O vítreo apresenta remodelamento de seus componentes durante a patogênese de doenças como descolamento de retina, buraco de mácula e membrana epirretínica e os fatores que levam a essas alterações ainda não são completamente conhecidos. As células gliais de Müller exercem um papel regulatório importante na retina, principalmente na produção de TGF-beta, e participam na formação de membranas contráteis em doenças da interface vítreo retínica. O TGF-beta está aumentado tanto na retina como no vítreo, em condições de doença, tendo o papel importante de ativar a capacidade contrátil na interface do tecidos estudados. MicroRNAs são moléculas de RNA fita simples de 19-25 nucleotídeos, endógenos, não codificantes e potentes reguladores pós-transcricional da expressão gênica, portanto potenciais alvos terapêuticos eficientes para o tratamento das doenças da interface vítreo-retínica. Em linhagem de células de Müller de rato (rMC-1), o tratamento com TGF- &#946 1 em concentração de 5 ng/ml leva a diminuição da expressão gênica de let7-b e let7-c, após 24 horas de tratamento. Já em linhagem humana de Müller ( MIO-M1), a expressão gênica de let-7b e let-7c foi alterada com 10 ng/ml de TGF- &#946 1 e TGF- &#946 2. TGF- &#946 2 induziu a uma queda da expressão de let-7b e let-7c, que variou entre 70% e 40 % , após 24 e 48 horas de tratamento. Investigamos os possíveis alvos desses miRNAs, COL1A1, COL1A2 e HAS2, proteínas que possuem relação com as alterações da interface vítreo-retiniana. Entretanto, a análise da expressão de RNAm de COL1A1 e COL1A2 após a estimulação de MIO-M1 com TGF- &#946 1 e TGF- &#946 2 não apresentou alterações. No estudo com gene reporter de luciferase validamos COL1A2 como um novo alvo de let-7b na célula de Müller. Nos estudos funcionais observamos que o let-7c mimético diminui a contração do gel de colágeno, Dessa forma, neste estudo concluímos que o micro-ambiente das células de Müller nas doenças da interface vítreo-retiniana, pode alterar a expressão dos miRNAs da família let7 e, consequentemente, levar à formação de membranas densas e contráteis. / Vitreous remodeling occurs during disease pathogenesis, such as retinal detachment, macular hole and epiretinal membrane, and the factors that lead to these alterations are still not fully determined. Müller glial cells regulate TGF-beta production in the retina and participate in the formation of contractile membranes in vitreoretinal interface.TGF-beta is increased in both vitreous and retina in disease conditions, and are key participants in activating contractible capacity. MicroRNAs are short (19- 25 nucleotides), endogenous, non-coding RNA, involved in post-transcriptional gene regulation, that have a potential role as new therapeutic targets for vitreoretinal interface diseases. In rat Müller cell line (rMC-1), treatment with 5ng /ml of TGF-&#946 1 induced a downregulation of let7-b and let7-c expression after 24 hours. In Müller human line (MIO-M1), let-7b and let-7c expression were altered with 10 ng / ml of TGF-&#946 1 and TGF-&#946 2. TGF-&#946 2 induced a downregulation ranging from 70% to 40%, after 24 and 48 hours of treatment. We investigated the possible targets of these miRNAs: COL1A1, COL1A2 and HAS2, proteins that are related to vitreoretinal interface alterations. However, the analysis of COL1A1 and COL1A2 mRNA expression after stimulation of MIO-M1 with TGF-&#946 1 and TGF-&#946 2 did not show any changes. The luciferase gene reporter analysis revealed COL1A2 as a let-7b target in the Müller cell. In the functional studies we observed that the let-7c mimic decreases collagen gel contraction. In summary, we conclude that the microenvironment of Müller cells in vitreoretinal interface diseases may alter the expression of the miRNAs of the let7 family and, consequently, lead to the formation of dense and contractile membranes.

