Lors de la transcription de gènes, il y a production d'un pré-ARN messager qui subira plusieurs étapes de maturation pour former l'ARN messager (ARNm) pouvant coder pour une protéine. Une de ces étapes est l'épissage qui consiste à exciser des portions (appelés les introns) pour juxtaposer les autres séquences (les exons), formant ainsi l'ARNm.Lors de l'épissage alternatif, un exon peut parfois être excisé menant à la formation d'un autre isoforme d'ARNm, donc possiblement d'une autre protéine, à partir d'un seul gène. Il est maintenant estimé que plus de 97% des pré-ARNm humains multi-exoniques subissent l'épissage alternatif, permettant ainsi une augmentation considérable de la quantité de protéines codées par les 30 000 gènes humains. Certaines des protéines produites par épissage alternatif peuvent avoir des activités très différentes, voire antagonistes. Ceci est souvent le cas dans l'apoptose, soit la mort cellulaire programmée. Le gène bcl-x, par exemple, peut mener à la formation de deux isoformes majoritaires. Lorsque le site d'épissage 5' proximal est utilisé, il y a formation de Bcl-x[indice inférieur L], codant pour une protéine ayant une activité anti-apoptotique, donc favorisant la survie de la cellule. Par contre, lorsque le site d'épissage 5' distal est utilisé, il y a formation d'un ARNm ayant perdu un exon de 189 nucléotides (nt) qui code pour Bcl-x[indice inférieur s], qui favorisera l'apoptose. L'épissage alternatif de ce pré-ARNm, comme les autres, est évidement bien contrôlé par des séquences présentes sur l'ARN, des facteurs protéiques liant ces séquences et des signaux cellulaires régulant l'activité de ceux-ci. Ma thèse consistait à analyser ces trois aspects et mon travail a mené à la découverte de deux protéines liant le pré-ARNm de bcl-x afin de réguler son épissage, ainsi que d'une région nécessaire à la signalisation cellulaire par la protéine kinase C (PKC). Au début, j'ai aidé à l'identification des hnRNP F et H qui lient une région riche en guanidine, nommée B2G, présente dans l'exon alternatif. Ces protéines lient cet élément et ainsi augmenterait la formation de l'isoforme Bcl-x[indice inférieur s]. Ces résultats sont présentés en annexe. Par la suite, j'ai identifié une région contenant deux éléments antagonistes, soit B1AC et B1u, située 10 nt en amont du site Bcl-x[indice inférieur s]. B1AC augmente l'utilisation de ce site, tandis que B1u fait le contraire, par la liaison de hnRNP K. Cette protéine est souvent fortement exprimée dans certains types de cancer, en accord avec son activité d'inhibition de l'isoforme pro-apoptotique. Ceci est présenté dans la première partie de ma thèse. La deuxième section de ma thèse consiste à l'identification d'une grande région nommée SB1 (361 nt) située au début de l'exon 2. De façon basale, cet élément inhibe le site Bcl-x[indice inférieur s]. Cependant, lors du blocage de l'activité de la PKC par la staurosporine, l'inhibition est perdue entrainant ainsi une forte augmentation de cet isoforme. L'inhibition de la PKC dans les lignées cellulaires cancéreuses n'entraîne pas cet effet sur l'épissage de Bcl-x, suggérant que la voie de signalisation de PKC est déréglée ou non-couplée à la régulation du gène apoptotique Bcl-x dans les cancers. L'avancement de la compréhension de l'épissage alternatif de gènes clés impliqués dans certaines maladies, tel que bcl-x dans le cancer, est primordial pour une meilleure compréhension de celles-ci. Cette thèse présente ma participation dans l'étude de la régulation de l'épissage alternatif de bcl-x .

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interferes with hnRNP U for binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Overall, a mechanistic model has been revealed where hnRNP U competes with hnRNP L for C9/E3 binding to enhance the inclusion of the four-exon cassette, and this splice-enhancing effect is blocked by the AKT pathway via phosphorylation of hnRNP L. As to the latter aim, it is unknown about the mechanistic roles of caspase-9b besides the inhibitory effect on caspase-9a processing. In this study, caspase-9b has been demonstrated to have a dual function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway, which is independent from modulating caspase-9a activation. In particular, caspase-9b has been shown to activate the canonical arm and inhibit the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for caspase-9b contributes to the enhanced survival and anchorage-independent growth of NSCLC cells conferred by caspase-9b expression. Further mechanistic studies have demonstrated a direct association of caspase-9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif. Through this interaction, caspase-9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the survival, anchorage-independent growth and tumorigenicity of NSCLC cells. Overall, a novel tumorigenic mechanism has been identified, by which alternative mRNA processing regulates the NF-κB signaling independent of external agonist.

