京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23841号 / 薬科博第156号 / 新制||薬科||17(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

Pentavalent antimonials [Sb(V)] are the primary drug of choice against all forms of leishmaniasis. Emergence of antimony unresponsiveness is a major issue. There is a dire need of understanding antimony resistance mechanisms in Leishmania. One important mechanism is the down regulation of the trivalent antimony [Sb(III)] (the active form of Sb(V)) uptake system. To date, Leishmania aquaglyceroporin AQP1 is the only reported facilitator of Sb(III). Leishmania do not have promoters. They primarily regulate their genes at post-transcriptional and/or post-translational levels. We reported that mitogen activated protein kinase 2 (MPK2) positively regulated AQP1 stability through the phosphorylation of the threonine 197 (T197) residue of AQP1. The goal of this study was to elucidate the regulatory mechanism(s) of AQP1 in Leishmania in order to advance our understanding about the physiological role(s) of AQP1 in Leishmania biology. When Leishmania promastigotes were treated with the proteasome inhibitor MG132, SbIII accumulation was increased due to upregulation of AQP1. Alteration of lysine 12 of AQP1 to either alanine or arginine improved protein stability. Cells co-expressing a dominant-negative MPK2 mutant exhibited severely reduced AQP1 expression, which was reversed upon addition of MG132. Interestingly, the dominant-negative MPK2 mutant could not destabilize either AQP1K12A /AQP1K12R. Stabilization of AQP1 by MPK2 led to its relocalization from the flagellum to the entire surface of the parasite. Both altered AQP1K12A and AQP1K12R were restricted to the flagellum only. The data demonstrated that lysine12 was targeted for AQP1 proteasomal degradation playing an integral role in subcellular localization of AQP1 as well as its interaction with MPK2. This study also demonstrated that the stability of AQP1 mRNA in different Leishmania species was regulated by their respective 3’-untranslated regions. Cutaneous leishmaniasis causing species accumulated more antimonite and therefore, exhibited higher sensitivity to antimonials than species responsible for visceral leishmaniasis. This species-specific differential sensitivity to antimonite was found to be directly proportional to the expression levels of AQP1 mRNA. The differential regulation of AQP1 mRNA explained the distinct antimonial sensitivity of each species. This study will help us to identify new drugs for treatment in the future and also lead to a novel understanding of parasite biology aspects such as integral membrane protein trafficking and regulation.

RNA interference (RNAi) is a conserved pathway that plays key roles in heterochromatin formation, gene regulation and genome surveillance across a wide range of eukaryotes. One of the most utilised model organisms for studying the RNAi pathway is the fission yeast Schizosaccharomyces pombe. However, this species is somewhat atypical, in that it has not retained the ancestral role for RNAi in the silencing of mobile genetic elements. In contrast, the related fission yeast S. japonicus has a large and diverse retrotransposon complement that appears to give rise to abundant siRNAs. For this reason, we believe that S. japonicus may be a more suitable model for studying the role of RNAi in silencing mobile genetic elements, a function that is conserved in many higher eukaryotes. Functional analysis of the S. japonicus RNAi pathway proved more challenging than expected, as it was generally not possible to recover strains bearing deletions of core RNAi components (Ago1/Clr4/Rdp1/Arb1/Arb2). This suggests that a functional RNAi pathway may be required for viability in S. japonicus, unlike in S. pombe. However, disruption mutants were isolated for the sole Dicer ribonuclease Dcr1, at very low frequency. Analysis of these mutants revealed that disruption of Dcr1 impaired the generation of retrotransposon derived siRNAs, and caused de-repression of retroelement transcript accumulation and mobilisation in an element dependent manner. Surprisingly however, Dcr1 appeared dispensable for the maintenance of H3K9me2 at transposons, suggesting that, in contrast to S. pombe, silencing may occur principally at the post-transcriptional level. It is also possible that the isolated Dcr1 mutants represent rare survivors that are viable due to the presence of suppressor mutations elsewhere in the genome. I utilised my genome wide RNA sequencing data to help improve the annotation of the S. japonicus genome, with a specific focus on the retrotransposon complement. From this, I identified 12 new families of LTR retrotransposon, which increased the annotated retrotransposon complement by around 40% in S. japonicus. Finally, I characterised the integrative preference of the S. japonicus retrotransposon Tj1, and found that it shares characteristics associated with the S. cerevisiae retrotransposons Ty1 and Ty3, mostly integrating upstream of RNA PolIII transcribed tRNA genes. The findings of this work highlight some potentially key differences in the way the RNAi pathway functions across the fission yeast clade, both in terms of its importance for viability and its mode of action. The work undertaken here also contributes to the establishment of S. japonicus as a model for the study of RNA interference and genome regulation.

