abstract: Protein crystallization has become an extremely important tool in biochemistry since the first structure of the protein Myoglobin was solved in 1958. Survival of motor neuron protein has proved to be an elusive target in regards to producing crystals of sufficient quality for X-ray diffraction. One form of Survival of motor neuron protein has been found to be a cause of the disease Spinal Muscular Atrophy that currently affects 1 in 6000 live births. The production, purification and crystallization of Survival of motor neuron protein are detailed. The Fenna-Matthews-Olson (FMO) protein from Pelodictyon phaeum is responsible for the transfer of energy from the chlorosome complex to the reaction center of the bacteria. The three-dimensional structure of the protein has been solved to a resolution of 2.0Å with the Rwork and Rfree values being 16.6% and 19.9% respectively. This new structure is compared to the FMO protein structures of Prosthecocholoris aestuarii 2K and Chlorobium tepidum. The early structures of FMO contained seven bacteriochlorophyll-a (BChl) molecules but the recent discovery that there is an eighth BChl molecule in Ptc. aestuarii 2K and Cbl. tepidum and now in Pld. phaeum requires that the energy transfer mechanism be reexamined. Simulated spectra are fitted to the experimental optical spectra to determine how the BChl molecules transfer energy through the protein. The inclusion of the eighth BChl molecule within these simulations may have an impact on how energy transfer through FMO can be described. In conclusion, a reliable method of purifying and crystallizing the SMNWT protein is detailed, the placement of the 8th BChl-a within the electron density and the implications on energy transfer within the FMO protein when the 8th BChl-a is included from the green sulfur bacteria Pld. phaeum is discussed. / Dissertation/Thesis / Ph.D. Biochemistry 2010

The course of pathogenic bacterial infection is dependent on the interactions between the host immune response and the bacterial virulence mechanisms. Our lab previously discovered that the Survival of Motor Neuron (SMN) protein complex undergoes a change in subcellular localization during infection with invasive Shigella bacteria, forming novel cytoplasmic aggregates called "U bodies". Similar results were obtained with other intracellular bacterial pathogens suggesting that these U bodies are a fundamental entity in microbial pathogenesis. Notably, the SMN complex normally plays a key role in the assembly of the spliceosomal U snRNA. We have shown during infection that there are changes in U snRNA maturation and splicing patterns. Importantly, we have found that U bodies are downstream of a stress pathway involving the stress-inducible ATF3 protein. Altogether, intracellular bacterial infection induces novel cellular stress pathways that disrupt normal SMN complex function and leads to changes in U snRNA associated functions.

The course of pathogenic bacterial infection is dependent on the interactions between the host immune response and the bacterial virulence mechanisms. Our lab previously discovered that the Survival of Motor Neuron (SMN) protein complex undergoes a change in subcellular localization during infection with invasive Shigella bacteria, forming novel cytoplasmic aggregates called "U bodies". Similar results were obtained with other intracellular bacterial pathogens suggesting that these U bodies are a fundamental entity in microbial pathogenesis. Notably, the SMN complex normally plays a key role in the assembly of the spliceosomal U snRNA. We have shown during infection that there are changes in U snRNA maturation and splicing patterns. Importantly, we have found that U bodies are downstream of a stress pathway involving the stress-inducible ATF3 protein. Altogether, intracellular bacterial infection induces novel cellular stress pathways that disrupt normal SMN complex function and leads to changes in U snRNA associated functions.

Despite our understanding of the role of the survival motor neuron protein (SMN) in cytoplasmic small ribonucleoproteins (snRNP) assembly, it is unclear how loss of this protein causes motor neuron degeneration in Spinal Muscular Atrophy (SMA). It could be explained by defects in functions that are specific to tissues most affected in SMA. In neurons, SMN localizes to neuronal RNA granules, RNA-containing foci in axons. They regulate many aspects of mRNA fate which include transport along neurites, mRNA stability, and mRNA translation. Most recently, our work provided evidence for SMN’s role in mRNA translation. Specifically, we demonstrated that SMN associates with polyribosomes and may repress translation of specific mRNA targets. Our group demonstrated that SMA-causing mutations within the Tudor domain of SMN completely abolished this activity. This indicates the potential significance of this novel SMN function in the SMA pathology. To further investigate SMN’s function in regulating translation, our group performed a proteomic screen on polysome-containing sucrose gradient fractions. We identified and validated novel interacting partners for SMN that may act as co-factors to regulate translation. DDX5 (an RNA helicase) is an unexpected novel interacting partner as it is known for its role in micro-RNA processing. Moreover, we observe that FMRP, a recognized protein in translational complexes, is required for the presence of SMN and DDX5 in polysomal fractions. With these latest findings, we updated our model of the molecular mechanism by which SMN regulates translation. This work provides more insights on how SMN regulates translation, a newly uncovered role for SMN in motor neurons. Identification of the molecular targets that are misregulated due to loss of this function may reveal new information on the pathogenesis of SMA.

