Smu1 and RED play an important role for the activation of human spliceosomes Dissertation for the award of the degree „Doctor rerum naturalium“ of the Georg-August-Universität Göttingen within the doctoral program “Molecular Biology of Cells” of the Georg-August University School of Science (GAUSS) submitted by Sandra Maria Keiper from Berlin Göttingen, 2018 Members of the Examination Board: Prof. Dr. Reinhard Lührmann (1st Referee) Department of Cellular Biochemistry Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Heike Krebber (2nd Referee) Department of Molecular Genetics Georg-August-Universität Göttingen Prof. Dr. Patrick Cramer Department of Molecular Biology Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Markus Zweckstetter Department for NMR-based Structural Biology Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Jörg Großhans Department of Developmental Biochemistry University Medical Center Göttingen Prof. Dr. Ralph Kehlenbach Department of Molecular Biology University Medical Center Göttingen Date of oral examination: September 27th 2018 For Oskar Table of Contents Abstract .................................................................................................................................................................. 1 1. Introduction ........................................................................................................................................................ 4 1.1 Basic structure of pre-mRNAs ...................................................................................................................... 4 1.2 Pre-mRNA splicing mechanism ................................................................................................................... 5 1.3 Trans-splicing ............................................................................................................................................... 6 1.4 snRNPs – the major building blocks of the spliceosome .............................................................................. 7 1.4.1 The small nuclear RNAs ...................................................................................................................... 7 1.4.2 The snRNP-specific proteins ............................................................................................................... 8 1.5 Spliceosome assembly .............................................................................................................................. 10 1.6 Gene architecture and pre-mRNA splicing ................................................................................................. 13 1.6.1 Alternative splicing ............................................................................................................................. 13 1.6.2 Co-transcriptional splicing ................................................................................................................. 15 1.6.3 Exon-definition pathway .................................................................................................................... 16 1.6.4 Spliceosome assembly requires a minimum intron length ................................................................. 18 1.7 Dynamic RNA/RNA interaction network ..................................................................................................... 18 1.8 Dynamic protein composition of the spliceosome ...................................................................................... 20 1.9 Post-translational phosphorylation of proteins during splicing .................................................................... 22 1.10 The structure of the human B complex and the organization of the B-specific proteins ........................... 23 1.11 Aims ......................................................................................................................................................... 27 2. Materials and methods .................................................................................................................................... 29 2.1 Materials .................................................................................................................................................... 29 2.1.1 Chemicals .......................................................................................................................................... 29 2.1.2 Consumables..................................................................................................................................... 31 2.1.3 Chromatographic resins and columns ............................................................................................... 32 2.1.4 Commerical kits and buffers .............................................................................................................. 32 2.1.5 Machines ........................................................................................................................................... 32 2.1.6 Nucleotides ........................................................................................................................................ 34 2.1.7 Radiolabeled nucleotides .................................................................................................................. 34 2.1.8 Oligonucleotides ................................................................................................................................ 34 2.1.9 siRNAs .............................................................................................................................................. 36 2.1.10 Plasmids .......................................................................................................................................... 36 2.1.11 Antibodies ........................................................................................................................................ 37 2.1.12 Proteins, enzyme inhibitors and enzymes and ................................................................................ 38 2.1.13 Bacteria strains ................................................................................................................................ 38 2.1.14 Cell lines .......................................................................................................................................... 38 2.1.15 Commercial media ........................................................................................................................... 39 2.1.16 Buffers, media and solutions ........................................................................................................... 39 2.2 Methods ..................................................................................................................................................... 45 2.2.1 Molecular biology standard methods ................................................................................................. 45 2.2.1.1 PCR amplification ..................................................................................................................... 45 2.2.1.2 Restriction digest of DNA .......................................................................................................... 46 2.2.1.3 Ligation of digested insert and vector ....................................................................................... 46 2.2.1.4 Nucleic acid quantification ......................................................................................................... 47 2.2.1.5 PCI extraction ........................................................................................................................... 47 2.2.1.6 Proteinase K digestion .............................................................................................................. 47 2.2.1.7 Generation of truncated MINX, PM5 and PM5-10 constructs ................................................... 48 2.2.1.8 Generation of MINX-80-cleaved ................................................................................................ 50 2.2.1.9 In vitro transcription ................................................................................................................... 50 2.2.1.10 Denaturing polyacrylamide gel electrophoresis ....................................................................... 51 2.2.1.11 SYBR® Gold Nucleic Acid Gel Staining ................................................................................... 51 2.2.1.12 Agarose gel electrophoresis of nucleic acids .......................................................................... 51 2.2.2 Protein-biochemistry standard methods ............................................................................................ 52 2.2.2.1 Protein quantification ................................................................................................................ 52 2.2.2.2 Denaturing SDS polyacrylamide gel-electrophoresis (SDS-PAGE) .......................................... 52 2.2.2.3 Coomassie staining ................................................................................................................... 53 2.2.2.4 Western blot .............................................................................................................................. 53 2.2.2.5 Northern blot ............................................................................................................................