La progression tumorale dépend d'un dialogue entre les cellules cancéreuses et leur environnement. Parmi les cellules du microenvironnement du mélanome, les adipocytes ont longtemps été ignorés. Pourtant, ces cellules sont le composant majeur de l'hypoderme, la couche la plus profonde de la peau. Ainsi, elles sont proches du mélanome lors de la tumorigenèse et, lorsque la tumeur envahi les couches profondes de la peau, les deux types cellulaires entrent en contact. Il est donc important de comprendre l'impact des adipocytes sur la progression du mélanome, d'autant plus que des études épidémiologiques montrent que l'obésité est un facteur de mauvais pronostic pour ce cancer. Le surpoids et l'obésité sont en hausse constante avec près d'un tiers de la population mondiale affectée, faisant du lien entre l'obésité et le cancer un enjeu de santé publique majeur. Parmi les différents moyens de communication cellulaire, les vésicules extracellulaires (VE) jouent un rôle important dans le cancer. Les VE régulent la communication entre les cellules cancéreuses mais aussi entre les composants du microenvironnement et la tumeur. Les VE sécrétées par les adipocytes sont peu caractérisées et leur rôle sur la progression tumorale reste à élucider. Les VE adipocytaires pourraient être modifiées qualitativement et quantitativement en obésité car différents stress (inflammation, hypoxie...), connus pour modifier les VE, sont retrouvées dans le tissu adipeux d'individus obèses. Dans ce contexte, le premier objectif de ma thèse était de caractériser les VE adipocytaires et déterminer leur impact sur le mélanome dans un contexte normopondéral et d'obésité. Les résultats obtenus montrent que ces VE favorisent la migration et l'invasion des cellules de mélanome. Une analyse protéomique a révélé une signature spécifique dans ces VE, fortement enrichies en protéines du métabolisme des acides gras (AG).[...] / It is now clear that tumor progression is the result of a permanent dialog between cancer cells and the tumor microenvironment (TME). Among the cells found within the melanoma microenvironment, adipocytes had long been ignored. However, adipocytes are the main component of the hypodermis, the deepest skin layer, and are therefore close to melanoma from tumorigenesis and, as the tumor becomes aggressive and invades the deeper skin layers, the two cell types come into contact. Thus, understanding how adipocytes influence melanoma progression is of major importance, especially since epidemiological studies show that obesity is a poor prognosis factor for melanoma. As overweight and obesity are constantly rising and affect around a third of the World's population, the link between obesity and cancer is a major public health issue. Among the different ways in which cells communicate, extracellular vesicles (EV) play a particularly important role in cancer. Moreover, not only can tumor cells communicate with each other through EV, but the cellular components of the TME also use EV to communicate with cancer cells. Adipocyte-derived EV are poorly characterized and their role in tumor progression remains to be determined. In obesity, adipocyte EV may be qualitatively and quantitatively altered since various stresses (inflammation, hypoxia etc.), which are known to modify EV, are found in the adipose tissue of obese individuals. In this context, the first aim of my thesis was to characterize adipocyte EV and their impact on melanoma in lean and obese individuals. The results obtained show that EV secreted by adipocytes promote migration and invasion of melanoma cells. Analysis of their proteome revealed a protein signature specific to adipocyte EV, which was highly associated with fatty acid (FA) metabolism, a metabolic pathway involved in tumor aggressiveness. In melanoma treated with adipocyte EV, fatty acid oxidation (FAO) is increased and FAO inhibitors reverse their pro-invasive effect. Moreover, adipocytes secrete increased numbers of EV in obesity and, using equal numbers of EV from lean or obese subjects, their effect on tumor aggressiveness is increased and remains dependent on FAO. T[...]