Eukaryotic cells tightly regulate metabolism in order to sustain normal processes. Dysregulation of cellular metabolism is associated with multiple diseases including cancer. Polyamine metabolism is a tightly regulated process that is co-opted by multiple cancers for selective growth advantages. Polyamines are small organic molecules with two or more amino groups attached, whose biosynthesis is initiated by ornithine decarboxylase (ODC). Although much is known regarding the effects of polyamine metabolism and ODC on cellular processes, little is known regarding the intracellular signaling events that are regulated by polyamines. Clinical studies demonstrated that the ODC inhibitor difluromethylornithine (DFMO) was an effective chemopreventative strategy causing a reduction of colon adenomas in patients with prior colon polyps. However, the molecular mechanisms leading to this reduction are unknown. This dissertation provides mechanistic insight into the biological roles of the polyamines and show that these amines are regulators of multiple non-coding RNAs involved in cellular responses including effects on the let-7 microRNA family. Moreover, the polyamine modified translation factor eIF5A is demonstrated to regulate the oncofetal factor LIN28. This work also indicates that polyamines regulate the mTOR pathway and suggests alternative signaling nodes for polyamine-mediated regulation of cellular processes. Overall, these findings support the notion that polyamines are oncometabolites that are targetable and serve as a promising approach to manipulate oncogenic signaling for cancer therapy.

A la suite d’un criblage pangénomique, nous avons montré que l’inhibition d’un miARN de la famille let-7, miR-98, entraîne une forte accélération de la différenciation musculaire avec hypertrophie des myotubes. Lors de ma thèse, j’ai cherché à comprendre comment miR-98 retarde la différenciation du muscle strié squelettique. Des analyses transcriptomiques dans des myoblastes où ce miARN a été inhibé ont montré la perturbation de l’expression d’environ 240 gènes. Parmi eux, j’ai montré que le répresseur transcriptionnel E2F5 est important pour la régulation de la différenciation musculaire via miR-98, et est directement ciblé par ce miARN. L’inhibition de E2F5 permet de rétablir un niveau de différenciation normal malgré l’inhibition de miR-98. E2F5 est un régulateur important du cycle cellulaire. Sa fonction dans le muscle n’avait pas encore été explorée. Mes résultats montrent que E2F5 accélère le processus de différenciation musculaire. J’ai ensuite montré que E2F5 peut réguler directement des gènes répresseurs de la différenciation musculaire, comme ID1 et HMOX1, et indirectement des répresseurs de la voie TGF-β, expliquant son action sur la différenciation. Au final, miR-98 régule la différenciation du muscle strié squelettique en agissant directement sur l’expression de E2F5, et indirectement sur ses multiples cibles. Mes résultats ont ainsi mis en évidence une nouvelle voie de régulation de la différenciation musculaire : la voie let-7 – E2F5. / A genome-wide screen had previously shown that knocking-down miR-98, a miRNA of let-7 family, leads to a dramatic increase of terminal myogenic differentiation, with myotube hypertrophy. My PhD project aimed to understand how miR-98 delays skeletal muscle differentiation. Transcriptomic analysis of human myoblasts with knocked-down miR-98 revealed that approximately 240 genes were sensitive to miR-98 depletion. Among these potential targets, I have identified the transcriptional repressor E2F5, which turned out to be important for miR-98 regulation of muscle differentiation. Knocking down E2F5 and miR-98 simultaneously had almost fully restored normal differentiation. I have subsequently shown that E2F5, an important cell cycle regulator, is a direct target of miR-98 in muscle, where its function had never been investigated before. My results show that E2F5 is a positive regulator of the muscle differentiation process. E2F5 can directly repress inhibitors of muscle differentiation, such as ID1 and HMOX1, and indirectly regulate TGF-β pathway family members. In conclusion, miR-98 regulates skeletal muscle differentiation by directly regulating E2F5 expression, and thus controlling the expression of multiple E2F5 targets. My results have highlighted a new regulatory pathway of muscle differentiation: the let-7 – E2F5 pathway.