No description available.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease, which, before the approval of therapies, was the leading genetic cause of infant mortality. The primary features of this pathology are progressive muscle weakness and atrophy, due to the degeneration of α-motor neurons in the anterior horn of the spinal cord. SMA is caused by deletions or mutations in the Survival Motor Neuron gene (Smn1), which induce reduced levels of the SMN protein. Since 1999, this disease has been primarily associated with splicing defects caused by loss of SMN protein due to its role in ribonucleoparticle biogenesis. However, further research revealed that this mechanism alone is not sufficient to explain the pathogenesis of the disease. More recent findings revealed that deficient SMN levels lead to defective translation in primary motor and cortical neurons, and in multiple tissues at the late stage of disease in the severe Taiwanese mouse model of SMA. Furthermore, SMN protein has been confirmed to be a ribosome-associated protein in vitro, in mouse cell lines and in vivo, and to physiologically regulate the translation of a particular subset of transcripts (defined as SMN-specific transcripts), which are characterized by specific sequence features. Upon SMN loss, the translation of this subset of transcripts is defective. SMN protein is ubiquitously expressed and its levels vary at different developmental stages and tissues in physiological conditions, leading to the hypothesis that translational defects may vary accordingly. However, the effect of SMN loss on translation across different tissue types, SMA mouse models, and disease stages is yet to be clarified. To investigate the link between SMN loss and translational defects in SMA, I took advantage of ribosome profiling to obtain the translatome from multiple tissues, stages and disease mouse models. Given that SMN is ubiquitously expressed, brain, spinal cord and liver were collected to investigate if common features underly translational defects upon its loss in these tissues. Since little is known about how translational impairments are modulated over time, tissues were collected from various developmental and disease stages, ranging from the embryo to the post-natal early-symptomatic stage of SMA. Furthermore, translation defects were studied in multiple models of SMA have ranging from severe to mild (i.e., Taiwanese, Delta7 and Smn2b/-), allowing for the exploration of the heterogeneity of the SMA clinical phenotype. Hence, the tissues were collected from three SMA mouse models (i.e., Taiwanese, Delta7, and Smn2b/-), allowing for the investigation of translational impairments in conditions that range from severe to mild SMA. A wide range of computational approaches was adopted to analyze ribosome profiling data from multiple perspectives, including Principal Component Analysis (PCA), pipelines for the analysis of RiboSeq positional information, differential and Gene Ontology enrichment analysis, and network methodologies. This set of tools applies to the study of ribosome profiling data and allows to investigate the translational mechanisms underlying SMA. This multilevel analysis holds difficulties in the representation and interpretation of the obtained results due to the number of variables (i.e., tissue, stage, model, and disease condition). I hence developed an R package to support the visualization of changes occurring in omics data from complex experimental designs. Next, I focused on the identification of translational defects in SMA through pairwise differential analyses performed on each set of experiments. This allowed me to identify significantly altered transcripts within each comparison. Despite poor overlaps between the sets of translationally dysregulated transcripts across the different stages, tissues, and models, commonly enriched biological processes were found. The analysis of sequence features on translationally dysregulated transcripts across all the stages, tissues, and models revealed the presence of features similar to those already found on the SMN-specific transcripts. In addition, based on network methodologies, I investigated the system-wide effects of SMN loss on connectivity patterns at the translational level, by taking advantage of network-based methodologies to integrate all sets of experiments and unravel any relationships between genes at the translatome level. Causal-inference networks, coupled with differential network analysis, complemented the standard differential analysis by modeling how the fluctuations in reciprocal transcript-specific ribosome occupancy might influence each other. This allowed to detect disrupted relationships in the disease condition across the multiple tissues, stages and models. In summary, this thesis provides, to my knowledge, the first multi-tissue, -stage, and -model translatome analysis to investigate the mechanisms underlying SMA. Furthermore, results provided within this work confirm that translation dysregulation is a common feature of SMA pathology across multiple tissues, stages, and SMA models. This highlights that the presence of specific sequence features of translationally dysregulated transcripts is a common link between defective translational regulation and SMN loss. Moreover, the detection of disrupted connectivity patterns at the translatome level underlies that a strong remodeling occurs upon SMN loss, and further emphasizes the pivotal role of this protein in translation. These outcomes highlight the importance of further investigating the mechanisms underlying defective translation in SMA from a system perspective to provide a comprehensive understanding of this pathology and promote the development of effective therapeutic strategies.