Le complexe de liaison de la coiffe des ARN (CBC) joue un rôle essentiel dans leur maturation et déclenche une variété de réactions biochimiques, via son interaction avec différents partenaires. Deux complexes, CBC-ARS2-PHAX (CBCAP), et CBC-ARS2-ZC3H18-NEXT (CBCN), ont récemment été montré comme important pour cibler les ARN vers l'export (CBCAP) ou la dégradation (CBCN). Cependant, les mécanismes par lesquels la sélection se fait pour l'une voie ou l'autre reste mystérieuse. Ainsi, une question majeure qui reste à résoudre est de savoir quand et comment ces complexes sont recrutés sur les ARN. Dans ce travail, j'ai utilisé la procédure du iCLIP (Cross-Linking and Immuno-Precipitation), afin d'identifier les cibles de ces complexes sur l'ensemble du transcriptome humain. J'ai réalisé un iCLIP sur cinq composants de CBCAP et CBCN, et j'ai comparé les résultats à ceux obtenus avec RBM7, un composant de NEXT précédemment étudié par iCLIP. Mes résultats indiquent que: (i) CBP20, ARS2, PHAX et ZC3H18 se lient près de la coiffe des ARN, tandis que RBM7 et MTR4 se lient partout; (ii) CBP20, ARS2, PHAX et ZC3H18 s'associent à un large ensemble d'ARN transcrits par l'ARN polymérase II et montrent une faible sélectivité; (iii) la liaison de ces protéines varie avec l'état de maturation des ARN, avec le CBC enrichi sur les ARN matures, tout comme ARS2/PHAX/ZC3H18 et MTR4 (bien que dans une moindre mesure), tandis que RBM7 est préférentiellement lié sur les pre-mRNAs non épissés; (iv) une liaison différentielle de RBM7 et MTR4 sur les ARN, avec RBM7 enrichi sur les introns et les PROMPTs, et MTR4 plus présent sur les ARN mature. Bien que des expériences additionnelles soient requises, nous proposons que le CBCAP et le CBCN se lient à un même ensemble d'ARN, ce qui indique à la fois une compétition entre ZC3H18 et PHAX pour la liaison à ces ARN, et l'absence de voies de routage bien déterminées qui ciblerait les ARN vers l'une ou l'autre de ces protéines. Le devenir des ARN pourrait ainsi être déterminé par d'autres caractéristiques des ARN, ou encore par des protéines additionnelles. Ces facteurs pourraient s'allier aux protéines liées à la coiffe afin de favoriser la formation du CBCAP ou du CBCN. Dans le but d’identifier des facteurs additionnels, j'ai réalisé un screen d'interaction par spectrométrie de masse après purification de ARS2 ou CBP80. Ceci a été fait dans des conditions natives ou après un cross-link des complexes à la formaldéhyde, afin de stabiliser les interactions transitoires. Ceci a permis d'identifier de nouveaux partenaires de ARS2 et de CBP80, dont la majorité sont impliqués dans l'épissage des ARN. Des expériences additionnelles seront nécessaires pour valider ces interactions. / The cap-binding complex (CBC) plays a pivotal role in post-transcriptional processing events and orchestrates a variety of metabolic pathways, through association with different interaction partners. Two CBC sub-complexes, the CBC-ARS2-PHAX (CBCAP) and the CBC-nuclear exosome targeting (NEXT) complex (CBCN), were recently shown to target capped RNA either toward export or degradation, but the mechanisms by which they can discriminate between different RNA families and route them toward different metabolic pathways still remain unclear. A major question to be answered is how and when the different CBC subcomplexes are recruited to the RNP. Here, we used an individual nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) approach to identify the transcriptome-wide targets for 5 different components of the CBCAP and CBCN complexes, and compared results to the previously analysed NEXT-component RBM7. We report that: (i) CBP20, ARS2, PHAX and ZC3H18 bind close to the cap, while RBM7 and MTR4 bind throughout the mRNA body; (ii) CBP20, ARS2, PHAX and ZC3H18 associate with a broad set of RNA polymerase II (PolII)-derived RNAs and have only mild species preferences; (iii) binding varies with the RNA maturation stage, with the CBC being highly enriched on mature mRNA, ARS2/PHAX/ZC3H18/MTR4 less so, and RMB7 preferentially bound to pre-mRNAs; (iv) MTR4 and RBM7 show different specificities, with RBM7 being highly enriched on introns and promoter upstream transcripts (PROMPTs), while MTR4 is additionally present on mature RNAs. Although more experimental work is needed to fully support our model, we propose that CBCAP and CBCN bind overlapping sets of RNAs, indicating a competition between the proteins ZC3H18 and PHAX, and the lack of a strict RNA sorting mechanism. RNA fate may therefore be determined by additional RNA features and/or by other RNA-binding proteins, which may synergize with the cap and drive the formation of one specific CBC subcomplex instead of another. In an attempt to identify yet unknown factors that may interact with cap-bound CBCAP and CBCN, we performed a protein interaction screen leveraging affinity capture-mass spectrometry (ACMS), using ARS2 and CBP80 as bait proteins. As a complementary approach, we also employed a formaldehyde-based chemical cross-linking strategy, aimed at stabilizing weak/transient interactions. Although we failed to detect any transient interactions involving the CBC, we identified several potential CBC80 and ARS2 interactors, the majority of which are involved in pre-mRNA splicing. Additional quantitative experiments are required to validate our ACMS results and confirm the existence of such protein interactions in vivo.

No description available.