La découverte de l’ARN interférence et des micro-ARN a permis de définir un principe majeur de régulation de l’expression des gènes, et a produit de nouveaux outils pour la médecine. Chez les mammifères, l’étude des fonctions des micro-ARN a été restreinte au cytoplasme, bien qu’ils soient aussi présents dans le noyau.Cette thèse présente une série d’expériences visant à caractériser les facteurs moléculaires requis pour l’activité nucléaire des micro-ARN. Nous avons débuté ce projet en explorant les partenaires ARN-dépendant de la protéine AGO2 par immunoprécipitation et spectrométrie de masse quantitative. Parmi les interactants ARN-dépendants, nous nous sommes concentrés sur trois protéines nucléaires abondantes : SFPQ, PSPC1 et NONO qui forment la famille drosophila behavior and human splicing (DBHS). Nous avons démontré que le complexe RISC nucléoplasmique est associé aux protéines SFPQ, PSPC1 et NONO dans plusieurs lignées cellulaires murines et humaines, d’une manière qui dépend de SFPQ. Des expériences de type HITS-CLIP de la protéine AGO2 et/ou de la protéine SFPQ dans des cellules souches nous ont permis de montrer que SFPQ se lie préférentiellement aux 3’UTR longs en utilisant deux motifs spécifiques. En effet, SFPQ contrôle significativement environ 20% de l’activité de liaison de AGO2, ce qui est répercuté au niveau transcriptomique. Cependant, cette activité concerne uniquement les sites de liaison de SFPQ proches (<500 nucléotides) de AGO2. De plus, nous avons observé que cette régulation s’étend aux ARNm cytoplasmiques. Ce résultat suggère que la liaison et l’agrégation de la protéine SFPQ à l’ARN programme la structure du 3’UTR et donc les possibilités de ciblage par les miARN dans le noyau, et ceci d’une manière qui semble préservée dans le cytoplasme. Enfin, nous avons montré en particulier que l’expression de SFPQ contrôle le programme de ciblage par let-7a, et module la transition des cellules souches vers l’état différencié.
Ces résultats contribuent à la diversité des mécanismes de régulation de l’activité des miARN. Dans la deuxième partie du projet, nous avons exploré les partenaires ARN-indépendant de la protéine nucléaire AGO2. Nous avons découvert que la protéine AGO2 interagit avec le complexe CCR4-NOT1 et l’exosome nucléaire d’une manière indépendante de l’ARN. Nous proposons une série d’expériences visant à confirmer ces résultats. Brièvement, l’hypothèse de travail qui semble la plus cohérente avec les données actuelles est la liaison directe de l’exosome au module CNOT2-CNOT3 du complexe CCR4-NOT1. Ce modèle permettrait d’expliquer le mécanisme d’extinction des gènes par les miARN nucléaires qui reposerait donc sur leur interaction avec les complexes CCR4-NOT1 et exosome. Son mode opératoire comprendrait des protéines de liaison à l’ARN et des micro-ARN pour sélectionner les cibles. / The discovery of RNA interference and micro-RNA has unravelled a fundamental principle of gene expression regulation, and has produced new tools for medicine. In mammals, the study of micro-RNA functions have been confined to the cytoplasm, although there is a growing body of evidence about their presence in the nucleus.This thesis present a set of experiments directed towards understanding the molecular factors required for nuclear miRNA activity.We started this project by exploring the RNA-dependent interactors of AGO2 by immunoprecipitation followed by quantitative mass spectrometry analysis. We focused on three partners: SFPQ, PSPC1 and NONO which are abundant nuclear proteins and belong to the DBHS family (drosophila behavior and human splicing). We demonstrated that the nucleoplasmic RISC complex associates with DBHS proteins in multiple murine and human cell lines in an SFPQ-dependent manner.HITS-CLIP experiments of AGO2 and SFPQ proteins in mouse embryonic stem cells showed that SFPQ preferentially binds long 3’UTR using two specific motifs. Using these motifs, SFPQ significantly controls about 20% of local AGO2 binding activity and target mRNA stability as we observe by transcriptomic analysis. SFPQ mode of action appears to be local and SFPQ motifs are functional