No câncer, alterações em microRNAs (miRNAs), pequenos RNAs que regulam a tradução protéica, exerce efeito oncogênico (oncomiR). Os oncomiRs regulam genes chave para a proliferação celular e apoptose, sendo importantes para a biologia do câncer. O carcinoma papilífero de tiróide apresenta alterações genéticas alinhadas na via MAPK (RET>RAS>BRAF>ERK). Observamos que a indução do oncogene RET/PTC diminui a expressão de let-7 em células foliculares tiroidianas. Na linhagem TPC-1 (com RET/PTC-1), a introdução de let-7 diminui a proliferação celular e a fosforilaçãode ERK, indicando papel de gene supressor tumoral. No carcinoma anaplásico, avaliamos o papel da introdução do cluster miR-17-92 na linhagem ARO. Observamos que in vitro miR-17-92 atua de forma oncogênica aumentando proliferação e viabilidade celular de ARO. No entanto, estas células apresentam diminuição no crescimento em soft-agar. No xenotransplante, os tumores de ARO-miR-17-92 apresentam menor volume e expressam MMP-9 de forma reduzida, indicando também um papel de gene supressor tumoral para o cluster. / In cancer, alteration in microRNA, small RNAs (~22nt) that regulate post-transcriptionally protein levels, exerts oncogenic role (oncomiR). OncomiRs control genes involved in cell proliferation and apoptosis, influencing cancer biology. Papillary thyroid cancer displays activating genetic alterations in MAPK signaling pathway (RET>RAS>BRAF>ERK). Using conditional induction of oncogenes in thyroid cells, we observed that RET/PTC decreases let-7 miRNA expression. In papillary thyroid cancer cell TPC-1 (with RET/PTC-1) we observed that let-7 introduction inhibits cell proliferation and ERK phosphorylation, indicating tumor suppressor role for let-7. In anaplastic thyroid cancer, we evaluate the role of introduction of miR-17-92 cluster in ARO cell line. We observed in vitro that miR-17-92 increases ARO cell proliferation and viability, acting as oncogene. However, these cells show impaired soft agar growth. In xenotransplant, ARO-miR-17-92 tumors are smaller in volume and express reduced levels of MMP-9, indicating a tumor suppressor role for the cluster.

Das COP9 Signalosom ist ein hochkonservierter Proteinkomplex bestehend, aus acht Untereinheiten. In der vorgelegten Promotionsarbeit konnte ein bislang unbekannter Regulationsmechanismus der Biogenese des COP9 Signalosoms identifiziert werden. Die siRNA-vermittelte Reduktion der CSN1-Expression führte zu einer Reduktion der Expression aller CSN-Untereinheiten. Die Transfektion von His-CSN1 in siCSN1-Zellen induzierte die CSN-Neusynthese und ferner einen Anstieg der c-Myc- und STAT1 Expression. Durch die Stimulation der Zellen mit IFN alpha bzw. IFN gamma konnte die de novo Synthese des CSN-Komplexes induziert werden. Die siRNA-vermittelte Inhibition von STAT1, c-Myc bzw. Lin28B führte ebenso wie die Behandlung der Zellen mit AG9 bzw. AG490, pharmakologischen Inhibitoren der JAK-Kinasen, zu einer Reduktion der Proteinexpression der CSN-Untereinheiten. Dabei standen signifikanten Veränderungen auf der Proteinebene geringfügige Änderungen auf der mRNA-Ebene gegenüber. Daher wurde ein post-transkriptioneller Mechanismus zur Regulation der Expression der CSN-Untereinheiten unter Beteiligung von miRNAs postuliert. Diese Regulation wird vermutlich durch die Aktivität von c-Myc und Lin28B verstärkt. Dies stellt einen neuen, bislang unbekannten Mechanismus für die Regulation der Biogenese des COP9 Signalosoms, vermutlich über den c-Myc/Lin28B/let-7-Weg, dar. Die Co-Transfektion der siCSN1-Zellen mit spezifischen komplementären Inhibitoren dieser miRNAs führte zu einer Induktion der Proteinexpression der CSN-Untereinheiten. Die Transfektion von let-7 miRNA-Mimics bewirkte eine Reduktion der Expression der CSN-Untereinheiten in den siCSN1-Zellen. Ferner konnten im Rahmen dieser Arbeit mittels der miRBase Sanger Datenbank und der Software MicroInspector Bindestellen für let-7 miRNAs an den mRNAs der CSN- und der proteasomalen Lid-Untereinheiten identifiziert werden. Der gezeigte Regulationsmechanismus könnte auch für die Biogenese des proteasomalen Lids von Bedeutung sein. / The COP9 signalosome is a highly conserved protein complex composed of eight subunits. In this study a novel, regulatory mechanism of CSN biogenesis