京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21923号 / 生博第408号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 片山 高嶺, 教授 藤田 尚志, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22356号 / 医博第4597号 / 新制||医||1042(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 萩原 正敏, 教授 岩田 想, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13361号 / 論生博第24号 / 新制||生||59(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 片山 高嶺, 教授 永尾 雅哉, 教授 荒木 崇 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Deep sequencing of mRNAs (RNA-Seq) is now the preferred method for transcriptome-wide quantification of gene expression. Yet many mRNA isoforms, such as those eliminated by nonsense-mediated decay (NMD), are inherently unstable. Thus a significant drawback of steady-state RNA-Seq is that it provides marginal information on the flux through alternative splicing pathways. Measurement of such flux necessitates capture of newly made species prior to mRNA decay. One means to capture nascent mRNAs is affinity purifying either the exon junction complex (EJC) or activated spliceosomes. Late-stage spliceosomes deposit the EJC upstream of exon-exon junctions, where it remains associated until the first round of translation. As most mRNA decay pathways are translation-dependent, these EJC- or spliceosome-associated, pre-translational mRNAs should provide an accurate record of the initial population of alternate mRNA isoforms. Previous work has analyzed the protein composition and structure of pre- translational mRNPs in detail. While in the Moore lab, my project has focused on exploring the diversity of mRNA isoforms contained within these complexes. As expected, known NMD isoforms are more highly represented in pre-translational mRNPs than in RNA-Seq libraries. To investigate whether pre-translational mRNPs contain novel mRNA isoforms, we created a bioinformatics pipeline that identified thousands of previously unannotated splicing events. Though many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.

No description available.

Les mutations non sens introduisent un codon stop prématuré dans une phase ouverte de lecture. Ce type de mutation est retrouvé chez environ 11% des patients atteints de maladies génétiques et dans de nombreux cancers. En effet, entre 5 et 40% des mutations affectant des gènes suppresseurs de tumeurs sont des mutations non sens. La conséquence de la présence d’une mutation non sens dans un gène est la dégradation rapide de l’ARN messager correspondant, par l’activation d’un mécanisme de surveillance des ARN appelé NMD (pour nonsense-mediated mRNA decay) conduisant à une absence d’expression du gène mutant. Dans le cas des cancers, l’absence d’expression d’un gène suppresseur de tumeurs tel que TP53, perturbe un ensemble de processus biologiques dont l’apoptose, facilitant ainsi la progression tumorale.En utilisant un système de criblage moyen débit permettant d’identifier des molécules capables de ré-exprimer des gènes porteurs d’une mutation non sens en inhibant le NMD et/ou en activant la translecture, plusieurs molécules ont été identifiées. La translecture est un mécanisme naturel conduisant à l’incorporation d’un acide aminé à la position du codon stop prématuré au cours de la traduction. Parmi les molécules identifiées, je me suis intéressée à un extrait végétal nommé H7 et au composé CNSM1 (pour corrector of nonsense mutation 1) qui permettent une ré-expression très efficace du gène TP53 lorsqu’il est porteur d’une mutation non sens. J’ai caractérisé ces composés en montrant notamment la ré-expression du gène TP53 porteur d’une mutation non sens dans différentes lignées cellulaires issues de différents cancers. J’ai montré également la très faible toxicité de ces molécules, validant leur potentielle utilisation en clinique. Mon étude a aussi permis de montrer que la protéine p53 synthétisée est fonctionnelle puisqu'elle est capable d’induire l’activation transcriptionnelle d’un de ses gènes cibles, le gène TP21.En permettant la ré-expression du gène suppresseur de tumeur mutant, des molécules comme CNSM1 ou H7 restaurent la capacité des cellules à entrer en apoptose et pourraient aussi réduire certaines résistances à la chimiothérapie.De plus, par une approche d’édition du génome, j’ai confirmé le lien existant entre le blocage du cytosquelette et l’inhibition du NMD. J’ai aussi identifié deux protéines impliquées dans le réarrangement du cytosquelette qui pourraient être ciblées pour inhiber le NMD en thérapie et ré-exprimer une protéine tronquée fonctionnelle. L’utilisation de H7 ou de CNMS1 pourrait ainsi être couplée à une inhibition du NMD pour optimiser la correction des mutations non sens. Ces molécules correctrices de mutations non sens représentent de nouvelles approches thérapeutiques ciblées du cancer et des maladies rares liées aux mutations non sens. / Nonsense mutations generate premature termination codons (PTC) within an open reading frame. This type of mutation is found in about 11% of patients with genetic disorders. Concerning cancer, 5 to 40% of mutations affecting tumor-suppressing genes are nonsense mutations. The presence of a PTC in a gene leads to rapid degradation of its mRNA mediated by the RNA surveillance mechanism named NMD (Nonsense-mediated mRNA decay) preventing the synthesis of truncated proteins. In cancer, the absence of expression of tumor suppressing genes such as TP53 interferes with many biological pathways including apoptosis enabling tumor progression.A screening system that allows identifying molecules capable of re-expressing genes harboring nonsense mutations by inhibiting the NMD system and/or by activating readthrough has been developed in the lab. Readthrough is a natural mechanism, which occurs during translation, leading to the incorporation of an amino acid at the PTC position. Among the molecules that have been identified thanks to the screen, a natural extract named H7 and a compound named CNSM1 efficiently rescues the expression of the nonsense-mutated TP53 gene carrying a PTC.CNSM1 and H7 induces the expression of full-length proteins from PTC-containing genes indicating that these compounds are capable of activating readthrough. I validated the screen results on several cancer cell lines harboring an endogenous nonsense mutation in TP53 gene and showed that the function of p53 was restored in the presence of CNSM1 or H7. I also investigated the cellular toxicity related with the use of CMNS1 on cultured cells and the in vivo effect of H7 in a mouse model harboring a nonsense mutation in dystrophin gene. My results demonstrate that these compounds have a mild cellular toxicity. In addition, using a genome editing approach I confirmed the relationship between the cytoskeletal blockage and the NMD inhibition. I identified two proteins that are implicated in the cytoskeletal rearrangement, which might be targeted to induce NMD inhibition and then the expression of truncated but functional protein from the mutated mRNA. H7 or CNMS1 might be coupled to an NMD inhibition strategy to improve the nonsense mutation correction. Knowing CNSM1 and H7 are so far the most efficient molecule capable of rescuing the expression of PTC-containing genes, these compounds represents a realistic hope for a new-targeted therapy for pathologies associated with nonsense mutations.

