Abstract   The prostate gland, a male organ, situated right under the urine bladder, is involved in male reproduction. It can also be the place for more or less serious diseases such as inflammation, abnormal growth and cancer. Especially prostate cancer is very common in the Western world. Today PSA is the most widely used marker for detection of prostate cancer. Unfortunately, this method is not specific enough. Therefore, there is a need for a better marker for screening of malignant prostate cancer. The marker should be specific both for the organ prostate and for the cancer disease. One promising marker is the prostasome, a small vesicle emanating from epithelial cells in the ejaculatory ducts in the prostate. The aim of this project was to set up an ELISA and test a number of antibodies for their ability to work as suitable capture or detection antibodies. As blocking agent different concentrations of BSA were tested. Biotin-Streptavidin conjugate was used in the detection step. Two surface proteins, PSCA and PSMA were used as capture antigens; they are specific for prostasomes. Clusterin, a prostasomal surface-bound protein, was used as antigen for the secondary antibody in the assay. With this experimental setup the detection limit was 2500ng/mL, which is probably not enough to detect prostasomes in cancer. The development of the ELISA did not reach its final stage, a ready-to-use assay, during this project. We have not yet the knowledge of optimal antibody concentrations and the other test parameters are also at experimental state.

Les PABPs sont des protéines liant les répétitions d’adénosines se trouvant à l’extrémité 3’ des transcrits d’acides ribonucléiques messagers (ARNm) et par conséquent pouvant interagir avec la majorité d’entres eux. Cette classe de protéine est retrouvée chez la majorité des eucaryotes, mais elle semble absente chez les procaryotes. Il existe deux classes de PABPs : cytoplasmique et nucléaire. Mis à part les PABPs cytoplasmiques, il existe une PABP avec une localisation majoritairement nucléaire appelée PABPN1. Chez l’humain, PABPN1 est impliquée dans plusieurs processus cellulaires : la polyadénylation nucléaire des ARNms, le clivage et la polyadénylation alternative, l’expression de certains longs ARNs non-codant (lncARN) et l’export des ARNms du noyau vers le cytoplasme. La protéine contient plusieurs domaines dont une région riche en alanine. Cette région est sujette à des expansions trinucléotidiques qui ont pour conséquence l’ajout d’alanines supplémentaires. Ces ajouts entrainent, à long terme, l’apparition de la dystrophie musculaire oculopharyngée (DMOP). L’influence des expansions trinucléotidiques chez les patients atteints de la maladie est encore mal connue. En utilisant des cellules exprimant le transgène GFP-PABPN1, nous avons observé une régulation de l’expression de la protéine endogène, ce qui suggère une autorégulation du gène PABPN1. De plus, nous avons observé la présence de deux populations de transcrits du gène : un premier transcrit mature (ARNm) et le deuxième contenant l’intron terminal de PABPN1 (pre-ARNm). Expérimentalement, nous avons observé que l’expression du transgène module le ratio pre-ARNm/ARNm de l’endogène. Aussi, en utilisant un gène rapporteur (GFP-6/7) qui se comporte comme l’endogène, nous avons démontré que l’autorégulation de PABPN1 nécessite la présence d’un intron terminal ayant une séquence non optimale pour l’épissage. En plus, nos expériences suggèrent que l’autorégulation est indépendante des répétitions d’adénosines présentes à la fin du transcrit (queue poly-A). Par la suite, nous avons remarqué la présence d’une séquence riche en adénosine (A-riche) à l’intérieur de l’exon terminal de PABPN1. Nous avons démontré que cette séquence est liée par PABPN1 in vitro et in cellulo qu’elle influence l’autorégulation en inhibant l’épissage du transcrit. De plus, nous avons mis en évidence que le mécanisme de l’autorégulation passe par une compétition entre des facteurs qui influencent l’épissage et la dégradation du pre-ARNm par l’exosome nucléaire.