was identified. We used stable transfected siCSN1 cells in which the protein and the mRNA expression of CSN subuntis were downregulated. Transfection of His-CSN1 in those siCSN1 cells led to the induction of the de novo Synthesis of the whole CSN complex. In addition the expression of the transcription factors STAT1 and c-Myc was elevated. The cells were treated with IFN alpha or IFN gamma, respectively. This resulted in the induction of the CSN de novo synthesis. Moreover, the siRNA-mediated inhibition of STAT1, c-Myc, Lin28B as well as treatment with the pharmacological inhibitors AG9 or AG490 led to a reduced protein expression of the analysed CSN subunits. We found that in all experiments there was a significant change on protein level in contrast to a marginal change on the RNA level. Based on our study we hypothesized that the CSN biogenesis ist regulated post-transcriptionally by miRNAs. The participation of miRNAs in the regulation of CSN biogenesis was further analysed. The siCSN1 cells were transfected with complementary hairpin inhibitors of let-7 miRNAs, leading to an induction of the CSN synthesis. The transfection of let-7 miRNA-mimics, which enhance the impact of miRNA on target mRNAs, resulted in an decrease of the CSN expression. These findings prove the involvement of miRNAs in CSN biogenesis, presumably via the c-Myc/Lin28B/let-7 pathway. Furthermore, using miRBase Sanger database and MicroInspector software, potential binding sites for let-7 miRNAs were detected within the mRNA sequences of CSN subunits as well as of subunits of the proteasomal lid. Therefore, there is evidence to suggest that this mechanism is crucial for the regulation of the biogenesis of the proteasomal lid as well.

No carcinoma papilífero de tiroide (CPT) ocorrem alterações na via MAPK (do inglês Mitogen-Activated Protein Kinase), sendo a mais frequente mutação a BRAFT1799A. A via MAPK é modulada pelo miRNA, como o let-7 que estão pouco expresso no CPT. A biogênese de let-7 é controlada por proteínas ligantes de RNA LIN28 que inibem o processamento das formas primária e precursora de let-7 (pri-let-7 e pre-let-7). Avaliamos a expressão gênica de LIN28, pri-let-7f, pre-let-7f e let-7f por rtPCR em tempo real, utilizando RNA total das linhagens TPC-1 e BCPAP de PTC e amostras tumorais de pacientes com CPT. A linhagem não tumoral Nthyori 3-1 foi utilizada para induzir LIN28. Observa-se redução de let-7f em 4 dos 5 pacientes analisados enquanto que LIN28A está aumentado em 2 pacientes com BRAFT1799A. Nota-se maior expressão de pri-let-7f em TPC-1 e menor expressão de pre-let-7f e let-7f. Observa-se maior expressão de LIN28A em TPC-1, enquanto que LIN28B está mais expresso em BCPAP com BRAFT1799A. A inibição de BRAFV600E em BCPAP diminui LIN28B e aumenta let-7f, enquanto que a indução de LIN28B reduz a expressão de let-7f. Concluímos que há relação inversa entre LIN28 e let-7f no CPT e LIN28 influencia a diminuição de let-7f podendo assim contribuir com tumorigênese tireoidiana. / In papillary thyroid cancer (PTC) changes in MAPK (Mitogen-Activated Protein Kinase) pathway are common being BRAFT1799A mutation the most frequent alteration. MAPK pathway is modulated by miRNA such as let-7, an under-expressed miRNA in PTC. The RNA binding protein LIN28 controls let-7 biogenesis, blocking primary and precursor let-7 (pri-let-7 e pre-let-7) processing. We evaluated LIN28, pri-let-7f, pre-let-7f and let-7f gene expression by real time RTPCR using total RNA of human PTC sample and PTC cell lines TPC-1 and BCPAP. Non-tumoral cell line Nthyori 3-1 was used to evaluate LIN28B influence in let-7f. We observed decreased let-7f expression in 4 out of 5 PTC patients, and increased LIN28A in 2 patients with BRAFT1799A mutation. TPC-1 cells express higher levels of pri-let-7f while pre-let-7f and let-7f are less expressed. We noted that LIN28A is more expressed in TPC-1, while LIN28B is more expressed in BCPAP cells with BRAFT1799A. The inhibition of BRAFV600E in BCPAP decreases LIN28B and increases let-7f, while the induction of LIN28B in Nthyori 3-1 reduces let-7f expression. We conclude there is an inverse association between LIN28 and let-7f in PTC, and that LIN28 influences let-7f reduction which could contribute to thyroid tumorigenesis.