Les travaux décrits dans ce mémoire visent à élucider le rôle de la protéine Pab2 dans la régulation des gènes méiotiques. La méiose est un phénomène conservé à travers l'évolution chez les organismes eucaryotes. Chez les eucaryotes unicellulaires comme la levure, la méiose accomplie [i.e. accomplit] autant un rôle de reproduction sexuelle qu'un rôle de protection de l'organisme. Chez la levure à fission Schizosaccharomyces pombe , le processus de méiose résultant de la reproduction de deux individus génère des spores, des structures résistantes aux conditions environnementales détrimentales. Lorsque la cellule enclenche la méiose, plusieurs centaines de gènes sont temporellement induits pour exprimer des gènes spécifiques aux processus méiotiques. Afin de s'assurer que des gènes méiotiques ne soient pas exprimés pendant la mitose au détriment de la cellule, des mécanismes de surveillance doivent rigoureusement surveiller l'expression de ces gènes. Afin d'éclaircir le rôle de Pab2 dans cette surveillance, nous avons utilisé une approche génétique combinée à des essais biochimiques. Des expériences d'immunoprécipitation de la chromatine combinées à des essais de substitution de promoteur ont démontré que la régulation de Pab2 sur les gènes méiotiques est posttranscriptionnelle. De plus, des expériences d'immunoprécipitation de la protéine Pab2 suivies d'analyse d'ARN ont démontré que la protéine Pab2 forme un complexe in vivo avec les ARN qu'elle régule. Des analyses dans le laboratoire ont démontré que Pab2 possède une interaction fonctionnelle et physique avec des composantes de l'exosome. Afin de déterminer l'implication de l'exosome dans la régulation des gènes méiotiques, nous avons utilisé différentes souches de levure [i.e. levures] possédant des délétions dans les gènes des composantes de l'exosome ou des facteurs coopérant avec ce complexe, en présence ou absence de Pab2. Ces expériences ont démontré que l'exosome nucléaire, particulièrement la sous-unité Rrp6, était majoritairement responsable de la dégradation des transcrits méiotiques, et ce sans l'aide de complexes coopérateurs comme le TRAMP. Afin de déterminer le facteur de sélectivité de la régulation par Pab2 et par l'exosome, nous avons utilisé des souches thermosensibles pour la protéine Mmi 1, un autre agent posttranscriptionnel impliqué dans la surveillance de l'expression des gènes méiotiques. Ces expériences ont démontré que Pab2 coopère avec Mmi1 dans la dégradation des transcrits méiotiques. Notamment, l'augmentation de l'expression des gènes méiotiques en absence de Pab2 est suffisante pour contourner la voie dépendante de meiRNA, qui est un ARN non-codant essentiel à l'initiation de la méiose. Ces résultats constituent l'évidence que la protéine Pab2 liant les queues poly(A) participe au maintient du silence de l'expression des gènes méiotiques pendant la mitose.

The RNA binding proteins Nrd1 and Nab3 function in transcription termination by RNA Pol II, acting via interactions with the CTD of the largest polymerase subunit, particularly on snRNA and snoRNA genes. They also participate in nuclear RNA surveillance and ncRNA degradation, functioning together with the exosome and the Trf-Air-Mtr4 polyadenylation (TRAMP) complexes. To better understand the signals for surveillance and ncRNA degradation, I applied an RNA-protein crosslinking approach in combination with Solexa sequencing. This approach identified in vivo binding sites for Nrd1, Nab3 and Trf4. Several million sequences were recovered and mapped to the yeast genome. This identified three classes of substrates: 1) Expected targets, including snRNAs, snoRNAs and characterized short ncRNAs. 2) Unknown but anticipated substrates, including several hundred previously uncharacterized ncRNAs that lie antisense to protein coding genes (asRNAs). 3) Unexpected targets, including many Pol III transcribed precursor RNAs. Bioinformatics analyses of the high-throughput sequencing data revealed that known binding motifs for Nrd1 and Nab3 were frequently recovered. Many recovered RNAs contained non-templated oligo(A) tails with an average of 2-5 nt length. This clearly distinguishes targets for surveillance machinery from polyadenylated mRNAs that get stabilized by polyadenylation (A70-90 in yeast). For a few selected, predicted asRNAs I was able to validate the crosslinking data by demonstrating that corresponding long RNAs are both detectable and increased by loss of Nrd1, Nab3, Trf4 or the exosome component Rrp6. Interestingly, loss of Nrd1 or Nab3 led to transcriptional read through on long asRNA transcripts. In addition, I have identified pre-TLC1 (telomerase RNA) as a target for the surveillance machinery. Processing of this long ncRNA was only poorly characterized in yeast but I could demonstrate that its transcription termination and maturation is mainly dependent on actions of the Nrd1-Nab3-Sen1, TRAMP4 and exosome complexes. It was previously reported that Nrd1-Nab3 acts only on short RNAs, due to the association with Ser5 phosphorylated CTD. My findings suggest that action of Nrd1- Nab3 is not exclusively on Ser5 phosphorylated form of the CTD. Unexpectedly the Pol II associated factors Nrd1 and Nab3 bound Pol III precursor transcripts. Surveillance of Pol III transcripts was dependent on Nrd1 and Nab3 since depletion of Nrd1 or Nab3 led to accumulation of pre-tRNAs. In addition, I could demonstrate that pre-RNase P RNA is oligoadenylated in vivo, which was dependent on Nrd1, Nab3 and Trf4. Together, my findings suggest a revised model of nuclear RNA surveillance in which Nrd1-Nab3 not only acts in co-transcriptional RNA recognition on Pol II transcripts but also post-transcriptionally on Pol III RNAs. The TRAMP complex is recruited to the defective RNA by the Nrd1-Nab3 complex, which remains associated with the RNA through the process of polyadenylation, until the exosome degrades the aberrant transcript.