DGCR8 (DiGeorge critical region 8) protein constitutes part of the Microprocessor complex together with Drosha, and is involved in the nuclear phase of microRNA (miRNA) biogenesis. DGCR8 recognises the hairpin RNA substrates of precursor miRNAs through two double-stranded RNA (dsRNA) binding motifs and acts as a molecular anchor to direct Drosha cleavage at the base of the pri-miRNA hairpin. Recent characterisation of the RNA targets of the Microprocessor by HITSCLIP of DGCR8 protein revealed that this complex also binds and regulates the stability of several types of transcripts, including mRNAs, lncRNAs and retrotransposons. Of particular interest is the binding of DGCR8 to mature small nucleolar RNA (snoRNA) transcripts, since the stability of these transcripts is dependent on DGCR8, but independent of Drosha. This raises the interesting possibility that there could be alternative DGCR8 complex/es using different nucleases to process a variety of cellular RNAs. We performed mass spectrometry experiments and revealed that DGCR8 copurifies with subunits of the nucleolar exosome, which contains the exonuclease RRP6. We demonstrated DGCR8 and the exosome form a nucleolar complex, which degrade the mature snoRNAs tested within this study. Interestingly, we also show that DGCR8/exosome complex controls the stability of the human telomerase RNA component (hTR/TERC), and absence of DGCR8 creates a concomitant telomere phenotype. In order to identify the RNA targets of the DGCR8/Exosome complex on a global scale we performed iCLIP of endogenous and overexpressed RRP6 (wild-type and a catalytically inactive form). Thus, intersection of CLIP datasets from DGCR8 and RRP6 identified common substrates; accordingly snoRNAs were the most represented. In addition, we identified the cellular RNA targets of the RRP6 associated human exosome. The use of a catalytically inactive form of RRP6 stabilised important in vivo interactions that are highly dynamic and transient and also highlighted the role of RRP6-mediated trimming of 3’flanks of immature non-coding RNAs. We will present a global view of the RNA-binding capacity of the RRP6-associated exosome. In sum, we identified a novel function for DGCR8, acting as an adaptor to recruit the exosome to structured RNAs and induce their degradation. Moreover, we have identified DGCR8-depenedent substrates of the exosome and have demonstrated the requirement of RRP6 for 3’ processing of ncRNAs.

No description available.

While enteroviruses, including poliovirus, are conventionally released upon cell lysis, recent studies show that phosphatidylserine-enriched infectious extracellular vesicles (IEVs) shed by infected cells can transport clusters of enteroviruses from cell to cell, resulting in increased infectivity. Combining structural and biochemical analyses, we focused on IEVs shed from poliovirus-infected cells, a classical prototype for studying enteroviruses. Transmission cryo-electron microscopy, cryo-electron tomography and computational reconstruction, present the first three-dimensional structures of well-preserved IEVs and purified exosomes. We observed that single-membraned IEVs present a wide size range in diameter. Clusters of virions can be either densely packed within a protein-coated irregularly shaped IEV, or concentrated at one or both ends of an IEV, forming a polar structure. In addition to virions, IEVs often contain internal vesicles, “ramen-noodle”-like structures with strong density, and partially assembled virion-like structures. Viral replication complex components, including viral proteins polymerase 3D, 3CD, 3A, 3AB, 2BC, 2C and (+) and (-) stranded RNAs were detected in IEVs. Furthermore, (-) stranded RNA templates are protected by the IEVs, not packed in viral capsids. The transported viral replication components (viral proteins and RNAs) and virions within IEVs initiate a stronger and faster viral replication in recipient cells than free virions. Both cryo-electron tomographic and mass spectrometry data also showed that virions and “ramen-noodle”-like structures were also observed in purified CD9 positive exosomes from poliovirus-infected cells. Viral protein 3AB, detected on the membrane of IEVs, can invaginate membranous structures to engulf large proteins into a closed lumen. Our study demonstrates that IEVs can transport viral replication complex components to initiate a rapid onset of viral replication, as part of a novel viral transmission mechanism. Viral protein 3AB may contribute to forming IEVs throughout the infection. / 2019-06-12T00:00:00Z

