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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Spinal muscle atrophy (SMA) is an autosomal recessive neurodegenerative disease which is characterized by the loss of α motor neurons from the anterior horn of the spinal cord, resulting in progressive muscle atrophy. The loss of functional Survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene is the cause of SMA. A potential treatment strategy for SMA is to upregulate levels of the SMN protein originating from the copy gene SMN2 which can compensate in part for the absence of the functional SMN1 gene. I have shown a novel therapeutic strategy for SMA treatment through the activation of the p38 pathway by the bacterial antibiotic anisomycin which stabilizes and increases SMN mRNA levels in vitro. Activation of the p38 pathway by anisomycin leads to cytoplasmic accumulation of HuR protein which binds to the 3’UTR of SMN transcript resulting in increased SMN levels. This opens up a novel potential therapeutic strategy for SMA. I have also identified and demonstrated a significant induction of SMN protein levels in vitro and in vivo upon treatment with FDA approved drug celecoxib, which also activates the p38 pathway. Celecoxib mitigates disease severity along with increasing the lifespan of SMA mice. Sodium valproate, trichostatin A and aclarubicin, all agents which effectively enhance SMN2 expression, have been recently shown to activate STAT5 in SMA-like mouse embryonic fibroblasts and human SMN2-transfected NSC34 cells. Given that prolactin is also known to activate the STAT5 signalling pathway, can cross blood brain barrier and is FDA approved, we elected to assess its impact on SMN levels. In this manner, I have demonstrated a significant induction in SMN mRNA and protein levels in neuronal NT2 and MN-1 cells upon treatment with prolactin. I have also demonstrated that activation of the STAT5 pathway by prolactin is necessary for this transcriptional upregulation of the SMN gene. I have found that prolactin treatment induces SMN expression in brain and spinal cord samples and that it ameliorates the disease phenotype, improving motor neuron function and increasing survival in the SMA mouse model. Presently there is no cure for SMA. This study will help in the identification and characterization of potential therapeutic compounds for the treatment of SMA.

Spinal muscle atrophy (SMA) is an autosomal recessive neurodegenerative disease which is characterized by the loss of α motor neurons from the anterior horn of the spinal cord, resulting in progressive muscle atrophy. The loss of functional Survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene is the cause of SMA. A potential treatment strategy for SMA is to upregulate levels of the SMN protein originating from the copy gene SMN2 which can compensate in part for the absence of the functional SMN1 gene. I have shown a novel therapeutic strategy for SMA treatment through the activation of the p38 pathway by the bacterial antibiotic anisomycin which stabilizes and increases SMN mRNA levels in vitro. Activation of the p38 pathway by anisomycin leads to cytoplasmic accumulation of HuR protein which binds to the 3’UTR of SMN transcript resulting in increased SMN levels. This opens up a novel potential therapeutic strategy for SMA. I have also identified and demonstrated a significant induction of SMN protein levels in vitro and in vivo upon treatment with FDA approved drug celecoxib, which also activates the p38 pathway. Celecoxib mitigates disease severity along with increasing the lifespan of SMA mice. Sodium valproate, trichostatin A and aclarubicin, all agents which effectively enhance SMN2 expression, have been recently shown to activate STAT5 in SMA-like mouse embryonic fibroblasts and human SMN2-transfected NSC34 cells. Given that prolactin is also known to activate the STAT5 signalling pathway, can cross blood brain barrier and is FDA approved, we elected to assess its impact on SMN levels. In this manner, I have demonstrated a significant induction in SMN mRNA and protein levels in neuronal NT2 and MN-1 cells upon treatment with prolactin. I have also demonstrated that activation of the STAT5 pathway by prolactin is necessary for this transcriptional upregulation of the SMN gene. I have found that prolactin treatment induces SMN expression in brain and spinal cord samples and that it ameliorates the disease phenotype, improving motor neuron function and increasing survival in the SMA mouse model. Presently there is no cure for SMA. This study will help in the identification and characterization of potential therapeutic compounds for the treatment of SMA.

