.

La traduction est une étape critique de la régulation de l'expression des gènes. Chez l'oursin, la fécondation induit une augmentation de la synthèse protéique, qui dépend essentiellement de la traduction d'ARN messagers maternels. Cette synthèse protéique est indispensable au déroulement des cycles cellulaires du développement précoce. Le développement embryonnaire de l'oursin constitue ainsi un modèle de choix pour étudier la régulation de la traduction. Dans le cadre de cette thèse, le contrôle de la traduction a été étudié chez l'oursin dans deux situations : le contexte physiologique de la fécondation et le contexte de l'induction de l'apoptose. Nous nous sommes interrogés d'abord sur les mécanismes régulateurs impliqués dans la synthèse protéique après fécondation. L'une des étapes limitantes de la traduction est l'initiation. Dans ce cadre, le facteur d'initiation eIF2 joue un rôle clé. eIF2 est responsable de l'apport de la méthionine initiatrice au niveau du ribosome. Lorsque la sous-unité alpha d'eIF2 est phosphorylée, la synthèse protéique globale est inhibée et la traduction sélective de certains ARNm est stimulée. Dans les ovules non fécondés d'oursin, eIF2alpha est physiologiquement phosphorylé et la fécondation provoque sa déphosphorylation. En micro-injectant dans les ovules non fécondés un variant d'eIF2alpha mimant l'état phosphorylé, nous avons montré que la déphosphorylation d'eIF2alpha est nécessaire pour la première division mitotique chez l'oursin. Nous nous sommes intéressés au lien entre la phosphorylation d'eIF2alpha et l'induction de l'apoptose chez l'oursin. En effet, la traduction d'ARNm codant pour des protéines pro- ou anti- apoptotiques influence directement la survie des cellules. L'embryon d'oursin possède la machinerie nécessaire pour le déclenchement de l'apoptose, après induction par l'agent génotoxique MMS. Le traitement au MMS des embryons provoque la phosphorylation d'eIF2alpha. Dans cette situation, nous avons trouvé que la kinase GCN2 est impliquée dans la phosphorylation d'eIF2alpha. En fin, dans le but d'étudier comment la machinerie traductionnelle module le recrutement polysomal, nous avons analysé le traductome en réponse à la fécondation et après le traitement au MMS. Nous avons effectué une approche de séquençage à haut-débit des transcrits purifiés par gradients de polysomes. L'analyse de ces transcrits nous permettra d'appréhender le réseau des gènes régulés au niveau traductionnel lors de la fécondation et de l'induction de l'apoptose dans les embryons d'oursin.

The expression of numerous plant mRNAs is attenuated by RNA sequence elements located in the 5' and 3' untranslated regions (UTRs). For example, in plants and many higher eukaryotes, roughly 35% of genes encode mRNAs that contain one or more upstream open reading frames (uORFs) in the 5' UTR. For this dissertation I have analyzed the pattern of conservation of such mRNA sequence elements. In the first set of studies, I have taken a comparative transcriptomics approach to address which RNA sequence elements are conserved between various families of angiosperm plants. Such conservation indicates an element's fundamental importance to plant biology, points to pathways for which it is most vital, and suggests the mechanism by which it acts. Conserved motifs were detected in 3% of genes. These include di-purine repeat motifs, uORF-associated motifs, putative binding sites for PUMILIO-like RNA binding proteins, small RNA targets, and a wide range of other sequence motifs. Due to the scanning process that precedes translation initiation, uORFs are often translated, thereby repressing initiation at the an mRNA's main ORF. As one might predict, I found a clear bias against the AUG start codon within the 5' untranslated region (5' UTR) among all plants examined. Further supporting this finding, comparative analysis indicates that, for ~42% of genes, AUGs and their resultant uORFs reduce carrier fitness. Interestingly, for at least 5% of genes, uORFs are not only tolerated, but enriched. The remaining uORFs appear to be neutral. Because of their tangible impact on plant biology, it is critical to differentiate how uORFs affect translation and how, in many cases, their inhibitory effects are neutralized. In pursuit of this aim, I developed a computational model of the initiation process that uses five parameters to account for uORF presence. In vivo translation efficiency data from uORF-containing reporter constructs were used to estimate the model's parameters in wild type Arabidopsis. In addition, the model was applied to identify salient defects associated with a mutation in the subunit h of eukaryotic initiation factor 3 (eIF3h). The model indicates that eIF3h, by supporting re-initation during uORF elongation, facilitates uORF tolerance.