Through a genome wide study of spliceosome recruitment in Saccharomyces cerevisiae, we were able to identify a set of protein-coding genes that despite the fact that they contain no introns and their mRNA is not known to be spliced; their loci were occupied by the spliceosome. Bioinformatic analysis revealed the existence of splicing signals on these genes. Detailed analysis of BDF2, a representative gene, revealed that the spliceosome negatively regulates its mRNA levels through an unconventional one-step splicing reaction that cleaves BDF2 mRNA and targets the cleavage products for degradation. In an effort to clarify the mechanism of spliceosome recruitment to BDF2 locus, we identified that Bdf1, the redundant to Bdf2 factor, is required for the recruitment of the spliceosome at BDF2 and the subsequent down-regulation of its mRNA levels. The above led us to propose a new role for the spliceosome in the regulation of gene expression. Finally, we investigated the generality of this regulatory mechanism is S. cerevisiae and identified a set of genes which can be differentially spliced and whose physiological expression could be potentially regulated by the spliceosome.

Background: Acute kidney injury (AKI) is a highly prevalent clinical disorder with significant mortality and no current treatment. The Burns Lab has previously shown that endothelial colony forming cells (ECFCs) release exosomes highly enriched in pro-survival micro-RNA-486-5p. In our mouse model of AKI, intravenous (i.v.) injection of ECFCs or their exosomes protects against kidney ischemic injury, associated with reduction in PTEN, a target of miR-486-5p. Mechanisms mediating recruitment and retention of exosomes are unclear. The interaction of CXC chemokine receptor type 4 (CXCR4) with stromal cell-derived factor (SDF)-1α promotes ECFC adhesion and migration in hypoxic endothelial cells. Whether exosomal miR-486-5p is critical to the prevention of ischemic injury is unclear. The current study aimed to investigate biodistribution and targeting mechanisms of ECFC-derived exosomes, to investigate the delivery and therapeutic potential of miR-486-5p alone, and to determine whether sex differences alter the treatment efficacy. Methods: ECFC-derived exosomes were isolated from cultured media by differential centrifugation and characterized using nanoparticle tracking analysis and immunoblot. Kidney ischemic injury was induced in male and female FVB mice by bilateral renal vascular clamping (30 min). Exosomes (20 µg) or Invivofectamine-mimic complex containing miR-486-5p (1mg/kg) were injected at the start of kidney reperfusion via tail vein. Organs were removed and assays were performed to identify structure and function. In vitro cell studies were also used when necessary. Results: ECFC-derived exosomes preferentially target the ischemic kidney, its endothelium and tubular epithelium, which correlates with increases in miR-486-5p. The transfer of exosomes may be mediated by macropinocytosis by target cells. The SDF-1α/CXCR4 axis plays a role in targeting exosomes to the site of injury. miR-486-5p alone has a similar therapeutic efficacy in preventing ischemia/reperfusion injury as ECFC-exosomes in the mouse model of AKI. Both male and female mice respond to both therapies, however female mice are protected against ischemia reperfusion injury. Conclusions: These results suggest that the protective effects of ECFCs or their exosomes in ischemic AKI may be largely mediated by pro-survival miR-486-5p. These data provide further support for the promising therapeutic potential of ECFC-derived exosomes and miR-486-5p in human AKI.

Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. Previous studies have demonstrated that the sexually transmitted pathogen, Neisseria gonorrhoeae, can induce or inhibit host cell death. N. gonorrhoeae is a mucosal pathogen and, in females, initiates infection in epithelial cells of the ectocervix and endocervix. Mucosal epithelial cells of the female genital tract are the first line of defense, and thus their cellular fate can alter the early immune response to invading pathogens such as N. gonorrhoeae. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In the present study, we demonstrate that N. gonorrhoeae stimulation induces a transient increase in cIAP2 protein levels in human cervical epithelial cells. High intracellular protein levels were observed during early N. gonorrhoeae stimulation and were followed by a marked intracellular decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes, which are nano-sized vesicles that carry protein and coding RNA as cargo from one cell to another. We also observed that depletion of cIAP2 in N. gonorrhoeae stimulated cells resulted in cell death resembling necroptosis, an inflammatory form of cell death. Furthermore, inhibition of cIAP2 led to an increase in interleukin-1β production. Exosomes have been found to have important roles in cell communication during microbial infection. Here, we demonstrate that N. gonorrhoeae induces exosome production and alters exosome content. We also demonstrate that exosomes elicit cytokine responses in uninfected naïve cells. Collectively, these studies highlight an additional mechanism for epithelial cells to orchestrate the immune response in the female genital tract during N. gonorrhoeae infection.

O exossomo é um complexo constituído por até 11 subunidades (Rrp4p, Rrp6p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Csl4p, Mtr3p) que possui atividade exorribonucleolítica 3`→5` e está envolvido no processamento e degradação de vários tipos de RNAs na célula eucariótica. O complexo tem sido estudado em diversos organismos, como leveduras, insetos, plantas, humanos e também em várias espécies de archaea. Apesar da conservação da estrutura do exossomo ao longo da evolução e de oito subunidades do exossomo eucariótico apresentarem domínios de RNase, apenas duas proteínas, Rrp6p e Rrp44p têm atividade catalítica. A despeito da importância do exossomo para a célula, ainda não está claro o papel de cada subunidade na atividade do complexo. Neste trabalho foram utilizados mutantes da subunidade Rrp43p a fim de avaliar como mutações pontuais nesta subunidade afetam a montagem e estabilização do complexo do exossomo de Saccharomyces cerevisiae. Ensaios de purificação do exossomo com TAP-Rrp43p revelaram que os mutantes co-purificam Mtr3p e Rrp44p menos eficientemente. Além disso, os mutantes também apresentam atividade exorribonucleolítica 3`→5` reduzida, indicando que o defeito na montagem do complexo pode afetar a sua atividade enzimática. / The exosome is a protein complex comprised of up to eleven subunits (Rrp4p, Rrp6p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Csl4p and Mtr3p) that has 3`→5` exoribonucleolytic activity and is involved in degradation and processing pathways of several kinds of RNA in eukaryotes. This complex has also been identified in several organisms, such as yeast, insects, plants, humans and also many species of archaea. Despite the overall structure conservation of the complex throughout evolution and eight of the eukaryotic exosome subunits displaying RNase domains, only two proteins, Rrp6p and Rrp44p have catalytic activity. Although the exosome has been shown to be involved in many different aspects of RNA metabolism, the role that each subunit plays in the activity of the complex has not yet been determined. In this work we used of TAP-purified exosome complexes to study the effect of Rrp43p mutations on the assembly and stabilization of the complex in Saccharomyces cerevisiae. Co-immunoprecipitation assays revealed that Rrp43p mutants co-purify Mtr3p and Rrp44p subunits less efficiently. Besides, Rrp43p mutants also present decreased activity, indicating that an assembly defect may affect its enzymatic activity

Background: Exosomes have great potential in regenerative medicine through the transfer of their bioactive cargos, such as RNA. tRF RNA and tiRNA are tRNAderived non-coding RNA. Here, we sought to identify the tRF/tiRNA profile in human mesenchymal stem cell (hMSC) exosomes. Methods: Bone marrow hMSCs were cultured with/without osteogenic differentiation medium and exosomes were harvested. RNA was extracted from: 1) control cells (Cell-NT); 2) control exosomes (EXO-NT); 3) differentiated cells (Cell-OM); 4) exosomes produced by differentiated cells (EXO-OM). RNA was sequenced to profile the small RNA with a focus on tRF/tiRNA. Results: tRF/tiRNA was highly enriched in hMSC exosomes. Less diversity was seen in the tRF/tiRNA profile in exosomes than that in parent cells. Selective tRF/tiRNA were packed into MSC exosomes and their profile is dependent on the cell maturation status. Conclusions: Our results suggest that tRF/tiRNA may play a role in mediating the function of exosomes in tissue regeneration.