In mammals, a major fraction of the genome is transcribed as non-coding RNAs. An increasing amount of evidence has accumulated showing that non-coding RNAs play important roles both for normal cell function and in disease processes such as cancer or neurodegeneration. Interpreting the functions of non-coding RNAs and the molecular mechanisms through which they act is one of the most important challenges facing RNA biology today. In my Ph.D. thesis, I have been investigating the role of 7SK, one of the most abundant non-coding RNAs, in the development and function of motoneurons. 7SK is a highly structured 331 nt RNA transcribed by RNA polymerase III. It forms four stem-loop (SL) structures that serve as binding sites for different proteins. Larp7 binds to SL4 and protects the 3' end from exonucleolytic degradation. SL1 serves as a binding site for HEXIM1, which recruits the pTEFb complex composed of CDK9 and cyclin T1. pTEFb has a stimulatory role for transcription and is regulated through sequestration by 7SK. More recently, a number of heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified as 7SK interactors. One of these is hnRNP R, which has been shown to have a role in motoneuron development by regulating axon growth. Taken together, 7SK’s function involves interactions with RNA binding proteins, and different RNA binding proteins interact with different regions of 7SK, such that 7SK can be considered as a hub for recruitment and release of different proteins. The questions I have addressed during my Ph.D. are as follows: 1) which region of 7SK interacts with hnRNP R, a main interactor of 7SK? 2) What effects occur in motoneurons after the protein binding sites of 7SK are abolished? 3) Are there additional 7SK binding proteins that regulate the functions of the 7SK RNP? Using in vitro and in vivo experiments, I found that hnRNP R binds both the SL1 and SL3 region of 7SK, and also that pTEFb cannot be recruited after deleting the SL1 region but is able to bind to a 7SK mutant with deletion of SL3. In order to answer the question of how the 7SK mutations affect axon outgrowth and elongation in mouse primary motoneurons, we proceeded to conduct rescue experiments in motoneurons by using lentiviral vectors. The constructs were designed to express 7SK deletion mutants under the mouse U6 promoter and at the same time to drive expression of a 7SK shRNA from an H1 promoter for the depletion of endogenous 7SK. Using this system we found that 7SK mutants harboring deletions of either SL1 or SL3 could not rescue the axon growth defect of 7SK-depleted motoneurons suggesting that 7SK/hnRNP R complexes are integral for this process. In order to identify novel 7SK binding proteins and investigate their functions, I proceeded to conduct pull-down experiments by using a biotinylated RNA antisense oligonucleotide that targets the U17-C33 region of 7SK thereby purifying endogenous 7SK complexes. Following mass spectrometry of purified 7SK complexes, we identified a number of novel 7SK interactors. Among these is the Smn complex. Deficiency of the Smn complex causes the motoneuron disease spinal muscular atrophy (SMA) characterized by loss of lower motoneurons in the spinal cord. Smn has previously been shown to interact with hnRNP R. Accordingly, we found Smn as part of 7SK/hnRNP R complexes. These proteomics data suggest that 7SK potentially plays important roles in different signaling pathways in addition to transcription. / Bei Säugetieren wird ein großer Teil des Genoms als nicht-kodierende RNAs transkribiert. Es gibt immer mehr Hinweise darauf, dass nicht-kodierende RNAs eine wichtige Rolle sowohl für die normale Zellfunktion als auch bei Krankheitsprozessen wie Krebs oder Neurodegeneration spielen. Die Interpretation der Funktionen nicht-kodierender RNAs und der molekularen Mechanismen, über die sie wirken, ist eine der wichtigsten Herausforderungen, denen die RNA-Biologie heute gegenübersteht. In meiner Promotionsarbeit habe ich die Rolle von 7SK, einer der am häufigsten vorkommenden nicht-kodierenden RNAs, bei der Entwicklung und Funktion von Motoneuronen untersucht. 7SK ist eine RNA, die aus 331 Nukleotiden besteht und deren Struktur bekannt ist. Sie wird von der RNA-Polymerase III transkribiert. Sie bildet vier Stem-Loop (SL)-Strukturen, die als Bindungsstellen für verschiedene Proteine dienen. LARP7 bindet an SL4 und schützt das 3'-Ende vor exonukleolytischem Abbau. SL1 dient als Bindungsstelle für HEXIM1, das den P-TEFb-Komplex rekrutiert, der aus CDK9 und Cyclin T1 besteht. P-TEFb hat eine stimulierende Rolle für die Transkription und wird durch Sequestrierung durch 7SK reguliert. In jüngerer Zeit wurde eine Reihe von heterogenen nukleären Ribonukleoproteinen (hnRNPs) als 7SK-Interaktoren identifiziert. Eines davon ist hnRNP R, von dem gezeigt wurde, dass es eine Rolle bei der Entwicklung von Motoneuronen spielt, indem es das Axonwachstum reguliert. Durch die Interaktion mit P-TEFb und RNA-bindenden Proteinen kann 7SK als Drehscheibe für die Rekrutierung und Freisetzung verschiedener Proteine betrachtet werden. Die Fragen, mit denen ich mich während meiner Doktorarbeit beschäftigt habe, lauten wie folgt: 1) Welche Region von 7SK interagiert mit hnRNP R, einem Hauptinteraktor von 7SK? 2) Welche