Many viruses can produce multiple proteins from a single mRNA sequence by encoding the proteins in overlapping genes. One mechanism that causes the ribosomes of infected cells to decode both genes is –1 programmed ribosomal frameshifting. In this process, structural elements of the viral mRNA signal the ribosome to shift reading frames at a specific point. Although –1 frameshifting has been recognized since 1985, the mechanism is not well understood. I have developed a stochastic model of mRNA translation that includes the possibility of a –1 frameshift at any codon. The transition probabilities between states of the model are based on the energetics of local molecular interactions. The model reproduces observed translation rates as well as both the location and efficiency of frameshift events in the HIV-1 gag-pol sequence. In this work, the model is used to predict changes in the frameshift efficiency due to mutations in the viral mRNA sequence or variations in relative tRNA abundances. The model is sensitive to the size of the translating ribosome and to assumptions about the unfolding pathway of the stimulatory structure. As knowledge in the field of RNA structure prediction grows, that knowledge can be incorporated into the model developed here to make improved predictions. The single-ribosome translation model has been extended to polysomes by including initiation and termination rates and an exclusion principle, and allowing the stimulatory structure to refold on an appropriate timescale. The predicted frameshift efficiency for a given mRNA can be tuned by varying the ribosome density on the mRNA. This finding affects the interpretation of frameshift efficiencies measured in the lab. In the parameter regime where translation is initiation-limited, the frameshift efficiency also depends on the structure refolding rate, which determines the availability of the downstream structure for stimulating –1 frameshifts. Furthermore, there is a trade-off between frameshift efficiency and protein synthesis rate.

H1 and core histone mRNA levels have been examined in the presence of portein synthesis inhibitors with different mechanisms of action. Total HeLa cell RNAs were analyzed by Northern Blot hybridization using cloned human histone genes as probes. Inhibition of DNA replication resulted in a rapid decline in histone mRNA levels. However, in the presence of cycloheximide or puromycin, H1 and core mRNAs did not decrease in parallel with DNA synthesis, but were stabilized and accumulated. Inhibition of DNA synthesis with hydroxyurea after the inhibition of protein synthesis did not lead to a decline in histone mRNA levels. These results suggest that synthesis of a protein(s) - perhaps a histone protein(s) - is required for the coordination of DNA synthesis and histone mRNA levels.

Spinal muscular atrophy (SMA) is a devastating disorder caused by deletions and mutations in the survival of motor neuron (SMN1) gene and is marked by motor neuron loss and muscle weakness. While its genetic basis is clear, the underlying molecular mechanisms remain elusive. Decreased levels of the survival of motor neuron (SMN) protein, encoded by the SMN1 gene, are implicated in SMA pathology. Despite splicing has been under the spotlight as a major mechanism impaired in SMA, recent evidence suggests that SMN deficiency also disrupts protein translation in vivo in a mouse model of severe SMA, complicating SMA's molecular landscape. This thesis examines the impact of SMN protein loss on translation in SMA mouse models across tissues, post-natal and pre-natal disease stages, focusing on both mild (Chapter 1) and severe forms of SMA (Chapter 2) respectively. To tackle this question, in this thesis, I took advantage of multiple cutting-edge and sequencing-based techniques (ribosome profiling and RNA-seq) coupled with biochemical and molecular biology-based assay (polysome profiling, co-sedimentation profiles, qPCR, and western blotting), which applied to study in molecular detail the translational defects in the brain, spinal cord and liver at asymptomatic, pre-symptomatic and early symptomatic stages of SMA. Polysome profiling in control mice (Smn2B/+) reveals a gradual increase in SMN association with ribosomes/polysomes during postnatal development, indicating dynamic SMN function in protein translation during post-natal development. In SMA condition, where SMN protein levels drop, this binding reveals a tissue-specific decrease in the spinal cord and liver. Through ribosome profiling, numerous alterations in translation were identified at the pre-symptomatic stage of the disease, suggesting that translational defects are features of the early stages of SMA. Importantly these alterations are independent of transcriptional and splicing changes. Although no gene was found to be in common, I found that genes altered in at least 2 tissues are involved in the same processes. The dysregulated mRNAs exhibit rare codons at the beginning of coding sequences in all three tissues, as shown in the case of the severe model of SMA. 4 From these common processes I have identified specific mRNA targets that play key roles in the organization of the extracellular matrix I validated the presence of translational changes in Col1a1, Col1a2, and Spp1 highlighted effects of SMN deficiency on translational regulation, in the absence of transcriptional alterations. Validation studies in both mice and SMA patient-derived fibroblasts further underscored the potential of translational dysregulation and drop in Col1a1 protein expression during SMA progression. Finally, prenatal studies have revealed distinct translational changes in embryonic tissues from Taiwanese mice. Despite no alterations in global translation, a drop in SMN association with ribosomes/polysomes and tissue-specific differences in ribosome occupancy were observed. Also, in this case, dysregulated mRNAs exhibit rare codons at the beginning of coding sequences. These findings shed light on the unique molecular landscape of prenatal development in the context of SMN deficiency. In summary, this study provides insights into translation dysregulation in SMA pathology, emphasizing tissue-specific effects and developmental stage-dependent alterations. By elucidating the complex relationship between SMN protein function and translational dynamics, it lays the groundwork for targeted therapeutic strategies and biomarkers to improve SMA management. Ongoing investigations into prenatal development and translation dynamics are crucial for a comprehensive understanding of SMA pathogenesis and effective treatment development.