Les petites particules ribonucléoprotéiques nucléaires (snRNPs) sont les composants majeurs du splicéosome, la machinerie responsable de l'épissage des pré-messagers. La biogenèse des snRNPs est un processus complexe qui fait intervenir de nombreux facteurs comme les protéines SMN et ICln. Au cours de ma thèse, je me suis intéressé à l'étude du rôle de ces deux protéines dans la maturation et la production des snRNPs du splicéosome.Dans la première partie de mon travail, les modifications internes des snRNAs ont été caractérisées dans des cellules dont les corpuscules de Cajal sont dispersés à cause d'une déficience de la protéine SMN. En effet, en plus de son rôle dans les étapes précoces de formation des snRNPs, la protéine SMN est également requise pour la formation des corpuscules de Cajal, structures nucléaires qui concentrent les scaRNAs impliqués dans le processus de modification post-transcriptionnelle des ARNs. Nous avons pu ainsi montrer que la protéine SMN et les corps de Cajal ne sont pas essentiels à la production des résidus 2'-O-methyl et pseudouridine dans les snRNAs majeurs et mineurs.La deuxième partie de mon travail a porté sur l'étude des relations fonctionnelles entre les protéines ICln et SMN in vivo en utilisant l'organisme modèle S. pombe. Après avoir caractérisé un homologue de la protéine humaine dans la levure fissipare, nous avons montré que la protéine ICln n'est pas essentielle mais est importante pour une croissance optimale des cellules de levure. Notre étude montre aussi que la modulation de l'activité de la protéine ICln ne permet pas de compenser les défauts dans la production de snRNPs observés dans les cellules portant un allèle muté de SMN. Finalement, l'utilisation d'une approche génomique montre que la délétion du gène ICln entraine des défauts différentiels d'épissage, indiquant que le choix des sites et la cinétique d'épissage sont fortement dépendants de la concentration des composants de base du splicéosome. / Small nuclear ribonucleoproteins (snRNPs) are the major components of the spliceosome, the machinery responsible for the splicing of pre-messenger RNAs. The biogenesis of snRNPs is a complex process that involves many factors such as the SMN and ICln proteins. During my thesis, I studied the role of these two proteins in the maturation and the production of the spliceosome snRNPs.The goal of the first part of my work was to characterize the internal modifications of snRNAs in SMN-deficient cells carrying disrupted Cajal bodies. Indeed, in addition to its role in the early stages of snRNPs assembly, the SMN protein is also required for the formation of Cajal bodies which are nuclear structures carrying the scaRNAs involved in the post-transcriptional modification process of RNAs. We could show that the SMN protein and Cajal bodies are not essential for the formation of 2'-O-methyl and pseudouridine residues in the major and minor snRNAs.In the second part of my work, the functional relationships between the ICln and SMN proteins were examined in vivo using the S. pombe model organism. We first identified a fission yeast homologue of the human ICln protein and found that the ICln protein is not essential but important for optimal growth of yeast cells. Our study also showed that the modulation of the activity of the ICln protein does not compensate for defects in the production of snRNPs observed in yeast cells carrying a SMN mutated allele. Finally, the use of a genome-wide approach allowed us to show that deletion of the ICln gene resulted in differential splicing defects, indicating that the choice of splice sites and the kinetics of splicing are strongly dependent on the concentration of the basic components of the spliceosome.