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E‑binding protein (4E‑BP)‑1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator‑activated receptor (PPAR)γ expression. The small interfering RNA‑mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

Hepatocellular carcinoma (HCC) is a type of liver cancer with low survival rate, not least due to the difficulty of diagnosing it in an early stage. The objective of this thesis is to build a random forest classification method based on microRNA (and messenger RNA) expression profiles from patients with HCC. The main purpose is to be able to distinguish between tumor samples and normal samples by measuring the miRNA expression. If successful, this method can be used to detect HCC at an earlier stage and to design new therapeutics. The microRNAs and messenger RNAs which have a significant difference in expression between tumor samples and normal samples are selected for building random forest classification models. These models are then tested on paired samples of tumor and surrounding normal tissue from patients with HCC. The results show that the classification models built for classifying tumor and normal samples have high prediction accuracy and hence show high potential for using microRNA and messenger RNA expression levels for diagnosis of HCC. / Hepatocellulär cancer (HCC) är en typ av levercancer med mycket låg överlevnadsgrad, inte minst på grund av svårigheten att diagnosticera i ett tidigt skede. Syftet med det här projektet är att bygga en klassificeringsmodell med random forest, baserad på uttrycksprofiler av mikroRNA (och budbärar-RNA) från patienter med HCC. Målet är att kunna skilja mellan tumörprover och normala prover genom att mäta uttrycket av mikroRNA. Om detta mål uppnås kan metoden användas för att upptäcka HCC i ett tidigare skede och för att utveckla nya läkemedel. De mikroRNA och budbärar-RNA som har en signifikant skillnad i uttryck mellan prover från tumörvävnad och intilliggande normal vävnad väljs ut för att bygga klassificaringsmodeller med random forest. Dessa modeller testas sedan på parade prover av tumörvävnad och intilliggande vävnad från patienter med HCC. Resultaten visar att modeller som byggs med denna metod kan klassificera tumörprover och normala prover med hög noggrannhet. Det finns således stor potential för att använda uttrycksprofiler från mikroRNA och budbärar-RNA för att diagnosticera HCC.

No description available.

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...