O T3 aumenta a poliadenilação e estabilidade do GH mRNA. O citoesqueleto (Cy) participa da estabilidade e tradução de mRNAs, pois fatores, como o EF 1a, ligam alguns transcritos a ele, aumentando sua estabilidade e tradução. Cy também participa dos processos secretores celulares. Observamos que no hipotiroidismo (Tx), há um desarranjo do Cy nos somatotrofos que é revertido pela administração aguda de T3. Neste estudo avaliamos a ligação do EF 1a e do GH mRNA ao Cy e, deste aos polissomos, na hipófise, e a expressão do IGF-I mRNA hepático, em ratos controle e Tx tratados com T3 ou salina, e sacrificados após 30 min. Observamos redução da F-actina, da ligação do EF 1a e GH mRNA a ela, do GH mRNA nos polissomos, e da expressão de IGF-I mRNA hepático, nos ratos Tx, o que indicou redução da síntese e secreção do GH. A administração de T3 estimulou esses processos, aumentando a estabilidade, tradução do GH mRNA e a secreção de GH, o que ocorreu em paralelo ao rearranjo do Cy, indicando uma ação não genômica do T3. / T3 increases GH mRNA poly-A tail and stability. Cytoskeleton (Cy) plays a part on mRNA stability and translation, since factors, like EF 1a, can bind some transcripts to it, improving stability and translation efficiency. Cy is also involved in cellular secretory process. We showed that somatotropes Cy is disrupted in hypothyroidism (Tx), and rearranged by acute T3 treatment. In this study we investigated the binding of EF 1a and GH mRNA to Cy and of GH mRNA to polysomes in pituitary, as well as the liver IGF-I mRNA content, in control and Tx rats treated with T3 or saline, and killed 30 min thereafter. We observed that Tx reduced F-actin content, EF 1a and GH mRNA binding to it, GH mRNA recruitment to polysomes, in pituitary, and IGF-I mRNA expression in liver, which indicates that GH synthesis and secretion are impaired. Acute T3 treatment stimulated all these process, indicating that stability, translation of GH mRNA and GH secretion were restored. These events occurred in parallel to the Cy rearrangement, which strongly indicates a non genomic effect of T3.

O T3 aumenta a poliadenilação e estabilidade do GH mRNA. O citoesqueleto (Cy) participa da estabilidade e tradução de mRNAs, pois fatores, como o EF 1a, ligam alguns transcritos a ele, aumentando sua estabilidade e tradução. Cy também participa dos processos secretores celulares. Observamos que no hipotiroidismo (Tx), há um desarranjo do Cy nos somatotrofos que é revertido pela administração aguda de T3. Neste estudo avaliamos a ligação do EF 1a e do GH mRNA ao Cy e, deste aos polissomos, na hipófise, e a expressão do IGF-I mRNA hepático, em ratos controle e Tx tratados com T3 ou salina, e sacrificados após 30 min. Observamos redução da F-actina, da ligação do EF 1a e GH mRNA a ela, do GH mRNA nos polissomos, e da expressão de IGF-I mRNA hepático, nos ratos Tx, o que indicou redução da síntese e secreção do GH. A administração de T3 estimulou esses processos, aumentando a estabilidade, tradução do GH mRNA e a secreção de GH, o que ocorreu em paralelo ao rearranjo do Cy, indicando uma ação não genômica do T3. / T3 increases GH mRNA poly-A tail and stability. Cytoskeleton (Cy) plays a part on mRNA stability and translation, since factors, like EF 1a, can bind some transcripts to it, improving stability and translation efficiency. Cy is also involved in cellular secretory process. We showed that somatotropes Cy is disrupted in hypothyroidism (Tx), and rearranged by acute T3 treatment. In this study we investigated the binding of EF 1a and GH mRNA to Cy and of GH mRNA to polysomes in pituitary, as well as the liver IGF-I mRNA content, in control and Tx rats treated with T3 or saline, and killed 30 min thereafter. We observed that Tx reduced F-actin content, EF 1a and GH mRNA binding to it, GH mRNA recruitment to polysomes, in pituitary, and IGF-I mRNA expression in liver, which indicates that GH synthesis and secretion are impaired. Acute T3 treatment stimulated all these process, indicating that stability, translation of GH mRNA and GH secretion were restored. These events occurred in parallel to the Cy rearrangement, which strongly indicates a non genomic effect of T3.