NUCLEOLIN: A NUCLEOLAR RNA-BINDING PROTEIN INVOLVED IN RIBOSOME BIOGENESIS Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Julia Fremerey aus Hamburg Düsseldorf, April 2016 2 Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. A. Borkhardt Korreferent: Prof. Dr. H. Schwender Tag der mündlichen Prüfung: 20.07.2016 3 Die vorgelegte Arbeit wurde von Juli 2012 bis März 2016 in der Klinik für Kinder- Onkologie, -Hämatologie und Klinische Immunologie des Universitätsklinikums Düsseldorf unter Anleitung von Prof. Dr. A. Borkhardt und in Kooperation mit dem ‚Laboratory of RNA Molecular Biology‘ an der Rockefeller Universität unter Anleitung von Prof. Dr. T. Tuschl angefertigt. 4 Dedicated to my family TABLE OF CONTENTS 5 TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................... 5 LIST OF FIGURES ......................................................................................................10 LIST OF TABLES .......................................................................................................12 ABBREVIATION .........................................................................................................13 ABSTRACT ................................................................................................................19 ZUSAMMENFASSUNG ..............................................................................................21 1 INTRODUCTION .................................................................................................23 1.1 The nucleolus, the site of ribosome biogenesis ..........................................23 1.2 Ribosome biogenesis in eukaryotes ............................................................24 1.2.1 The human ribosome and ribosomal proteins ........................................................... 27 1.2.2 Ribosomal RNA and the precursor ribosomal RNA transcript ................................... 28 1.2.3 Ribosome biogenesis factors ..................................................................................... 31 1.2.3.1 Phosphorylation of ribosome biogenesis factors by protein kinases ................ 34 1.2.3.2 RNA-helicases involved in ribosome biogenesis .............................................. 35 1.2.4 Structure and function of small nucleolar RNAs ........................................................ 37 1.2.5 The role of ribosome biogenesis in cancer and genetic diseases ............................. 40 1.3 RNA-binding proteins ....................................................................................44 1.3.1 RNA-binding domains ................................................................................................ 46 1.3.2 RNA-binding proteins in human diseases.................................................................. 49 1.3.3 Identification of RNA-protein interaction .................................................................... 50 1.4 Nucleolin, a nucleolar RNA-binding protein ................................................53 1.4.1 Localization of Nucleolin ............................................................................................ 55 1.4.2 The role of Nucleolin in ribosome biogenesis ............................................................ 56 1.4.3 The role of Nucleolin in cancer .................................................................................. 58 1.5 Aim of the thesis ............................................................................................60 2 MATERIAL AND METHODS ...............................................................................61 TABLE OF CONTENTS 6 2.1 Material ...........................................................................................................61 2.1.1 Media, Buffer and Solution ........................................................................................ 63 2.1.2 Cells ........................................................................................................................... 68 2.1.3 Cell Culture Media and Antibiotics ............................................................................. 68 2.1.4 Chemicals and Enzymes ........................................................................................... 69 2.1.5 Commercial Kits ......................................................................................................... 72 2.1.6 Plasmids of the Gateway Cloning System ................................................................. 72 2.1.7 DNA and RNA oligos ................................................................................................. 73 2.1.7.1 Primer ................................................................................................................ 75 2.1.7.2 siRNAs .............................................................................................................. 76 2.1.8 Inhibitors .................................................................................................................... 76 2.1.9 Antibodies and Peptides ............................................................................................ 77 2.1.10 Recombinant Protein ............................................................................................. 77 2.2 Methods ..........................................................................................................78 2.2.1 Generation of stable and inducible Flp-In T-REx HEK293 cells ................................ 78 2.2.1.1 The Gateway Recombination Cloning Technology ........................................... 79 2.2.1.2 Transfection of HEK293 cells using Lipofectamine 2000 ................................. 81 2.2.1.3 Cultivation of Flp-In T-REx HEK293 cells ......................................................... 82 2.2.1.4 Nucleolin constructs .......................................................................................... 82 2.2.2 SDS-PAGE and Western Blot analysis ...................................................................... 83 2.2.3 NoD, a nucleolar localization signal prediction tool ................................................... 84 2.2.4 Immunohistochemistry and RNA Fluorescent In-Situ Hybridization .......................... 85 2.2.5 TRIzol - Extraction of RNA ......................................................................................... 86 2.2.6 The 5’ and 3’ end labeling of RNA ............................................................................. 86 2.2.7 Deprotection of a 2’-ACE protected RNA oligo .......................................................... 88 2.2.8 Inhibition of the Casein Kinase II phosphorylation reaction ....................................... 89 2.2.9 Photoactivatable-ribonucleoside enhanced crosslinking and immunoprecipitation .. 89 2.2.9.1 4SU labeling and UV crosslinking at 365 nm .................................................... 92 2.2.9.2 Cell lysis and Immunoprecipitation ................................................................... 93 2.2.9.3 Dephosphorylation and radiolabeling of the crosslinked RNA .......................... 93 2.2.9.4 Radiolabeling of the 5’ end of the RNA size markers using γ 32P-ATP............. 94 2.2.9.5 SDS-PAGE and transfer to a nitrocellulose membrane .................................... 94 2.2.9.6 Proteinase-K digest and RNA isolation ............................................................. 95 2.2.9.7 The 3’- and 5’-adapter ligation .......................................................................... 95 2.2.9.8 cDNA library preparation ................................................................................... 97 2.2.9.9 Analysis of the PAR-CLIP library ...................................................................... 98 2.2.10 RNA Immunoprecipitation-sequencing .................................................................. 99 2.2.10.1 Cell lysis and Immunoprecipitation ................................................................. 100 2.2.10.2 RNA extraction, library preparation and Next-Generation Sequencing .......... 100 2.2.11 DREME: A motif discovery algorithm to identify binding motifs .......................... 101 2.2.12 Biochemical and structural binding studies ......................................................... 102 2.2.12.1 Electrophoretic mobility shift assay ................................................................. 103 TABLE OF CONTENTS 7 2.2.12.2 Isothermal-Titration Calorimetry ...................................................................... 104 2.2.12.3 Size-Exclusion Chromatography ..................................................................... 106 2.2.12.4 Crystallography ............................................................................................... 107 2.2.13 Mass-Spectrometry ............................................................................................. 108 2.2.13.1 Cell lysis, Immunoprecipitation and FLAG-peptide elution ............................. 110 2.2.13.2 SDS-PAGE and silver staining ........................................................................ 110 2.2.13.3 Liquid chromatography-electrospray ionization Mass-Spectrometry .............. 111 2.2.13.4 Data analysis of Mass-Spectrometry using SAINTexpress and SAM ............ 112