No câncer, alterações em microRNAs (miRNAs), pequenos RNAs que regulam a tradução protéica, exerce efeito oncogênico (oncomiR). Os oncomiRs regulam genes chave para a proliferação celular e apoptose, sendo importantes para a biologia do câncer. O carcinoma papilífero de tiróide apresenta alterações genéticas alinhadas na via MAPK (RET>RAS>BRAF>ERK). Observamos que a indução do oncogene RET/PTC diminui a expressão de let-7 em células foliculares tiroidianas. Na linhagem TPC-1 (com RET/PTC-1), a introdução de let-7 diminui a proliferação celular e a fosforilaçãode ERK, indicando papel de gene supressor tumoral. No carcinoma anaplásico, avaliamos o papel da introdução do cluster miR-17-92 na linhagem ARO. Observamos que in vitro miR-17-92 atua de forma oncogênica aumentando proliferação e viabilidade celular de ARO. No entanto, estas células apresentam diminuição no crescimento em soft-agar. No xenotransplante, os tumores de ARO-miR-17-92 apresentam menor volume e expressam MMP-9 de forma reduzida, indicando também um papel de gene supressor tumoral para o cluster. / In cancer, alteration in microRNA, small RNAs (~22nt) that regulate post-transcriptionally protein levels, exerts oncogenic role (oncomiR). OncomiRs control genes involved in cell proliferation and apoptosis, influencing cancer biology. Papillary thyroid cancer displays activating genetic alterations in MAPK signaling pathway (RET>RAS>BRAF>ERK). Using conditional induction of oncogenes in thyroid cells, we observed that RET/PTC decreases let-7 miRNA expression. In papillary thyroid cancer cell TPC-1 (with RET/PTC-1) we observed that let-7 introduction inhibits cell proliferation and ERK phosphorylation, indicating tumor suppressor role for let-7. In anaplastic thyroid cancer, we evaluate the role of introduction of miR-17-92 cluster in ARO cell line. We observed in vitro that miR-17-92 increases ARO cell proliferation and viability, acting as oncogene. However, these cells show impaired soft agar growth. In xenotransplant, ARO-miR-17-92 tumors are smaller in volume and express reduced levels of MMP-9, indicating a tumor suppressor role for the cluster.

Le Myélome Multiple (MM) est une hémopathie maligne caractérisée par la prolifération de plasmocytes tumoraux médullaires. MYC occupe un rôle central dans l'oncogenèse du MM car son activation est responsable de la progression du stade précurseur de MGUS en MM symptomatique. Dans ce travail, nous rapportons que l’expression de LIN28B est corrélée à celle de MYC et est associée à un mauvais pronostic dans le MM. Nous montrons que l'axe LIN28B/let-7 module l'expression de l’ARNm de MYC, lui-même cible de let-7. De plus, la perturbation de l'axe LIN28B/let-7 induit une régulation la prolifération des lignées cellulaires de MM in vitro et in vivo. L'analyse par séquençage d’ARN de modèles de KO par utilisation de la technologie CRISPR a montré que l'axe LIN28B/let-7 régule les voies de signalisation de MYC et du cycle cellulaire dans MM. Nous avons de plus établi une preuve de principe thérapeutique de la possibilité de cibler MYC par l’emploi de LNA-GapmeR contenant une séquence analogue à let-7b. Dans un modèle de xénogreffe murin, nous montrons que des niveaux élevés d'expression de let-7, par administration de LNA-GapmeR let-7b, répriment la croissance tumorale en régulant l’expression de MYC. Ces résultats révèlent un nouveau mécanisme de ciblage thérapeutique de MYC via l'axe LIN28B/let-7 dans MM. Nous nous sommes ensuite intéressés à évaluer de nouvelles formes de biomarqueurs moléculaires dans le MM par étude des miARN contenus dans les exosomes circulants. Nous avons examiné le rôle pronostique des miARN exosomaux dans une cohorte de 156 échantillons de patients uniformément traités pour un MM au diagnostic. Après analyse du profil de miARN exosomaux par séquençage de nouvelle génération, nous avons utilisé technique de qRT-PCR pour étudier la corrélation entre le niveau d’expression de 22 miARN et la survie sans progression (SSP) et la survie globale (SG). Deux miARN, à savoir let-7b et miR-18a, étaient significativement associés à la SSP et SG en analyse univariée, et étaient statistiquement significatifs après ajustement pour le système international de stratification du risque (ISS) et les marqueurs cytogénétique en analyse multivariée. Nos résultats confirment le niveau d’expression des miARN let-7b et miR-18a