Effekte treten in Motoneuronen auf, wenn die Bindung von hnRNP R an 7SK inhibiert wird? 3) Gibt es zusätzliche 7SK-bindende Proteine, die die Funktionen des 7SK RNPs regulieren? Mit Hilfe von in vitro und in vivo Experimenten fand ich heraus, dass hnRNP R sowohl die SL1- als auch die SL3-Region von 7SK bindet, und dass P-TEFb nach der Deletion der SL1-Region nicht rekrutiert werden kann, aber in der Lage ist, an eine 7SK-Mutante mit Deletion von SL3 zu binden. Um die Frage zu beantworten, wie sich die 7SK-Mutationen auf Axonwachstum in primären Motoneuronen der Maus auswirken, führten wir Rettungsexperimente an Motoneuronen unter Verwendung lentiviraler Vektoren durch. Die Konstrukte wurden so konzipiert, dass sie 7SK-Deletionsmutanten durch den U6-Promotor der Maus exprimieren und gleichzeitig eine 7SK-shRNA von einem H1-Promotor für die Depletion von endogenem 7SK transkribieren. Mit diesem System fanden wir heraus, dass 7SK-Mutanten, die Deletionen von SL1 oder SL3 beherbergen, den Axon-Wachstumsdefekt von 7SK-depletierten Motoneuronen nicht retten konnten, was darauf hindeutet, dass 7SK/hnRNP R-Komplexe für diesen Prozess von Bedeutung sind. Um neue 7SK-Bindungsproteine zu identifizieren und ihre Funktionen zu untersuchen, führte ich Pulldown-Experimente durch, bei denen ich ein biotinyliertes RNA-Antisense-Oligonukleotid verwendete, das an die U17-C33-Region von 7SK bindet und dadurch Aufreinigung endogener 7SK-Komplexe erlaubt. Nach der Massenspektrometrie der gereinigten 7SK-Komplexe identifizierten wir eine Reihe neuer 7SK-Interaktoren. Einer davon ist der Smn-Komplex. Ein Mangel des Smn-Komplexes verursacht die Motoneuronerkrankung Spinale Muskelatrophie (SMA), die durch den Verlust der unteren Motoneuronen im Rückenmark gekennzeichnet ist. Es wurde bereits gezeigt, dass Smn mit hnRNP R interagiert. Dementsprechend fanden wir Smn als Teil des 7SK/hnRNP R-Komplexes. Diese Proteom-Daten deuten darauf hin, dass 7SK neben der Transkription möglicherweise auch in anderen Signalwegen wie der spliceosomalen snRNP Biogenese eine wichtige Rolle spielt.

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Les particules ribonucléoprotéiques (RNP) sont impliquées dans divers mécanismes cellulaires : les UsnRNP, la SRP, les RNP à boîtes C/D et H/ACA dans la modification des ARN et la maturation des ARN ribosomiques, et la télomérase dans le maintien des extrémités chromosomiques. L'assemblage de ces RNP est un processus complexe faisant intervenir de nombreux facteurs, dont le complexe SMN. Un déficit de l’une des protéines de ce complexe conduit à l’amyotrophie spinale. Il est essentiel à la survie cellulaire et est nécessaire à l’assemblage des UsnRNP et de la SRP. Il est suggèré que le complexe SMN joue un rôle dans la biogenèse des RNP à boîtes C/D et H/ACA. Nous avons montré des interactions in vitro et des associations in cellulo entre le complexe SMN et la protéine NUFIP (un facteur d’assemblage de ces RNP). Ces résultats suggèrent l’existence d’un lien fonctionnel entre le complexe SMN et NUFIP dans l'assemblage des RNP à boîtes C/D et H/ACA et de la snRNP U4. Des interactions in vitro entre le complexe SMN et la protéine NAF1 (un facteur d’assemblage des RNP à boîtes H/ACA) ont révélés, que le complexe SMN est capable de s’associer avec la RNP à boîtes H/ACA en formation. Si le complexe SMN intervient dans l’assemblage des RNP, on peut supposer que cet assemblage soit défectueux dans la SMA. Nous avons montré que certains ARN sont accumulés dans la moelle épinière et le cerveau de souris SMA. La télomérase est réactivée dans les cellules cancéreuses. En collaboration avec l’équipe de J-M Vignaud (CHU central, Nancy), nous avons montré une augmentation du taux des protéines cœur des RNP à boîtes H/ACA et de NUFIP dans les cellules tumorales / Ribonucleoprotein particles (RNPs) are involved in various cellular mechanisms in eukaryotic cells: UsnRNP, SRP, C/D and H/ACA box RNPs in RNA modifications and rRNA maturation and telomerase in the synthesis of the chromosome extremities. RNP assembly is a very complex process, which involves numerous factors. One of these factors is the SMN complex. Decreased level of one of its components leads to spinal muscular atrophy. It is essential for cell survival and necessary for UsnRNP and SRP assembly. It is suggested that the SMN complex plays a role in C/D and H/ACA RNP biogenesis. We showed in vitro interactions and in cellulo associations between the SMN complex and the protein NUFIP (an assembly factor of these RNP). These results suggest the existence of a functional link between the SMN complex and NUFIP in the assembly of the C/D and H/ACA box RNPs and the U4 snRNP. In vitro interactions between the SMN complex and the protein NAF1 (an assembly factor of the H/ACA boxes RNPs) revealed, that the SMN complex is capable of joining with the H/ACA boxes RNPs in formation. If the SMN complex intervenes in the RNPs assembly, we can suppose that this assembly is defective in the SMA. We showed that any ARN is accumulated in the spinal cord and the brain of SMA mouse. The telomerase is reactivated in cancer cells. In association with the team of J-M Vignaud (CHU central, Nancy), we showed an increase of H/ACA box RNP proteins and NUFIP in these cancer cells