Aus dem Biomedizinischen Centrum (BMC) der Ludwig-Maximilians-Universität München Lehrstuhl Anatomie III – Zellbiologie Vorstand: Prof. Dr. Michael Kiebler The function of paraspeckle components in pluripotency maintenance and differentiation Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München Vorgelegt von Markus Grosch aus Rosenheim 2020 Gedruckt mit Genehmigung der Medizinischen Fakultät Der Ludwig-Maximilians Universität München Betreuer: Prof. Dr. Michael Kiebler Zweitgutachter: Prof. Dr. Olivier Gires Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 15.5.2020 Table of Contents Table of Contents Abstract ........................................................................................................................................................................ 7 1. Introduction ........................................................................................................................................................... 10 1.1 Exordium ......................................................................................................................................................... 10 1.2 The early stages of human embryonic development................................................................................. 10 1.3 Modeling embryonic development with pluripotent stem cells.............................................................. 11 1.3.1 Extrinsic factors regulating PSC maintenance .................................................................................... 12 1.3.2 Naïve and primed ESCs ......................................................................................................................... 12 1.3.3 Germ layer differentiation of pluripotent stem cells .......................................................................... 13 1.4 RNA binding proteins regulate pluripotency-differentiation transition ................................................ 13 1.4.1 Alternative splicing is crucial for pluripotency maintenance ........................................................... 14 1.4.2 The alternative polyadenylation profile changes during stem cell differentiation ........................ 14 1.5 Long non-coding RNAs are new players in embryonic development ................................................... 15 1.6 Membraneless organelles are phase-separated entities ............................................................................ 17 1.7 Composition and function of paraspeckles ................................................................................................ 18 1.7.1 Molecular mechanism of paraspeckles ................................................................................................ 20 1.7.1.1 RNA retention ...................................................................................................................................... 20 1.7.1.2 Protein sequestration ........................................................................................................................... 20 1.7.1.3 Chromatin binding .............................................................................................................................. 21 1.7.2 Paraspeckles in development and disease ........................................................................................... 22 1.7.2.1 Paraspeckles in development ............................................................................................................. 22 1.7.2.2 Paraspeckles in disease........................................................................................................................ 23 1.7.2.2.1 Paraspeckles in cancer ...................................................................................................................... 23 1.7.2.2.2 Paraspeckles during viral infection ................................................................................................ 24 1.7.2.2.3 Paraspeckles in neurodegenerative diseases ................................................................................. 24 1.8 DBHS proteins are involved in transcriptional and post-transcriptional gene regulation .................. 25 1.8.1 Molecular functions of DBHS proteins ................................................................................................ 26 1.8.2 Physiological roles of DBHS proteins................................................................................................... 27 1.8.3 DBHS proteins in disease ....................................................................................................................... 27 1.9 Aims and impact of this work ...................................................................................................................... 28 2. Materials and Methods ........................................................................................................................................ 30 2.1 Chemicals and kits ......................................................................................................................................... 30 Table of Contents 2.2 PSC culture ...................................................................................................................................................... 30 2.3 Fibroblast reprogramming ............................................................................................................................ 30 2.4 Spontaneous differentiation .......................................................................................................................... 30 2.5 Mesenchymal stem cell (MSCs), adipocyte and osteocyte differentiation ............................................. 31 2.6 Cardiomyocyte differentiation ..................................................................................................................... 31 2.7 Nephron differentiation ................................................................................................................................ 31 2.8 Definitive endoderm, lung progenitor and hepatocyte differentiation .................................................. 32 2.9 Neuronal stem cell differentiation ............................................................................................................... 32 2.10 Astrocyte differentiation ............................................................................................................................. 33 2.11 Motor neuron differentiation ...................................................................................................................... 33 2.12 Cortical neuron differentiation ................................................................................................................... 33 2.13 Somatic cell lines........................................................................................................................................... 34 2.14 Derivation of primary murine mesenchymal stem cells ......................................................................... 34 2.15 Derivation of primary murine astrocytes .................................................................................................. 34 2.16 Derivation of primary murine cardiomyocytes ....................................................................................... 35 2.17 Derivation of primary murine hepatocytes .............................................................................................. 35 2.18 Animal data ................................................................................................................................................... 35 2.19 Oil Red O staining ........................................................................................................................................ 35 2.20 Alizarin Red staining ................................................................................................................................... 35 2.21 Immunofluorescence staining .................................................................................................................... 36 2.22 Single-molecule fluorescence in situ hybridization (smFISH) ................................................................ 36 2.23 Chemicals used for DNA binding .............................................................................................................. 37 2.24 Image analysis for paraspeckle counting .................................................................................................. 37 2.25 Image analysis for NEAT1_2 single-molecule counting ......................................................................... 37 2.26 Quantification of nucleus size .................................................................................................................... 38 2.27 Flow cytometry analysis .............................................................................................................................. 38 2.28 SmFISH combined with flow cytometry ................................................................................................... 39 2.29 RNA extraction and quantitative RT-PCR (RT-qPCR) ............................................................................ 39 2.30 Western blot .................................................................................................................................................