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Related Mutations in the Sf3b1 Protein on Pre-Mrna Splicing in Vitro

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Related Mutations in the Sf3b1 Protein on Pre-Mrna Splicing in Vitro Investigation of the effects of the splicing inhibitor Pladienolide B and cancer- related mutations in the SF3b1 protein on pre-mRNA splicing in vitro Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program Molecular Biology of the International Max Planck Research School for Molecular Biology and the Georg-August University School of Science (GAUSS) submitted by Sebastian Ludwig from Leverkusen Göttingen 2019 Members of the Examination Board: Prof. Dr. Reinhard Lührmann (1st Referee) Department of Cellular Biochemistry Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Ralf Ficner (2nd Referee) Department of Molecular Structural Biology Georg-August-Universität Göttingen Prof. Dr. Patrick Cramer Department of Molecular Biology Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Henning Urlaub Research Group Bioanalytical Mass Spectrometry Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Ralph Kehlenbach Department of Molecular Biology Georg-August-Universität Göttingen Prof. Dr. Detlef Dönecke Department of Molecular Biology Georg-August-Universität Göttingen Affidavit Herewith I declare, that I prepared the Doctoral Thesis “Investigation of the effects of the splicing inhibitor Pladienolide B and cancer-related mutations in the SF3b1 protein on pre- mRNA splicing in vitro” on my own and with no other sources and aids than quoted. Göttingen, For my family. Index Affidavit .................................................................................................................. 3 Abstract ................................................................................................................ 10 1 Introduction .................................................................................................... 13 1.1 Pre-mRNA processing ............................................................................. 13 1.2 Identification of splice sites ...................................................................... 14 1.3 Chemical mechanism of pre-mRNA splicing ........................................... 15 1.4 Stepwise assembly of the human spliceosome ....................................... 16 1.5 Exon definition ......................................................................................... 19 1.6 The spliceosome and its building blocks ................................................. 20 1.6.1 Uridine-rich small nuclear ribonucleoproteins ................................... 20 1.7 The RNA network of the spliceosome ..................................................... 24 1.8 Protein composition of the spliceosome .................................................. 26 1.9 The role of RNA helicases during splicing ............................................... 28 1.9.1 Proofreading of spliceosome assemblies ......................................... 30 1.10 Alternative splicing ............................................................................... 30 1.10.1 Regulating alternative splicing ....................................................... 32 1.11 Structure of SF3b and the U2 snRNP .................................................. 33 1.12 Spliceosomal mutations in cancer ........................................................ 37 1.13 Small molecule inhibitors of splicing that interact with SF3b1 .............. 40 1.14 Aim of this thesis .................................................................................. 41 2 Material .......................................................................................................... 43 2.1 Chemicals ............................................................................................... 43 2.2 Chromatography materials and consumables ......................................... 44 2.3 Cell culture materials ............................................................................... 44 2.4 Machines ................................................................................................. 44 2.5 Nucleotides ............................................................................................. 45 2.5.1 Radiolabeled nucleotides .................................................................. 45 2.5.2 Oligonucleotides ............................................................................... 45 2.6 Commercial kits ....................................................................................... 46 2.7 Enzymes ................................................................................................. 46 2.8 Antibodies ............................................................................................... 46 2.9 Plasmids .................................................................................................. 46 2.10 Cell lines .............................................................................................. 47 2.10.1 Bacterial cell lines .......................................................................... 47 2.10.2 Human cell lines ............................................................................ 47 2.11 Buffers and Media ................................................................................ 47 2.11.1 Media ............................................................................................. 47 2.11.2 Buffers ........................................................................................... 48 3 Methods ......................................................................................................... 52 3.1 General Molecular Biology methods ....................................................... 52 3.1.1 Quantification of nucleic acids solutions ........................................... 52 3.1.2 Phenol-Chloroform-Isoamyl extraction .............................................. 52 3.1.3 Proteinase K digestion ...................................................................... 53 3.1.4 Protein precipitation .......................................................................... 53 3.1.5 SDS-PAGE electrophoresis .............................................................. 53 3.1.6 Coomassie staining of proteins ......................................................... 54 3.1.7 Western Blot ..................................................................................... 54 3.1.8 Restriction digest of plasmid DNA .................................................... 55 3.1.9 SYBR gold staining of RNA .............................................................. 55 3.1.10 Agarose gel electrophoresis .......................................................... 56 3.1.11 Extraction of DNA from agarose gels ............................................ 56 3.1.12 Ligation of DNA fragments ............................................................ 56 3.1.13 Generation of chemically competent E.coli .................................... 56 3.1.14 Transformation of chemically competent E.coli ............................. 57 3.1.15 Purification of plasmid DNA from E.coli ......................................... 57 3.2 Construct design ..................................................................................... 57 3.2.1 Design of the ZDHHC16-MS2 plasmid ............................................. 57 3.2.2 Design of MINX-exon complex plasmid and its derivatives .............. 58 3.2.3 Design of lentiviral plasmids and transient expression vectors ......... 58 3.2.4 Sequencing of plasmid DNA ............................................................. 59 3.3 Cell culture .............................................................................................. 59 3.3.1 General cell maintenance ................................................................. 59 3.3.2 Cell counting ..................................................................................... 60 3.3.3 Transfection of HEK293T cells ......................................................... 60 3.3.4 Preparation of HeLa S10 nuclear extract .......................................... 61 3.3.5 Preparation of small scale nuclear extracts from K562 cells ............. 61 3.4 In vitro splicing reactions ......................................................................... 62 3.4.1 Template generation for in vitro transcription .................................... 62 3.4.2 In vitro transcription .......................................................................... 62 3.4.3 In vitro splicing reactions .................................................................. 63 3.4.4 In vitro assembly of cross exon complexes ...................................... 64 3.4.5 Analysis of splicing complexes by native electrophoresis ................. 64 3.4.6 Denaturing polyacrylamide gel-electrophoresis ................................ 65 3.4.7 MS2-affinity selection of splicing complexes ..................................... 65 3.5 Other Complex and protein purifications ................................................. 66 3.5.1 Purification of MS2-MBP ................................................................... 66 3.5.2 Anti-FLAG purification of 17S U2 particles ....................................... 67 3.5.3 Crosslinking of 17S U2 snRNPs and crosslink identification ............ 68 3.5.4 Affinity purification of 17S U2 from HeLa nuclear extract .................. 68 3.5.5 Anti-FLAG affinity purification of exon complexes............................. 68 3.6 Mass spectrometry .................................................................................
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