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Identification and Characterization of Small Molecule Inhibitors of Pre-Mrna Splicing That Block Spliceosome Assembly at Novel Stages

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Identification and Characterization of Small Molecule Inhibitors of Pre-Mrna Splicing That Block Spliceosome Assembly at Novel Stages Identification and characterization of small molecule inhibitors of pre-mRNA splicing that block spliceosome assembly at novel stages Dissertation for the award of the degree "Doctor rerum naturalium" of the Georg-August University Göttingen within the doctoral program Biology of the Georg-August University School of Science (GAUSS) submitted by Anzhalika Sidarovich from Soligorsk, Belarus Göttingen 2015 Thesis committee Prof. Dr. Reinhard Lührmann Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Holger Stark Research group of 3D Electron Cryo-Microscopy, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Heike Krebber Department of Molecular Genetics, Institute for Microbiology and Genetics, Georg August University Göttingen Members of the Examination Board Reviewer: Prof. Dr. Reinhard Lührmann Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen Second Reviewer: Prof. Dr. Holger Stark Research group of 3D Electron Cryo-Microscopy, Max Planck Institute for Biophysical Chemistry, Göttingen Additional Reviewer: Prof. Dr. Heike Krebber Department of Molecular Genetics, Institute for Microbiology and Genetics, Georg August University Göttingen Further members of the Examination Board Prof. Dr. Markus Zweckstetter Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Ralf Ficner Department for Molecular Structural Biology Institute for Microbiology and Genetics, Georg August University Göttingen Prof. Dr. Kai Tittmann Department for Molecular Enzymology Georg August University Göttingen Date of the oral examination: 17.04.2015 “I wonder about turtles.” “What do you wonder about turtles?” Angela asked him. “When they pull in their heads,” he said, “do their spines buckle or contract?” Kurt Vonnegut, Cat’s Cradle For my family with love Affidavit I hereby declare that my thesis entitled "Identification and characterization of small molecule inhibitor of pre-mRNA splicing that block spliceosome assembly at novel stages" has been written independently and with no other sources and aids than quoted. This thesis (wholly or in part) has not been submitted elsewhere for any academic award or qualification. Anzhalika Sidarovich February 2015 Göttingen Table of Contents Abstract .................................................................................................................................. 1 1 Introduction ...................................................................................................................... 3 1.1 Pre-mRNA splicing ......................................................................................................... 3 1.1.1 Structure of pre-mRNAs ........................................................................................................... 3 1.1.2 Intron recognition sites ............................................................................................................ 4 1.1.3 Mechanism of pre-mRNA splicing ........................................................................................ 4 1.1.4 Composition of the spliceosome ........................................................................................... 5 1.1.5 Spliceosome assembly ............................................................................................................... 9 1.1.5.1 Exon definition, an alternative early assembly pathway .................................................. 11 1.1.6 RNA-RNA interactions in the spliceosome..................................................................... 12 1.1.7 RNA-based catalysis of pre-mRNA splicing ................................................................... 14 1.1.8 Protein composition of the spliceosome ......................................................................... 15 1.1.8.1 non-snRNP spliceosomal proteins .............................................................................................. 16 1.1.8.2 Dynamics of the spliceosome’s protein composition ......................................................... 18 1.1.9 Posttranslational modification of proteins during splicing .................................... 19 1.1.10 Structure of the spliceosome and spliceosomal snRNPs ....................................... 21 1.1.11 Small molecule inhibitors of pre-mRNA splicing ...................................................... 22 1.2 Aim of this study .................................................................................................................23 2 Materials and methods ............................................................................................... 25 2.1 Material ..................................................................................................................................25 2.1.1 Chemicals ..................................................................................................................................... 25 2.1.2 Antisera, monoclonal and polyclonal antibodies ........................................................ 27 2.1.3 Enzymes and enzyme inhibitors, and MS2-MBP protein ......................................... 28 2.1.4 Nucleotides.................................................................................................................................. 28 2.1.5 Oligonucleotides ....................................................................................................................... 29 2.1.6 Plasmids ....................................................................................................................................... 29 2.1.7 Cell lines ....................................................................................................................................... 29 2.1.8 Buffers ........................................................................................................................................... 30 2.1.9 Commercial kits ........................................................................................................................ 33 2.1.10 Chromatography materials and consumables ........................................................... 33 2.1.11 Small Molecule Screening Apparati ................................................................................ 34 2.1.12 Apparati ..................................................................................................................................... 34 2.2 Methods ..................................................................................................................................36 2.2.1 Molecular biology standard methods .............................................................................. 36 2.2.1.1 in vitro transcription ......................................................................................................................... 36 2.2.1.2 Concentration determination of nucleic acids ....................................................................... 37 2.2.1.3 Agarose gel electrophoresis of nucleic acids .......................................................................... 37 2.2.1.4 Phenol-chloroform-isoamyl alcohol extraction .................................................................... 38 2.2.1.5 Proteinase K digestion ..................................................................................................................... 38 2.2.1.6 Denaturing Urea polyacrylamide gel electrophoresis ....................................................... 38 2.2.1.7 RNA silver staining ............................................................................................................................ 39 2.2.1.8 Radioactive labeling of the 5´-end of DNA oligonucleotides ........................................... 39 2.2.1.9 Northern blotting ............................................................................................................................... 39 2.2.2 Standard protein biochemical methods .......................................................................... 40 2.2.2.1 TCA precipitation of protein .......................................................................................................... 40 2.2.2.2 Acetone precipitation of protein.................................................................................................. 40 2.2.2.3 Denaturing SDS polyacrylamide gel electrophoresis. ........................................................ 40 2.2.2.4 Coomassie staining of SDS-PAGE gels ....................................................................................... 41 2.2.2.5 Silver staining of SDS-PAGE gels .................................................................................................. 41 2.2.3 Immunological Methods ........................................................................................................ 42 2.2.3.1 Immunoblotting (Western blotting) .......................................................................................... 42 2.2.3.2 Immunoprecipitation........................................................................................................................ 43 2.2.4 Special Methods ........................................................................................................................ 43 2.2.4.1 Preparation of HeLa cell nuclear extract.................................................................................. 43 2.2.4.2
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