only when proximal to AGO2 binding sites in a window of 500 nucleotides.Moreover, although SFPQ is only nuclear, we observe that SFPQ has an effect on cytoplasmic mRNAs, which suggests that SFPQ binding and aggregation to mRNA in the nucleus programs the 3’UTR for miRNA targeting, in a way that appears preserved in the cytoplasm.In particular, we found that SFPQ controls let-7 targeting program and modulates embryonic stem cell differentiation into neuron cells.These results contribute to expand the diversity of miRNA regulatory mechanisms.In the second part of the project, we explored the RNA-independent interactors of AGO2. We discovered that nuclear AGO2 interacts with CCR4-NOT1 complex and the RNA exosome in an RNA-independent manner.We propose a set of experiments to confirm these results, based on a new working model. Briefly, the most likely hypothesis to explain the current data is the direct binding of RNA exosome to the CNOT2-CNOT3 module of CCR4-NOT1 complex.This model would explain the silencing mechanism of genes by nuclear miRNAs, whose activity would depend on the RISC complex interaction with CCR4-NOT1 complex and RNA exosome.Hence, the RNA exosome would use both RNA binding proteins and miRNAs to select targets for degradation, based on CCR4-NOT1 mode of action.

In mammals, circadian rhythms are driven by a cell-autonomous core-clock mechanism consisting of over a dozen core-clock genes forming transcription-translation feedback loops. The core-clock mechanism also drives the rhythmic expression of downstream genes called clock-controlled genes, which are thought to be important for driving rhythmic biochemical and physiological processes. Mathematical models predict that for a gene to be rhythmically expressed, synthesis, degradation, or a combination of the two must be rhythmic. The purpose of this project was to investigate the contribution of synthesis and degradation of RNA to rhythmic gene expression. To systematically understand the contribution of synthesis, degradation, and other RNA dynamics to rhythmic gene expression, I used metabolic labeling and a novel computational pipeline to analyze transcriptomic data in synchronized NIH3T3 cells. I identified 685 rhythmically expressed RNAs with a period of 24-hour in my dataset, of those 389 were rhythmically synthesized and 24 were rhythmically degraded. Low amplitude degradation rhythms were detected more broadly in the 685 rhythmically expressed RNAs, but these were not statistically significant. Although synthesis was the primary driver of rhythmic 24-hour RNA expression, core-clock gene RNAs were regulated by both synthesis and degradation, presumably to sustain high amplitude of rhythmic expression. I also identified rhythmic RNA expression with a period of 12 and 8 hours; interestingly, degradation primarily drove rhythmic expression of these RNAs. Overall this dissertation revealed RNA dynamics that drive rhythmic gene expression. This will provide insights into how diverse circadian clock mechanisms ultimately drive tissue-specific rhythmic gene expression. / Doctor of Philosophy / Almost all organisms on Earth have an internal timekeeping system, called a circadian clock, that enables them to anticipate and respond to the day/night cycle. The circadian clock regulates diverse body processes such as the sleep/wake cycle, eating and digestion, body temperature, and blood pressure. Disruptions to the circadian clock are detrimental to health and wellbeing. Many organisms, including humans, have a core circadian clock mechanism that drives rhythmic gene expression in thousands of genes that are thought to be ultimately responsible for rhythmic biological and physiological processes. My project investigates how the core clock mechanism drives rhythmic gene expression. I found that rhythmic synthesis of RNA was a primary driver of rhythmic gene expression, but the genes involved in the core circadian clock mechanism itself was regulated by multiple rhythmic processes. Understanding rhythmic gene expression control points is integral for understanding how circadian gene expression can be changed or interrupted.

