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Structure-Functional Studies on Lncrna MEG3 Tina Uroda

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Structure-Functional Studies on Lncrna MEG3 Tina Uroda Structure-functional studies on lncRNA MEG3 Tina Uroda To cite this version: Tina Uroda. Structure-functional studies on lncRNA MEG3. Biomolecules [q-bio.BM]. Université Grenoble Alpes, 2019. English. NNT : 2019GREAV014. tel-02569435 HAL Id: tel-02569435 https://tel.archives-ouvertes.fr/tel-02569435 Submitted on 11 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE LA COMMUNAUTÉ UNIVERSITÉ GRENOBLE ALPES Spécialité : Biologie Structurale et Nanobiologie Arrêté ministériel : 25 mai 2016 Présentée par Tina URODA Thèse dirigée par Marco MARCIA préparée au sein du Laboratoire laboratoire européen de biologie moléculaire dans l'École Doctorale Chimie et Sciences du Vivant Caractérisation structurale et fonctionnelle de l'ARN long non codant MEG3 Structure-functional studies on lncRNA MEG3 Thèse soutenue publiquement le 9 mai 2019, devant le jury composé de : Monsieur MARCO MARCIA DIRECTEUR DE RECHERCHE, EMBL GRENOBLE, Directeur de thèse Monsieur ALBERTO INGA PROFESSEUR, UNIVERSITE DE TRENTE - ITALIE, Rapporteur Madame MAITE HUARTE DIRECTRICE DE RECHERCHE, UNIVERSITE DE NAVARRE- PAMPELUNE-ESPAGNE, Rapporteur Monsieur CLAIRE VOURC'H PROFESSEUR, UNIVERSITE GRENOBLE ALPES, Président Madame REINI F. LUCO DIRECTRICE DE RECHERCHE, CNRS DELEGATION LANGUEDOC- ROUSSILLON, Examinateur Monsieur JANOSCH HENNIG DIRECTEUR DE RECHERCHE, EMBL HEIDELBERG ALLEMAGNE, Examinateur Table of Contents Table of Contents ............................................................................................................................ 1 List of Abbreviations ....................................................................................................................... 5 List of Figures .................................................................................................................................. 9 List of Tables ................................................................................................................................. 12 Abstract ......................................................................................................................................... 13 Résumé en Français ...................................................................................................................... 15 1. Introduction ............................................................................................................................. 17 Résumé en Français ...................................................................................................................... 19 1.1. Non-coding RNAs ............................................................................................................ 21 1.1.1. Long non-coding RNAs ............................................................................................. 22 1.1.1.1. Structural studies on lncRNAs ........................................................................... 22 1.2. LncRNA MEG3 ................................................................................................................. 23 1.2.1. MEG3 evolutionary conservation ............................................................................ 23 1.2.2. Genomic organization of the human MEG3 locus ................................................... 24 1.2.3. MEG3 expression in physiological and pathological conditions .............................. 27 1.2.4. MEG3 cellular localization ....................................................................................... 27 1.2.5. MEG3 functions........................................................................................................ 28 1.2.5.1. Development via Polycomb gene silencing ....................................................... 28 1.2.5.2. Tumor suppression ............................................................................................ 29 1.2.5.2.1. Tumor suppression via p53 pathway ......................................................... 31 1.3. Scientific aims ................................................................................................................. 32 2. Materials and Methods ............................................................................................................. 35 1 Résumé en Français ...................................................................................................................... 37 2.1. Cloning and mutagenesis ............................................................................................... 39 2.2. RNA production and purification ................................................................................... 40 2.3. Native gel electrophoresis .............................................................................................. 41 2.4. Analytical ultracentrifugation......................................................................................... 41 2.5. Dynamic light scattering ................................................................................................. 42 2.6. Size-exclusion chromatography coupled to multi-angle laser light scattering .............. 42 2.7. Size-exclusion chromatography coupled to small angle x-ray scattering ...................... 42 2.8. In vitro secondary structure probing (in vitro SHAPE) ................................................... 42 2.9. Atomic force microscopy ................................................................................................ 45 2.10. Electron microscopy ..................................................................................................... 46 2.10.1. Negative staining ................................................................................................... 46 2.10.2. Data collection and 2D processing ........................................................................ 46 2.10.3. Cryo electron microscopy ...................................................................................... 47 2.11. Mammalian cell lines .................................................................................................... 47 2.12. In vivo structure probing .............................................................................................. 47 2.13. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay .............. 49 2.14. Luciferase assay ............................................................................................................ 49 2.15. EdU incorporation and flow cytometry assay .............................................................. 49 2.16. Sequence and structural alignments ............................................................................ 50 2.17. Quantitative real-time PCR (qRT-PCR) ......................................................................... 50 3. Results ....................................................................................................................................... 53 Résumé en Français ...................................................................................................................... 55 3.1. MEG3 production ........................................................................................................... 57 2 3.1.1. Homogeneity and purity of MEG3 ........................................................................... 58 3.2. MEG3 secondary structure ............................................................................................. 60 3.2.1. In vitro secondary structure probing of MEG3 variant 1 ......................................... 60 3.2.2. In vitro secondary structure probing of MEG3 variant 9 ......................................... 62 3.2.3. In vitro secondary structure probing of MEG3 variant 3 ......................................... 66 3.2.2. Sequence and structural alignments ....................................................................... 66 3.2.3. In vivo chemical probing .......................................................................................... 69 3.3. MEG3 tertiary structure ................................................................................................. 72 3.3.1. Compaction of MEG3 by AUC .................................................................................. 72 3.3.2. Optimization of folding conditions by MALLS.......................................................... 73 3.3.3. Tertiary structure by SAXS ....................................................................................... 75 3.3.4. Tertiary structure by AFM ........................................................................................ 77 3.3.5. Structural studies with EM ....................................................................................... 82 3.4. Optimisation of functional assays .................................................................................. 87 3.5. Structure-function relationships ...................................................................................
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