La dystrophie myotonique de type 1 (DM1) est une maladie génétique caractérisée par une dégénérescence des muscles squelettiques accompagnée d’une myotonie. Cette maladie est due à une expansion instable de triplets CTG dans la région 3’ non traduite du gène DMPK. L’accumulation des ARNm DMPK mutés au sein de foci nucléaires conduit à la séquestration du facteur d’épissage MBNL1 et à des altérations de l’épissage alternatif de nombreux ARNm. En particulier, l’inclusion de l’exon 5 au sein du pré-ARNm de la troponine T cardiaque (hcTNT) est renforcée chez les patients DM1. Cette inclusion anormale participe aux anomalies cardiaques présentées par les patients. Les travaux de l’équipe, menés en collaboration avec l’équipe de Nicolas Sergeant à Lille, sur la régulation de l’épissage de l’ARNm hcTNT avaient établi l’existence de 8 sites MBNL1, dont 6 nouveaux, situés de part et d’autre de l’exon 5 et la présence de régions activatrices et inhibitrices de l’inclusion fixant des facteurs d’épissage dont l’identité n’était pas connue. L’un des objectifs de ma thèse était d’étudier l’importance fonctionnelle in cellulo de chacun des 8 sites MBNL1. J’ai ainsi pu montrer que chacun des 6 nouveaux sites participe à l’inhibition de l’inclusion de l’exon 5 par MBNL1. Les données obtenues nous ont amené à proposer un modèle dans lequel MBNL1 s’associe avec les triplets de sites MBNL1 situés de part et autre de l’exon 5 et entraine la formation d’une structure à longue distance via des interactions protéiques MBNL1-MBNL1. Cette structure isolerait l’exon 5 dans une boucle et limiterait la fixation du spliceosome. Par ailleurs, j’ai mis en œuvre une approche de purification de RNP formées en extrait nucléaire pour identifier d’autres facteurs régulant l’inclusion de l’exon 5. La protéine hnRNP H a ainsi pu être identifiée. Sa capacité à activer l’inclusion de l’exon 5 in cellulo et à entrer en compétition avec MBNL1 pour la régulation de l’inclusion de l’exon 5 via sa fixation sur des sites localisés dans l’exon 5 et en aval de cet exon a pu être confirmée. La seconde partie de ma thèse a porté sur l’étude de l’effet d’un stress oxydatif généré par 500 µM d’H2O2 sur le profil global d’épissage alternatif des pré-ARNm de cellules HeLa. Lors de ce travail, j’ai pu établir que la réponse des cellules HeLa au stress oxydatif implique deux phases de réponse : une phase précoce (1h-8h) caractérisée par un fort taux de mortalité associé à une forte augmentation du taux de d’entités oxygénées réactives (ROS) intracellulaire et une phase tardive (16h-24h) corrélée à une diminution du taux de ROS intracellulaires et une surexpression des ARN satellite III. Sur la base de ces données, une analyse globale du transcriptome par emploi de puces à exons (Affymetrix) a été réalisée à partir d’ARN totaux isolés 1h, 2h, 4h et 24h après le début du stress. Nous avons ainsi identifié des modulations d’expression et d’épissage spécifiques de chacune des deux phases. L’analyse des données par des outils bio-informatiques a permis de mettre en évidence des fonctions cellulaires bien définies qui sont plus particulièrement affectées lors d’un stress oxydant. Enfin, pour comprendre l’origine des variations d’épissage observées lors d’un stress oxydant, j’ai entrepris d’analyser