au sein des exosomes circulants permettent d’améliorer la stratification du risque chez les patients atteints de MM. Enfin, pour mieux comprendre le programme oncogénique piloté par MYC, nous avons étudié l’efficacité thérapeutique d’une librairie de petites sur des lignées cellulaires avec une forte expression de MYC, dans le MM. Les résultats ont permis d’identifier les rocaglates, une famille de composés inhibant l’initiation de la traduction, comme étant les plus actifs. L’étude du profil transcriptionnel par séquençage de l’ARN de lignées cellulaires de MM traitées par CMLD010509 ou DMSO a révélé l’activation d’un programme de transcription et l’inhibition d’un programme traductionnel, caractéristique de l’inactivation de HSF1 secondaire à l’inhibition de la traduction. Le profile traductionnel était étudié par spectrométrie de masse quantitative, permettant d’identifier un ensemble de protéines, tels que MYC, MDM2, CCND1, MAF et MCL-1, spécifiquement affectées par l’inhibition de la traduction liée au composé CMLD010509 dans le MM. Nous avons confirmé l’efficacité thérapeutique des rocaglates dans plusieurs modèles murins de MM. Ces résultats démontrent la possibilité de cibler le programme de traduction oncogénique lié à MYC dans MM. / MYC is a major oncogenic driver of Multiple Myeloma (MM) and yet almost no therapeutic agents exist that target MYC in MM. Here we report that the let-7 biogenesis inhibitor LIN28B correlates with MYC expression in MM and is associated with adverse outcome. We also demonstrate that the LIN28B/let-7 axis modulates the expression of MYC, itself a let-7 target. Further, perturbation of the axis regulates the proliferation of MM cells in vivo in a xenograft tumor model. RNA-sequencing and gene set enrichment analyses of CRISPR-engineered cells suggested that the LIN28/let-7 axis regulates MYC and cell cycle pathways in MM. We provide proof of principle for therapeutic regulation of MYC through let-7 with an LNA-GapmeR (locked nucleic acid-GapmeR) containing a let-7b mimic in vivo, demonstrating that high levels of let-7 expression repress tumor growth by regulating MYC expression. These findings reveal a novel mechanism of therapeutic targeting of MYC through the LIN28B/let-7 axis in MM. We next sought to establish new biomarkers in MM, enable to capture the molecular alterations of the disease. For this purpose, we examined the prognostic significance of circulating exosomal microRNAs (miRNAs) in a cohort of 156 patients with newly diagnosed MM, uniformly treated and followed. Circulating exosomal miRNAs were isolated and used to perform small RNA sequencing analysis on 10 samples and a qRT-PCR array on 156 samples. We studied the relationship between miRNA levels and patient outcomes including progression-free survival (PFS) and overall survival (OS). We identified miRNAs as the most predominant small RNAs present in exosomes isolated from the serum of MM patients and healthy controls by small RNA sequencing of circulating exosomes and used a qRT-PCR assay to measure the expression of 22 exosomal miRNAs. Two of them, namely let-7b and miR-18a, were significantly associated with both PFS and OS in the univariate analysis, and were still statistically significant after adjusting for the International Staging System (ISS), and adverse cytogenetics in the multivariate analysis. Our findings support the use of circulating exosomal let-7b and miR-18a improves the identification of patients with newly diagnosed MM with poor outcomes. Finally, to better understand the oncogenic program driven by MYC and investigate its potential as a therapeutic target, we screened a chemically diverse small molecule library for anti-MM activity in cell lines with high expression of MYC. The most potent hits identified were rocaglate-scaffold inhibitors of translation initiation. Expression profiling of MM cells revealed reversion of the oncogenic MYC-driven transcriptional program by CMLD010509, the most promising rocaglate. Proteome-wide, reversion correlated with selective depletion of short-lived proteins that are key to MM growth and survival, most notably MYC, MDM2, CCND1, MAF, and MCL-1. The efficacy of CMLD010509 in several mouse models of MM confirmed the therapeutic relevance of these findings in vivo and supports the feasibility of targeting the oncogenic MYC-driven translation program in MM with rocaglates.

