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CWC22 Supports Pre-Mrna Splicing and Exon Junction Complex Assembly

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CWC22 Supports Pre-Mrna Splicing and Exon Junction Complex Assembly CWC22 Supports Pre-mRNA Splicing and Exon Junction Complex Assembly I n a u g u r a l - D i s s e r t a t i o n zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Anna-Lena Steckelberg aus Frankfurt am Main Köln, 2014 Berichterstatter: PD Dr. Niels H. Gehring Prof. Dr. Karin Schnetz Tag der mündlichen Prüfung: 01.07.2014 Parts of this thesis have been published as Steckelberg AL, Boehm V, Gromadzka AM, Gehring NH ‘CWC22 Connects pre-mRNA Splicing and Exon Junction Complex Assembly’; Cell Rep. 2012 Sep 27; 2(3):454-61 Steckelberg AL, Gehring NH ‘Studying the composition of mRNPs in vitro using splicing- competent cell extracts’; Methods. 2014 Feb;65(3):342-9 Table of contents Table of contents 1 Abstract............................................................................................................ 1 1.1 Deutsche Zusammenfassung ............................................................................... 1 1.2 Summary ............................................................................................................ 3 2 Introduction ..................................................................................................... 5 2.1 Regulation of eukaryotic gene expression ............................................................ 5 2.1.1 5' end capping of mRNA ............................................................................................................. 5 2.1.2 3’ end processing of mRNA ....................................................................................................... 6 2.1.3 Pre-mRNA splicing ........................................................................................................................ 7 2.2 mRNP assembly connects different steps of gene expression ............................... 9 2.3 The exon junction complex is part of the splicing-dependent mRNP ................... 10 2.3.1 Structure of the EJC ................................................................................................................... 11 2.3.2 Assembly and disassembly of EJCs ....................................................................................... 14 2.3.3 The EJC regulates different steps of the mRNA life cycle.............................................. 18 2.3.4 EJC-independent functions of eIF4A3.................................................................................. 23 2.4 DExD/H-box helicases are key regulators of mRNA metabolism ......................... 24 2.4.1 Structure of DExD/H-box helicases ...................................................................................... 24 2.4.2 DExD/H-box proteins function as components of large RNP complexes ................. 26 2.4.3 The MIF4G-domain has evolved as a helicase interaction domain ........................... 27 3 Aims and Significance of the project ............................................................... 31 4 Results ........................................................................................................... 33 4.1 CWC22 is a direct interaction partner of eIF4A3 ................................................. 33 4.1.1 An in silico search identifies CWC22 as a putative eIF4A3 binding protein ............ 33 4.1.2 CWC22 interacts with eIF4A3 in cell extracts ................................................................... 35 4.1.3 eIF4A3 binds the MIF4G domain of CWC22 ...................................................................... 36 4.1.4 Pulldown experiments confirm a direct interaction of eIF4A3 and CWC22 ........... 36 4.1.5 CWC22 does not interact with the other EJC proteins .................................................. 38 4.1.6 CWC22 is no component of the post-splicing mRNP ...................................................... 40 4.1.7 CWC22, eIF4A3 and Y14 interact with the C-complex spliceosome .......................... 41 i 4.1.8 CWC22 uses multiple domains to interact with the spliceosome ............................. 42 4.1.9 Initial spliceosome interaction of CWC22 and eIF4A3 is independent of the EJC deposition site on spliced mRNA .......................................................................................................... 44 4.2 CWC22 recruits eIF4A3 to the spliceosome and mediates EJC assembly .............. 45 4.2.1 The CWC22/eIF4A3-interaction can be modeled with an existing eIF4G/eIF4A crystal structure ......................................................................................................................................... 45 4.2.2 CWC22-binding deficient eIF4A3 mutants form an EJC in vitro ................................. 49 4.2.3 CWC22-binding deficient eIF4A3 mutants fail to form splicing-dependent EJCs . 50 4.2.4 Recombinant CWC22(110-409) disturbs EJC assembly in vitro .................................. 52 4.2.5 Recombinant CWC22(110-409) disturbs EJC assembly in cell extracts .................... 53 4.2.6 CWC22 interaction with the spliceosome is independent of eIF4A3 ....................... 56 4.3 CWC22 is required for pre-mRNA splicing in human cells .................................... 58 4.3.1 siRNA knock down of CWC22 leads to an accumulation of pre-mRNA ................... 58 4.3.2 MIF4G and MA3 domains of CWC22 are required for pre-mRNA splicing ............. 60 4.3.3 The interaction between CWC22 and eIF4A3 is dispensable for splicing ............... 62 4.3.4 Over-expression of FLAG-CWC22(NK-171/2-DE) is toxic for HEK293 cells ............. 63 4.3.5 Over-expression of FLAG-CWC22(NK-171/2-DE) inhibits EJC assembly without impairing pre-mRNA splicing ................................................................................................................. 64 5 Discussion ....................................................................................................... 67 5.1 CWC22 links pre-mRNA splicing and EJC assembly .............................................. 67 5.2 The eIF4A3-CWC22 complex shows a conserved interaction pattern ................... 67 5.2.1 The CWC22/eIF4A3 structure confirms a eIF4G/eIF4A-like interaction .................. 67 5.2.2 The MIF4G domain of CWC22 regulates the conformation of eIF4A3 ..................... 69 5.2.3 The MIF4G-domain emerges as a common regulator of RNA helicases ................. 71 5.3 CWC22 mediates splicing-dependent EJC assembly ............................................ 72 5.3.1 CWC22 recruits eIF4A3 to the spliceosome ...................................................................... 72 5.3.2 The core EJC proteins are recruited to the spliceosome via different routes ....... 73 5.3.3 EJC assembly requires a rearrangement of the eIF4A3-CWC22 interaction .......... 73 5.3.4 The ATPase activity of eIF4A3 is dispensable for spliceosomal recruitment ......... 75 5.4 CWC22 is required for pre-mRNA splicing in human cells .................................... 77 5.4.1 MIF4G and MA3 domain are required for pre-mRNA splicing in humans .............. 77 5.4.2 N- and C-terminal domains of CWC22 regulate spliceosomal recruitment ........... 79 5.5 The roles of CWC22 in pre-mRNA splicing and EJC assembly can be functionally uncoupled ..................................................................................................................... 80 ii 5.5.1 Pre-mRNA splicing and EJC assembly are closely linked processes ........................... 80 5.5.2 EIF4A3 does not influence the splicing function of CWC22 ......................................... 80 5.5.3 The selective inhibition of EJC assembly is toxic in human cells ................................. 81 5.6 Concluding remarks .......................................................................................... 82 6 Material and Methods .................................................................................... 83 6.1 Plasmids ........................................................................................................... 83 6.1.1 Mammalian expression plasmids .......................................................................................... 83 6.1.2 Bacterial expression plasmids ................................................................................................ 83 6.1.3 Plasmids for in vitro transcription ........................................................................................ 83 6.2 Mammalian cell culture .................................................................................... 83 6.2.1 Mammalian cell lines ................................................................................................................ 83 6.2.2 Generation of stable cell lines ................................................................................................ 84 6.2.3 Transfection of plasmid DNA .................................................................................................. 85 6.2.4 Transfection of siRNA ............................................................................................................... 86 6.2.5 Cell viability assay ...................................................................................................................... 86 6.2.6 Preparation of cell lysates ....................................................................................................... 87 6.3 Molecular biology ............................................................................................. 87 6.3.1 Cloning ..........................................................................................................................................
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