les effets de ce stress sur le niveau d’expression et la localisation cellulaire des composants du spliceosome ou des facteurs qui s’associent pour réguler son activité / Myotonic distrophy of type 1 (DM1) is a genetic disease characterized by skeletal muscle degeneration associated to myotonia. DM1 results from the instable expansion of CTG repeats within the 3’ untranslated region of the DMPK gene. The accumulation of mutated DMPK mRNAs within nuclear foci leads to the sequestration of the MBNL1 splicing factor and causes splicing misregulation of numerous pre-mRNAs. Among altered events the increase of the inclusion of exon 5 in the human cardiac troponin T (hcTNT) mRNA is of particular importance, since it contributes to the cardiac symptoms presented by the patients. Through collaborative work with N. Sergeant’s team from Lille, the team has studied the molecular bases of hcTNT exon 5 inclusion regulation and mapped 8 MBNL1 binding sites, including 6 new ones, within intronic regions surrounding exon 5. They also identified positive and negative splicing regulatory elements of which protein partners remain unidentified. The first objective of my PhD thesis was to test the functional importance of each individual MBNL1 binding site. The obtained results established that the 6 newly identified MBNL1 binding sites are involved in splicing regulation by MBNL1 and lead us to propose a new regulation model in which MBNL1 binds on triplets of MBNL1 sites present on each side of exon 5 and form a long distance structure via MBNL1-MBNL1 protein interaction. The formation of this looping-structure is expected to isolate exon 5 and limit its recognition by the spliceosome. In addition I searched for protein partners of the identified regulatory elements by affinity chromatography. By this way, I identified hnRNP H as a positive regulator of exon 5 inclusion. Its capacity to compete with MBNL1 to regulate splicing in cellulo by binding on exonic and intronic binding sites was further confirmed. The second part of my PhD work corresponds to the study of the global impact of oxidative stress, generated by exposition of HeLa cells to 500 µM of H2O2, on alternative splicing. This allows us to establish that the response of HeLa cells to oxidative stress involve two distincts phases: an early one (1h-8h) characterized by poor survival rate and high intracellular ROS content and a late phase (16-24h), associated with a decrease of the intracellular ROS level and the overexpression of the long non coding sat III RNAs. Based on this observation, we performed a transcriptome global analysis by using exon arrays from Affymetrix on RNA samples isolated 1, 2, 4 or 24 hours after the induction of the oxidative stress. We identified changes of the gene expression level or mRNA splicing pattern specific of each of the response phases. Data computing by bio-informatic tools identified the most affected cellular processes and functions during the cell response to oxidative stress. In order to better understand the mechanisms underlying alternative splicing modulation during oxidative stress, I started to study the impact of oxidative stress on the expression level and the cellular localization of spliceosome components and most common splicing regulation factors