L’infection par le virus de l’herpès simplex de type 1 (HSV-1), un virus pathogène humain majeur, engendre la déstabilisation des centromères. Cette déstabilisation est induite par la protéine virale ICP0, et entraîne la dégradation par ICP0, via le protéasome, des protéines CENP-A, -B et CENP-C. Des résultats obtenus au laboratoire ont mis en évidence le phénomène iCDR (pour interphase Centromere Damage Response) qui implique la redistribution de la coïline, fibrillarine et SMN dans ces structures centromériques déstabilisées par ICP0 mais également par des drogues ou des siRNAs dirigés contre des constituants protéiques essentiels pour la stabilité des centromères. Il a été étudié leur interdépendance dans la réponse iCDR. Il a été ainsi démontré que la redistribution de SMN aux centromères déstabilisés est dépendante de : 1) la présence de la coïline aux centromères, et 2) de son interaction, via son domaine TUDOR, avec l’histone H3 méthylée sur la lysine K79 par l’enzyme Dot1L. L’équipe suggère donc l’hypothèse que ces protéines ont pour rôle de protéger l’ADN nu se trouvant aux centromères après dégradation des histones pour empêcher les cellules de rentrer en apoptose. Ces résultats ont mené à démontrer l’implication de certaines des protéines de l’iCDR et notamment la coïline, dans une réponse apoptotique générale suite à un stress UV. Ces protéines pourraient donc faire partie d’un mécanisme de contrôle qui serait défini comme un checkpoint centromérique / Infection by Herpes Simplex Virus type 1, a major pathogenic virus in human, has been shown to cause centromere destabilization. The infected cell protein 0 (ICP0) induces centromere destabilization and lead to proteasomal-dependent degradation of the proteins of the centromeres, CENP-A, -B and CENP-C. Recent data, obtained in our laboratory, highlights the interphase Centromere Damage Response (iCDR) phenomena. This phenomena involves centromeric accumulation and redistribution of the Cajal body-associated coilin and fibrillarin as well as the Survival Motor Neuron (SMN) proteins by ICP0 or by other drugs or siRNA targeting several constitutive centromere proteins known to play a major role in centromeres stabilization. Our data shows that SMN reditribution in the destabilized centromere is dependent of : 1) centromeric presence and accumulation of the coilin, 2) its interaction, via the TUDOR domain, with the methylated (Lys K79) histone H3. This methylation occurs in the presence of the Dot-1L enzyme. We hypothesize that these proteins play a critical role in safeguarding centromeric DNA to prevent the cells from apoptosis after Histone degradation. These observations, demonstrate the implication of certain iCDR proteins, more specifically the coilin, in the apoptotic response following a UV stress. In conclusion, these proteins could be part of a safeguard mechanism considered as a centromeric checkpoint

L'amyotrophie spinale infantile (SMA) est une maladie génétique autosomique récessive de l'enfant pour laquelle aucun traitement efficace n'existe. La SMA est caractérisée par la perte spécifique des motoneurones spinaux conduisant à une faiblesse musculaire sévère. Le décès des patients survient lorsque les muscles vitaux sont touchés. Cette maladie est causée par la mutation du gène Survival of Motor Neuron 1 (Smn1) conduisant à une diminution importante de l’expression de la protéine Survival of Motor Neuron (SMN). Tous les patients possèdent un ou plusieurs gènes copie de Smn1, le gène Smn2. Ces copies modulent la sévérité de la maladie en produisant une faible quantité de transcrits SMN complets, en particulier possédant l’exon 7, un exon alternatif qui code pour un domaine important pour que la protéine SMN soit fonctionnelle et stable. Des résultats récents, obtenus au laboratoire, indiquent que l'exercice physique retarde la mort des motoneurones, conduit à une augmentation du taux de maturation postnatale des unités motrices et déclenche l’expression du gène Smn2 chez des souris mimant la SMA de type II. Les premières données moléculaires suggèrent que les effets de l'exercice physique pourraient être relayés par la signalisation dépendante 1) des récepteurs au NMDA (Biondi et coll., J Neurosci, 2008) et/ou 2) du récepteur à IGF-1. Dans notre étude, nous avons d’abord testé les effets de l’activation directe des récepteurs