The essential protein Mtr4 is a conserved Ski2-like RNA helicase that maintains the integrity of nuclear RNA by promoting the 3' end decay of a wide variety of RNA substrates. Mtr4 activates the multi-protein exosome in RNA processing, surveillance, and turnover pathways by unwinding secondary structure and/or displacing associated proteins from RNA substrates. While Mtr4 may be able to promote decay independently, it is often associated with large multi-protein assemblies. Specifically, Mtr4 is the largest member of the TRAMP (Trf4/Air2/Mtr4 polyadenylation) complex which targets a plethora of RNA substrates for degradation by appending them with small (~5nt) poly(A) tails via the polymerase activity of Trf4. Mtr4 preferentially binds and unwinds RNAs with short poly(A) tails. Notably, the mechanism by which Mtr4 recognizes the length and identity of the RNA 3' end is coupled to the modulation of poly(A) polymerase activity of Trf4. The lack of structural data for Mtr4 and associated complexes severely limits the understanding of Mtr4 function. Particularly, it is unclear how Mtr4 senses RNA features, acts on RNA substrates, delivers RNA substrates to the exosome, and assembles into larger protein complexes. Presented here is the x-ray crystal structure of Mtr4 combined with detailed structural and biochemical analysis of the enzyme. The structure reveals that Mtr4 contains a four domain helicase core that is conserved in other RNA helicases and a unique arch-like RNA binding domain that is required for the in vivo processing of 5.8S rRNA. Furthermore, kinetic and in vivo analysis of conserved residues implicated in the poly(A) sensing mechanism demonstrates that ratchet helix residues regulate RNA unwinding and impact RNA sequence specificity. A comparison of the apo Mtr4 structure with the RNA/ADP bound structure (determined elsewhere) provides a view of the range of motion that individual domains of Mtr4 adopt upon substrate binding as well as the possible conformations that occur during RNA translocation. These studies provide an important framework for understanding the fundamental role of Mtr4 in exosome-mediated RNA decay, and more broadly describe common themes in architecture and function of the Ski2-like helicase family.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20672号 / 医博第4282号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 野田 亮, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

La mucoviscidose est une maladie génétique due à des mutations du gène CFTR, conduisant à une altération de la fonction de canal à ions chlorure de la glycoprotéine transmembranaire CFTR associée à une atteinte pulmonaire sévère. Plusieurs études récentes ont amené à reconsidérer l’utilisation des vecteurs adénoviraux (Ad) de sérotype 5 (Ad5) dans la mucoviscidose, lesquels induisent non seulement des réactions immunes anti-adénovirales mais aussi des effets cytopathiques indésirables. (1) Dans une première partie de notre étude, nous avons étudié l’entrée et le transit intracellulaire de l’Ad5/F35, vecteur chimérique portant les fibres de l’Ad sérotype 35 sur une capside de sérotype 5. Nous avons montré que la protéine fibre est déterminante dans l’internalisation et le trafic intracellulaire de ce vecteur. Le vecteur Ad5/F35 exprimant la fusion GFP-CFTR s’est révélé (i) être dépourvu de cytotoxicité, (ii) transduire efficacement les cellules épithéliales pulmonaires par voie apicale, et (iii) restaurer l’activité de canal à chlorure dans les cellules CFTR(-). Il constitue donc un vecteur de transfert du gène CFTR potentiellement utilisable en thérapie génique de la mucoviscidose. (2) Dans une seconde partie, nous avons exploré une stratégie alternative de transfert de la protéine CFTR par trogocytose. Nous avons fait l’hypothèse que le canal CFTR pouvait être véhiculé par des microvésicules ou microparticules membranaires (MP) émanant de la membrane cellulaire et libérées dans le milieu de culture. En utilisant un système d’expression stable de la protéine CFTR étiquetée par la protéine fluorescente GFP (GFP-CFTR) dans des cellules donneuses, nous avons pu démontrer que les MP sont capables de prendre en charge et délivrer la protéine GFP-CFTR à des cellules réceptrices, mais ce transfert n’est assuré que par une population réduite de MP (≤ 8 %), et la durée de vie du GFP-CFTR n’est que transitoire (≤ 24h). En fait, la majorité des MP transfèrent des molécules d’ARN messager ou polysomal GFP-CFTR. La protéine GFP-CFTR néosynthétisée à partir de ces ARNm est exprimée plus tardivement (> 48h) mais de façon prolongée (≥ 10 jours). La fonctionnalité du canal CFTR ainsi néosynthétisé est en cours d’évaluation. Les MP constituent donc un nouveau type de vecteurs de transfert non génique du CFTR qui pourraient être employés en thérapie de la mucoviscidose. / The cystic fibrosis (CF) is a genetic disease due to mutations of the CFTR gene, resulting in the alteration of the Cl channel function carried by the transmembranal glycoprotein CFTR, and associated with severe pulmonary complications. Several recent studies led the medical and scientific community to reconsider the use of adenovirus serotype 5 (Ad5)-based vectors as CFTR gene transfer vectors in CF gene therapy. Not only immune response against the vector itself and the ansduced cells have been observed, but also Ad5-induced nondesired cytopathic effects. (1) In the first part of our study, we analyzed the cell entry and traficking of Ad5/F35, a chimeric vector consisting of serotype 5 capsid carrying serotype 35 fibers. We showed that the fibre protein is the major, if not only, determinant of the internalisation and entry pathway of Ad5/F35. Ad5/F35-GFP-CFTR, expressing the fusin protein GF-CFTR, was found (i) to be devoid of detectable cytopathic effect, (ii) efficiently transduced airway epithélial cells via the apical pole, and (iii) restore the Cl channel function in CF cells. Ad5/F35 therefore represents a CFTR gene transfer vector with a great potential for gene therapy of CF. (2) In the second part of our study, we have investigated an alternative strategy based on the transfer of the mature CFTR protein via trogocytosis. We hypothesized that microvesicles or microparticules (MP) issued from the cell membranes and released into the culture medium could transport and achieve the cell-to-cell transfer of CFTR channel cargo. We engineered donor cells for stable expression of GFP-tagged CFTR protein (GFP-CFTR), and showed that donor cell-issued MP were capable of delivering GFP-CFTR protein to recipient cell. However, the GFP-CFTR protein was only transferred by a limited population of MP (≤ 8 %), and was only transient (≤ 24h). In fact, the major population of MP transferred mRNAGFP-CFTR or polysomal thereof. Interestingly, the GFPCFTR protein newly synthesized from this mRNAGFP-CFTR was expressed at late times after transfer (≥ 48 h) but in a prolonged manner (≥ 10 jours). The Cl canal function after MP-mediated CFTR transfer is being evaluated. MP represent a novel type of CFTR vectors which can be produced by specifically designed autologous donor cells, and which would overcome most of the inconveniences of gene therapy using viral or nonviral vectors.