Le récepteur de l'acide rétinoïque RAR est une protéine de la superfamille des récepteurs nucléaires liant le ligand acide rétinoïque (AR). En présence de son ligand, RAR induit la transcription de ses gènes cibles alors qu'en son absence la transcription est inhibée. Le mécanisme de régulation de RAR est altéré dans les lignées cellulaires humaines de carcinome mammaire dû à une baisse de capacité de synthèse de l'AR. Aussi, l'expression des microARN (miR) est perturbée dans le cancer du sein et un grand nombre de gènes ont été identifiés, après une analyse in-silico, comme des cibles prédites des miRs. Ces derniers peuvent être régulés pas des facteurs de transcription et ils sont capables d'inhiber la prolifération cellulaire et d'induire l'apoptose via la régulation de leurs cibles. Ainsi, les miRs peuvent jouer un rôle dans le mécanisme de régulation de RAR et être impliqués dans des boucles de régulation avec ce récepteur. Dans le cadre de ce travail, nous décrivons une approche développée pour prédire et caractériser des circuits de régulation au niveau transcriptionnel et post-transcriptionnel dans le cancer du sein. Nous nous sommes intéressés aux boucles de régulation de type feed-forward où RAR régule un miR et en commun ils régulent un ensemble de gènes codants pour des protéines dans les cellules tumorales mammaires MCF7 et SKBR3. Ces circuits ont été construits en combinant des données de ChIP-chip de RAR et des données de micro-puces d'ADN tout en utilisant des outils in-silico de prédiction des gènes cibles de miRs. Afin de proposer le modèle approprié de régulation, une analyse in-silico des éléments de réponse de l'AR (RARE) dans les promoteurs des miRs est réalisée. Cette étape permet de prédire si la régulation par RAR est directe ou indirecte. Les boucles ainsi prédites sont filtrées en se basant sur des données d'expression de miR existantes dans des bases de données et dans différentes lignées cellulaires, en vue d'éliminer les faux positifs. De plus, seuls les circuits pertinents sur le plan biologique et trouvés enrichis dans Gene Ontology sont retenus. Nous proposons également d'inférer l'activité des miRs afin d'orienter leur régulation par RAR. L'approche a réussi à identifier des boucles validées expérimentalement. Plusieurs circuits de régulation prédits semblent être impliqués dans divers aspects du développement de l'organisme, de la prolifération et de la différenciation cellulaire. De plus, nous avons pu valider que let-7a peut être induit par l'AR dans les MCF7. / The retinoic acid receptor (RAR) is a type of nuclear receptor that is activated by the ligand retinoic acid (RA). In the presence of ligand, RAR induces the transcription of its targets whereas in the absence of ligand the transcription is blocked. The mechanism of regulation of RAR is altered in breast cancer cell lines due to a reduced capacity to synthesize RA. Also aberrant patterns of microRNA (miR) expression have been reported in human breast cancer and a number of genes involved in breast cancer progression have been identified by in-silico analysis to be targets of miRs. The miRs could be controlled by transcription factors and via the regulation of their mRNA targets, the miRs could promote apoptosis and even inhibit cell proliferation. Hence, the miRs may play a role in the mechanism of regulation of RAR and could be involved in regulatory loops with this receptor. In this work, we describe an approach developed for the prediction and characterization of mixed transcriptional and post-transcriptional regulatory circuits in breast cancer. We concentrated in particular on feed-forward loops, in which RAR regulates a miR, and together with it, a set of joint target protein coding genes in human breast cancer cell lines MCF7 and SKBR3. These loops are constructed by combining ChIP-chip datasets of RAR with datasets of DNA microarrays and by using miR target prediction tools. In order to predict the appropriate model of regulation, in-silico analysis was performed to look for retinoic acid response element (RARE) in miR promoter. This step could identify if the regulation by RAR is direct or indirect. The regulatory loops will be then filtered, in order to reduce the number of false positive, based on databases designed to represent human miR expression profiles in different tissues or cell types. Moreover, only biologically relevant circuits enriched in Gene Ontology were retained. Also, we propose to infer miR activity in order to detect their regulation by RAR. This approach was able to find some existing experimental data. Several regulatory circuits seem to be involved in various aspects of organism development, proliferation and cell differentiation. Furthermore, we were able to validate the induction of let-7a by RA in MCF7 cells.