au NMDA (NMDAR) dans un contexte de SMA. Nous montrons qu’une activation adéquate de ces récepteurs dans plusieurs modèles souris mimant les SMA sévères accélère la maturation postnatale des unités motrices, limite l'apoptose dans la moelle épinière et active l’expression du gène Smn2 favorisant l'expression de la protéine SMN. Ces effets bénéfiques sont dépendants du niveau d’activation des NMDARs et suggèrent que l'accélération de la maturation postnatale des unités motrices, induite par le NMDA, est indépendante du niveau d’expression de la protéine SMN. De manière importante, l’activation pharmacologique des NMDARs augmente fortement la durée de vie de deux modèles différents de souris mimant la SMA de type sévère. L'analyse des cascades de signalisation intracellulaire a révélé une altération inattendue des profils d’activation des voies de signalisation ERK et AKT/CREB, qui se rééquilibrent quand les NMDARs sont activés (Branchu et coll., J Neurosci, 2010).Comme la kinase ERK est constitutivement suractivée dans la moelle épinière des souris mimant la SMA, nous avons ensuite examiné son rôle potentiel dans la régulation de l'expression des gènes Smn2. Nous avons démontré que l'inhibition pharmacologique de la voie de signalisation MEK/ERK/Elk-1, notamment avec un médicament anti-cancéreux actuellement en essai clinique de phase 2, est bénéfique pour les souris mimant la SMA de type I. Nous avons identifié une relation croisée entre les voies de signalisation ERK et AKT impliquant la modulation, calcium-dépendante, de l'activité CaMKII. Ainsi, l'inhibition pharmacologique de ERK durant la phase symptomatique de la maladie chez ces souris, entraîne l'activation de la voie CaMKII/AKT/CREB et conduit à une augmentation significative de l’expression de la protéine SMN dans les motoneurones suite à une augmentation de la transcription du gène Smn2. Ces modifications sont corrélées avec une augmentation remarquable de la durée de vie et de la mobilité des souris et une neuroprotection des motoneurones spinaux. De plus, l’inhibition de ERK dans des cellules musculaires différenciées provenant de patients atteints de SMA de type II induit également une augmentation de l’activité de la voie AKT/CREB et de l’expression de SMN (Branchu et coll., J Neurosci, en révision positive). Enfin, nous avons montré que l'exercice physique est capable de diminuer l'expression du récepteur à l'IGF-1 (IGF-1R), qui est surexprimé dans la moelle épinière des souris mimant la SMA sévère... / Spinal muscular atrophy (SMA) is a severe autosomal recessive disease in childhood for which no efficient therapy is currently available. SMA is characterized by the specific loss of spinal motor neurons leading to a severe muscular weakness and death when vital muscles are affected. This disease is caused by mutation of the survival of motor neuron 1 (Smn1) gene leading to a deficiency of the Survival of Motor Neuron (SMN) protein expression. All patients retain one or more copies of the Smn2 gene, which modulates the disease severity by allowing a small amount of full-length SMN transcripts and stable SMN protein to be produced. Recent results in our laboratory indicate that physical exercise delays motor neuron death, leads to an increase in the motor-units postnatal maturation rate and trigger Smn2 gene expression in motor neurons. Furthermore, on the one hand, exercise is capable of specifically enhancing the expression of the gene encoding NR2A, the major activating subunit of the NMDA receptor in motor neurons. This subunit is known to be dramatically down-regulated in the spinal cord of severe SMA-like mice. Accordingly, inhibiting NMDA-receptor activity abolishes the exercise-induced effects on muscle development, motor neuron protection and life span gain (Biondi et al., J Neurosci, 2008). Thus, we tried to restore NMDA-receptor function as a therapeutic approach to SMA treatment. We demonstrated that an adequate NMDA receptor activation in severe SMA-like mouse model significantly accelerated motor-unit postnatal maturation, counteracted apoptosis in the spinal cord, and induced a marked increase in SMN expression resulting from a modification of Smn2 gene transcription pattern. These beneficial effects are dependent on the level of NMDA receptor activation since a treatment with high doses of NMDA led to an