Les exosomes sont des nanovésicules de 30 à 100nm sécrétées dans le milieuextracellulaire par une grande majorité de types cellulaires. Entourés d’une bicouchelipidique similaire aux radeaux lipidiques, ils contiennent des protéines, de l’ARNm et desmicroARNs. Récemment, il a été montré que les exosomes pourraient participer auxdialogues moléculaires inter-organes, au même titre que les protéines solubles (hormones etcytokines). Au cours de cette thèse, nous avons émis l’hypothèse que le muscle squelettiquepourrait utiliser les exosomes comme mode de communication intercellulaire, en plus desmyokines qu’il sécrète. Nous avons montré en couplant des techniques de microscopieélectronique, de génomique, de biologie cellulaire et moléculaire, et d’analysesprotéomiques, que le muscle était capable de sécréter des exosomes, dont la compositionvariait au cours de la myogenèse. De plus, nous avons montré que les exosomes sécrétéspar les cellules prolifératrices et différenciées avaient des rôles distincts au cours de ladifférenciation myogénique, via le transfert des microARNs notamment.En parallèle, nous nous sommes intéressés aux exosomes sécrétés par le musclesquelettique en situation d’insulino-résistance induite par du palmitate. En utilisant unedouble approche in vitro et in vivo, nous avons montrés que les exosomes sécrétés par lacellule musculaire insulino-résistante ont une morphologie et une composition luminalemodifiée. Enfin, ces exosomes sécrétés sembleraient transmettre un signal délétère àd’autres cellules musculaires différenciées, et aux autres tissus insulino-sensibles, comme lepancréas. / Exosomes are nanovesicles from 30 to 100nm, secreted into the extracellular spaceby a large majority of cell types. Surrounded by a lipid bilayer similar to lipid rafts, theycontain proteins, mRNA and microRNAs. Recently, it has been shown that exosomes maybe involved in inter-organs dialogues, as well as soluble proteins (hormones and cytokines).In this thesis, we hypothesized that skeletal muscle could use exosomes asintercellular communication mode, in addition to myokines. We have shown by couplingelectron microscopy techniques, genomics, molecular and cellular biology, and proteomicanalyzes, that the muscle was able to secrete exosomes, whose composition varied duringmyogenesis. In addition, we have shown that exosomes secreted by proliferating anddifferentiated cells have distinct roles during the myogenic differentiation, especially throughthe transfer of microRNAs. In parallel, we are interested in exosomes secreted by insulin resistant skeletalmuscle, induced by palmitate. Using a dual approach in vitro and in vivo, we have shown thatexosomes secreted by insulin-resistant muscle cells have a morphology and a luminalcomposition modified. Finally, these exosomes secreted seem to transmit a deleterioussignal to other differentiated muscle cells, and other insulin-sensitive tissues such as thepancreas.