Le récepteur de l'acide rétinoïque RAR est une protéine de la superfamille des récepteurs nucléaires liant le ligand acide rétinoïque (AR). En présence de son ligand, RAR induit la transcription de ses gènes cibles alors qu'en son absence la transcription est inhibée. Le mécanisme de régulation de RAR est altéré dans les lignées cellulaires humaines de carcinome mammaire dû à une baisse de capacité de synthèse de l'AR. Aussi, l'expression des microARN (miR) est perturbée dans le cancer du sein et un grand nombre de gènes ont été identifiés, après une analyse in-silico, comme des cibles prédites des miRs. Ces derniers peuvent être régulés pas des facteurs de transcription et ils sont capables d'inhiber la prolifération cellulaire et d'induire l'apoptose via la régulation de leurs cibles. Ainsi, les miRs peuvent jouer un rôle dans le mécanisme de régulation de RAR et être impliqués dans des boucles de régulation avec ce récepteur. Dans le cadre de ce travail, nous décrivons une approche développée pour prédire et caractériser des circuits de régulation au niveau transcriptionnel et post-transcriptionnel dans le cancer du sein. Nous nous sommes intéressés aux boucles de régulation de type feed-forward où RAR régule un miR et en commun ils régulent un ensemble de gènes codants pour des protéines dans les cellules tumorales mammaires MCF7 et SKBR3. Ces circuits ont été construits en combinant des données de ChIP-chip de RAR et des données de micro-puces d'ADN tout en utilisant des outils in-silico de prédiction des gènes cibles de miRs. Afin de proposer le modèle approprié de régulation, une analyse in-silico des éléments de réponse de l'AR (RARE) dans les promoteurs des miRs est réalisée. Cette étape permet de prédire si la régulation par RAR est directe ou indirecte. Les boucles ainsi prédites sont filtrées en se basant sur des données d'expression de miR existantes dans des bases de données et dans différentes lignées cellulaires, en vue d'éliminer les faux positifs. De plus, seuls les circuits pertinents sur le plan biologique et trouvés enrichis dans Gene Ontology sont retenus. Nous proposons également d'inférer l'activité des miRs afin d'orienter leur régulation par RAR. L'approche a réussi à identifier des boucles validées expérimentalement. Plusieurs circuits de régulation prédits semblent être impliqués dans divers aspects du développement de l'organisme, de la prolifération et de la différenciation cellulaire. De plus, nous avons pu valider que let-7a peut être induit par l'AR dans les MCF7. / The retinoic acid receptor (RAR) is a type of nuclear receptor that is activated by the ligand retinoic acid (RA). In the presence of ligand, RAR induces the transcription of its targets whereas in the absence of ligand the transcription is blocked. The mechanism of regulation of RAR is altered in breast cancer cell lines due to a reduced capacity to synthesize RA. Also aberrant patterns of microRNA (miR) expression have been reported in human breast cancer and a number of genes involved in breast cancer progression have been identified by in-silico analysis to be targets of miRs. The miRs could be controlled by transcription factors and via the regulation of their mRNA targets, the miRs could promote apoptosis and even inhibit cell proliferation. Hence, the miRs may play a role in the mechanism of regulation of RAR and could be involved in regulatory loops with this receptor. In this work, we describe an approach developed for the prediction and characterization of mixed transcriptional and post-transcriptional regulatory circuits in breast cancer. We concentrated in particular on feed-forward loops, in which RAR regulates a miR, and together with it, a set of joint target protein coding genes in human breast cancer cell lines MCF7 and SKBR3. These loops are constructed by combining ChIP-chip datasets of RAR with datasets of DNA microarrays and by using miR target prediction tools. In order to predict the appropriate model of regulation, in-silico analysis was performed to look for retinoic acid response element (RARE) in miR promoter. This step could identify if the regulation by RAR is direct or indirect. The regulatory loops will be then filtered, in order to reduce the number of false positive, based on databases designed to represent human miR expression profiles in different tissues or cell types. Moreover, only biologically relevant circuits enriched in Gene Ontology were retained. Also, we propose to infer miR activity in order to detect their regulation by RAR. This approach was able to find some existing experimental data. Several regulatory circuits seem to be involved in various aspects of organism development, proliferation and cell differentiation. Furthermore, we were able to validate the induction of let-7a by RA in MCF7 cells.