acceleration of the motor unit maturation but favored the apoptotic process and decreased SMN expression. Thus, these results suggest that the NMDA-induced acceleration of motor-unit postnatal maturation occurred independently of SMN. The NMDA receptor activating treatment strongly extended the life span in two different severe SMA-like mouse models. The analysis of the intracellular signaling cascades that lay downstream the activated NMDA receptor revealed an unexpected competition between the MEK/ERK/Elk-1 and the AKT/CREB signaling pathways for Smn2 gene regulation. Actually, the reactivation of the AKT/CREB pathway, thought calcium influx and the phosphorylation of CaMKII, opposed to MEK/ERK/Elk-1 inhibition, induces an enhanced SMN expression (Branchu et al., J Neurosci, 2010). On the other hand, exercise is capable of strongly decreasing the expression of IGF-1 receptor (IGF-1R); which is over-expressed in the spinal cord of severe SMA-like mice. We report that this reduction is also correlated with a reactivation of the AKT/CREB pathway and a MEK/ERK/Elk-1 inhibition. Therefore we generated an IGF-1R+/- SMA-like mouse model to investigate the functional link between IGF-1R expression level and the intracellular signaling pathway triggered in SMA spinal cord. We provided the first evidence that reducing the IGF-1R expression level is neuroprotective for SMA motor neurons, accelerates motor-unit postnatal maturation and leads to a remarkable increase in SMN expression and lifespan. The analysis of the intracellular signaling cascades revealed the same competition for Smn2 gene regulation. However, the activation of AKT/CREB is calcium-independent. In addition, we showed a drastic reduction of STAT3 phosphorylation and SOCS-1 and -3 expressions, which are over-expressed in SMA spinal cord and known to positively modulate ERK phosphorylation and negatively AKT (Data not published). Taken together all these data suggest new perspectives to therapeutic strategy, based on specific pharmacological correction, for SMA...

L'Atrophie Musculaire spinale (SMA) est une maladie neurodégénérative causée par des mutations du gène SMN1 et caractérisée par la dégénérescence sélective des motoneurones alpha de la moelle épinière. les mécanismes moléculaires de la SMA ne sont aps clairs. cependant, deux hypothèses ont été retenues:D'une part, que la déficience en SMN entraine une perturbation de la biogenèse des snRNPs spliceosomales individuelles et par conséquent des défauts d'épissage. pendant ma thèse, nous avons montré que la déficience en SMN provoquait une diminution des particules tri-snRNPs majeures amis surtout mineures et que cela avait des conséquences sur l'épissage d'un sous-groupe de pré-ARNm contenant des introns mineurs.D'autre part, que la déficience en SMN entraine des altérations de transport d'ARN dans les axones, essentiels pour la survie des motoneurones. A part l'ARNm de la beta-actine et l'ARNm de cpg15 récemment identifié, ceux qui pourraient être transportés par SMN n'ont pas été décrits. nous avons donc identifié les ARN interagissant avec les isoformes a-SMN et SMN-fl dans des cellules neuronales, et montré que certains de ces ARN cibles colocalisent avec SMN dans les axones, suggérant qu'elle est impliquée dans leur transport. / Spinal Muscular Atrophy is a neurodegenerative disease caused by mutations in SMN1 gene. SMA is characterized by the loss of alpha-motoneurons of the spinal cord. However, the precise molecular mechanisms underlying the disease are still unkown. two hypotheses have been retained to explain SMA pathigenesis:In one hand, the fact that SMN deficiency leads to a perturbation of individual snRNPs biogenesis and consequently splicing defects. During my PhD, we have shown that SMN deficiency alters the levels of major, but mostly, minor tri-snRNPs. And that leads to splicing defects of a subset of pre-mRNA containing minor introns.In the other hand, that SMN deficiency causes alteration of axonal transport of RNAs crucial to motoneurons survival. Except beta-actin mRNA and the recently identified cpg mRNA, the RNA targets of SMN have not been described. We succeed to identify RNA targets of both a-SMN and SMN-fl isoformes in a neuronal cell line and colocalisation data of some of these targets suggested that SMN could be implicated in the transport of these RNAs.