La famille des microARN (miARN) let-7 comprend treize membres qui jouent des rôles critiques dans de nombreux processus biologiques, notamment la différenciation et le développement cellulaires. Plus spécifiquement, ils fonctionnent comme des suppresseurs de tumeurs en ciblant plusieurs oncogènes. La dérégulation des niveaux de miARN let-7 a été associée à diverses maladies humaines, y compris des cancers et des troubles neurodégénératifs. Il est bien établi que Drosha et Dicer, appartenant à la famille des RNases III, sont deux enzymes clés de la voie de maturation des miARN, et qu'un traitement défectueux par ces endoribonucléases pourrait affecter l'expression des gènes. Au cours des dix dernières années, plusieurs recherches ont permis d'identifier les caractéristiques structurales de l'ARN et les protéines qui régulent la voie de maturation des miARN. Cependant, les détails moléculaires menant à la régulation des niveaux d’expression des miARN nécessitent des investigations supplémentaires. L'objectif principal de cette thèse est d'étudier l'activité de clivage in vitro des endoribonucléases Drosha et Dicer envers leurs substrats let-7, en se concentrant sur la façon dont diverses caractéristiques de séquence et de structure affectent leurs activités. Tout d'abord, un criblage structural de type SHAPE suivi d'investigations thermodynamiques et cinétiques détaillées pour les treize pré-miARN de la famille let-7 ont été réalisés avec une enzyme Dicer purifiée in vitro. Cette étude a révélé que malgré les différences structurales des membres de la famille let-7, Dicer ne discrimine pas entre ses substrats, y compris les pré-miARN avec une extension de 1-nt et 2-nt à leur extrémité 3'. L'ensemble de ces travaux met en évidence la remarquable promiscuité de Dicer vis-à-vis divers pré-miARN de la famille let-7. Deuxièmement, le mécanisme enzymatique du clivage du pré-let-7a-1 a été examiné. Les résultats de la cinétique de l'état stable, de l'état pré-stable et de l'impulsion-chase sont conformes à l'opinion dominante, soutenue par de récentes structures de cryo-EM, selon laquelle le ou les changements de conformation d'un complexe enzyme-substrat dans une conformation catalytiquement productive sont importants pour l'activité de clivage. Troisièmement, nous avons étudié la séquence et les déterminants structuraux du clivage du pri-let-7 par le complexe microprocesseur (MP) composé de Drosha et de son partenaire obligatoire DGCR8. Sur la base d'études de clivage de plusieurs substrats pri-let-7 avec un complexe MP reconstitué in vitro, il a été constaté que le clivage du pri-let-7g donne des produits multiples. En utilisant des variantes de pri-let-7g, il a été révélé qu'un élément structural conservé de pri-let-7g favorise un clivage improductif, peut-être en raison du clivage de son substrat par la MP dans l'orientation inverse. Cette étude fournit un cadre pour des investigations futures dans l'étude du clivage de pri-let-7g par Drosha et éventuellement l'identification de nouveaux mécanismes de régulation. Dans l'ensemble, nos résultats donnent un aperçu de la façon dont les caractéristiques structurales des pri-miARN et des pré-miARN de la famille let-7 modulent le traitement par Drosha et Dicer et ouvrent la voie à de futures études visant à examiner le rôle des facteurs protéiques dans la régulation de la maturation des miARN let-7. / The let-7 family of microRNAs (miRNAs) comprises of thirteen members that play critical roles in many biological processes, including cell differentiation and development. More specifically, they function as tumor suppressors by targeting several oncogenes. Deregulation in let-7 miRNA levels has been associated with various human diseases, including cancers and neurodegenerative disorders. It is well established that Drosha and Dicer are the two key enzymes of the miRNA maturation pathway, and that faulty processing by these endoribonucleases could affect gene silencing. Thus, it is crucial to better understand how