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

During the maturation of pre-mRNA introns are removed and exons are spliced together, to form a primary transcript, a reaction that is catalyzed by the spliceosome. Alternative splicing is a complex reaction that mainly utilizes one of four mechanisms; exon skipping, 5’ splice site choice, 3’ splice site choice and intron retention. To achieve accurate splicing four sequence elements are essential, two of which are located in the splice sites themselves; 5’ splice sites and 3’ splice sites, but also the polypyrimidine tract and the branch point sequence. Alternative splicing can be regulated by histone or chromatin modulations, siRNA, transcription efficiency and various proteins, many of which belong to either the SR protein family or the hnRNP family of proteins. SR proteins usually promote exon inclusion, while hnRNP proteins usually promote exon skipping. There are also regulatory elements that are called exonic splicing enhancers or silencers depending on if they promote or inhibit the inclusion of the exon they reside in. These elements also exist in introns and are then called intronic splicing enhancers or silencers. The enhancer elements are most commonly targeted by SR proteins and the silencer elements are usually targeted by hnRNP proteins. This paper will mainly focus on the regulation of alternative splicing and the role of alternative splicing under abnormal conditions, such as when mutations cause disease.

MYC is overexpressed in most types of tumors, but a means to selectively decrease its expression is yet to be found. Our recent findings on modulation of BCL2 gene expression through protein interactions with the BCL2 i-motif have provided a basis for further investigation of MYC gene control. It is proposed that the MYC i-motif could function by a similar molecular switch mechanism as in BCL2.Binding sites for heterogeneous nuclear ribonucleoprotein K (hnRNP K) within the MYC promoter also exist in the i-motif-forming sequence. Circular dichroism and bromine footprinting confirmed that this DNA sequence is able to form an i-motif, and systematic mutation of the cytosine residues in this sequence has revealed a 5:5:5 loop configuration. Indeed, all loops of the i-motif, when folded into a 5:5:5 loop configuration, contain the hnRNP K consensus sequence (CCCT). Previous studies show that hnRNP K binds to this i-motif-forming sequence, but it was assumed to be single-stranded. Binding studies revealed that hnRNP K has more binding affinity to its consensus sequence in the i-motif compared to a mutant sequence where the i-motif cannot form. Further investigation of the MYC promoter revealed an additional two runs of cytosine seven bases downstream of the MYC i-motif. Biophysical studies showed that the additional two runs were not involved in i-motif formation, however recent studies describe their importance for transcriptional activation. We found that hnRNP K preferred the longer 5CT sequence compared to the i-motif forming 4CT sequence when using a competitive binding assay. Utilizing luciferase reporters containing either the 4CT or 5CT sequence validated that hnRNP K required both the i-motif and 5th CT element for maximum transcriptional activation. Competition binding studies and bromine footprinting showed that hnRNP K bound to the downstream 5th CT element and the central and lateral loops of the i-motif.Additionally, we found that co-overexpression of Sp1 and hnRNP K induced a 10-fold increase in luciferase activity in the 5CT reporter only. We hypothesize that Sp1 continuously primes the promoter to initiate transcription inducing more negative superhelicity and increasing the melting of duplex DNA. This increased melting grants hnRNP K’s three KH domains access to the i-motif loops and the 5Th CT element. Confirmation by ChIP analysis validated that Sp1 overexpression causes an increase in hnRNP K occupancy at the MYC promoter. These findings provide new insight into the mechanisms of MYC transcriptional control by the i-motif and G-quadruplex.Recently, our group has demonstrated that two small molecules IMC-48 and IMC-76 can interact with the i-motif and can be an effective means to modulate BCL2 expression. Based on these results with the BCL2 i-motif, we employed a similar strategy and screened and identified small drug-like molecules that interact with MYC i-motif, using a FRET high-throughput assay. We then further validated that IMC-16 stabilizes the MYC i-motif through the interactions with the loops of the i-motif. No stabilization by IMC-16 treatment was observed with the MYC G-quadruplex and the BCL2 and PDGFRβi-motifs demonstrating selectivity for the MYC i-motif.Finally, we investigated the effects of IMC-16 on MYC expression in three lymphoma cell lines all expressing different levels of MYC. In the case of both Daudi and RAJI Burkitt’s lymphoma cell lines we demonstrated that selectively stabilizing the i-motif by IMC-16 could increase MYC expression. Furthermore, we demonstrated that the MYC G-quadruplex stabilizing compound GQC-05 and IMC-16, which stabilizes the MYC i-motif, have antagonistic effects on MYC expression, providing further evidence of a molecular switch mechanism in the NHEIII1. Directly targeting MYC expression through the i-motif offers advantages over targeting the G-quadruplex, because of the reduced stability and dynamic nature of the i-motif, additionally the i-motif is only found in DNA. The use of such i-motif interactive compounds is the first step into the development of new innovative approaches to treat cancers.

During cellular stress, the regulation of protein synthesis is a key adaptive mechanism used by cells to survive. In response to various stresses, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an RNA binding protein principally found within the nucleus, is phosphorylated and consequently accumulates in the cytoplasm. Among other roles, cytoplasmic hnRNP A1 functions as an auxiliary translation factor for internal ribosome entry site (IRES)-mediated translation of specific mRNA, including the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL). To identify which kinases control the cytoplasmic accumulation of hnRNP A1, an RNAi-based kinome-wide screen was performed in hypertonically stressed U2OS cells, from which AMPK-related kinase 5 (ARK5) was identified as a potential regulator of hnRNP A1’s localization. Here we show that ARK5 directly phosphorylates hnRNP A1 and that the inhibition of ARK5 expression blocks the stress induced cytoplasmic accumulation of hnRNP A1, modulates expression of Bcl-xL protein and increases cell viability. Our data points to a novel role for ARK5 and provides further insight into the mechanisms regulating cellular stress response.

abstract: The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers. Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed. Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before. Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages. DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2014

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

No description available.