Drosha and Dicer respectively process the primary miRNAs (pri-miRNAs) and precursor miRNAs (pre-miRNAs) to yield mature miRNAs, and how these enzymes are regulated. In the last decade of miRNA research, several investigations have identified RNA structural features and RNA-binding proteins that regulate the miRNA maturation pathway, adding another layer of regulation in this pathway. However, the molecular detail of this regulation requires further investigations. The main goal of this thesis is to investigate the in vitro processing activity of Drosha and Dicer toward their let-7 substrates, focusing on how diverse sequence and structural features affect their activities. First, SHAPE structural probing followed by detailed thermodynamic and kinetic investigations for all thirteen pre-miRNAs of the let-7 family were performed with in vitro purified Dicer. Surprisingly, this study revealed that despite structural differences in the pre-let-7 members, Dicer does not discriminate between these substrates, including pre-miRNAs with a 1 nt and a 2-nt overhang at their 3'-end. Additional binding and cleavage investigations of pre let-7 substrates carrying 3'-end modifications (mono- and oligo-uridylation, mono- and oligo-adenylation) were performed to clarify how these modifications affect Dicer binding and cleavage activities. Together, this work highlights the remarkable substrate promiscuity of Dicer toward diverse pre-miRNAs of the let-7 family. Second, the enzymatic mechanism of pre-let-7 cleavage by Dicer was examined using pre-let-7a-1 as a model substrate. The results from the steady-state, pre-steady state and pulse-chase kinetics are consistent with the prevailing view, supported by recent cryo-EM structures, that the conformational change(s) of an enzyme-substrate complex into a catalytically productive conformation are important for cleavage activity. Third, the sequence and structural determinants of pri-let-7 processing by the Microprocessor (MP) complex composed of Drosha and its obligatory partner DGCR8 were investigated. Based on cleavage studies of several pri-let-7 substrates with an in vitro reconstituted MP complex, it was found that cleavage of pri-let-7g yields multiple products. Using pri-let-7g variants, it was revealed that a conserved structural element of pri-let-7g promotes unproductive cleavage, possibly as a result of the MP cleaving its substrate in the reverse orientation. This study provides the framework for future investigations in studying pri-let-7g processing by Drosha and possibly identifying novel mechanisms of regulation. Overall, our findings provide insights on how the structural features of pri-miRNAs and pre-miRNAs of the let-7 family modulate processing by Drosha and Dicer and pave the way for future studies aimed at examining the role of protein factors in regulating the maturation of let-7 miRNAs.

MicroRNAs (miRNAs) are a diverse and evolutionarily conserved class of non-coding RNAs that play a multitude of roles in many branches of eukaryotic biology. The regulation of miRNAs is dynamically controlled both spatially and temporally, and the expression of miRNAs can be modulated at the level of transcription or at points downstream of the miRNA maturation process. A relevant example of post-transcriptional miRNA regulation is the blockade of let-7 precursor miRNAs by Lin28 in embryonic stem cells. This pathway, which is initiated by the small RNA-binding protein Lin28, recruits the terminal uridyl transferase (TUTase) Zcchc11 to add a non-templated oligouridine tail to the miRNAs 3' end, and signals it for degradation by the cytoplasmic exonuclease Dis3l2. The Lin28/let-7 axis is essential for development and metabolic homeostasis, and is reactivated in a subset of human cancers. This thesis describes the biochemical mechanism underlying Lin28-mediated degradation of let-7, as well as a novel role for Zcchc11 and the related TUTase Zcchc6 in targeting mature developmental miRNAs in a Lin28-independent manner.