p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie der Johannes Gutenberg-Universität Mainz vorgelegt von Marina E. Borisova geboren am 04.05.1989 in Moskau, Russland Mainz, 2018 Table of content List of Publications ....................................................................................................... I Summary .................................................................................................................... II Zusammenfassung .................................................................................................... III Introduction ................................................................................................................. 1 1.1 Types of DNA damage and common repair mechanisms ...................... 1 1.1.1 Mismatch repair ............................................................................... 4 1.1.2 Base-excision repair ........................................................................ 4 1.1.3 Nucleotide-excision repair ................................................................ 5 1.1.4 Double-strand break repair .............................................................. 6 1.1.5 Replication stress ............................................................................ 8 1.2 The DNA damage response ................................................................... 9 1.2.1 Phosphorylation: ATM and ATR are central kinases of DDR ........... 9 1.2.2 Other PTMs in the regulation of DDR ............................................ 11 1.3 Interplay between RNA metabolism and DDR...................................... 13 1.3.1 General mechanism of transcription .............................................. 13 1.3.2 Regulation of transcription after UV-light-induced DNA damage ... 15 1.3.2 Splicing regulation after UV-light-induced DNA damage ............... 17 1.3.3 Regulation of mRNA stability and translation after UV-light-induced DNA damage ..................................................................................................... 18 1.4 Mitogen-activated protein kinases ........................................................ 19 1.4.1 p38 MAPK ...................................................................................... 19 1.4.2 p38 MAPK phosphatases .............................................................. 21 1.4.3 MAPK-activated protein kinase family ........................................... 22 1.4.4 JNK MAPK ..................................................................................... 23 1.5 14-3-3 protein family ............................................................................. 24 1.6 Aims of the study .................................................................................. 27 Results ...................................................................................................................... 28 2.1 Identification of p38-dependent phosphorylation sites .......................... 28 2.1.1 UV-light activates p38 MAPK signaling independently of canonical DNA damage signaling ...................................................................................... 28 2.1.2 p38 MAPK signaling has a broad regulatory role after UV-light ..... 28 2.1.3 p38 MAPK phosphorylates LXRQXS/T motif after UV-light ........... 31 2.1.4 MK2/3 are key transducers of p38-dependent signaling ................ 32 2.1.5 p38-MK2/3 signaling axis phosphorylates RNA-binding proteins ... 32 2.1.6 p38 MAPK promotes dissociation of RNA-binding proteins from chromatin ........................................................................................................... 35 2.2 14-3-3 “reads” p38-MK phosphorylation induced by UV-light ............... 37 2.3 UV-light-induced NELFE phosphorylation mediates 14-3-3 binding ..... 38 2.3.1. NELFE transiently interacts with 14-3-3 after UV-light exposure in an MK2-dependent manner .................................................................................... 39 2.3.2 NELFE phosphorylation on S115 regulates its binding to 14-3-3 ... 40 2.3.3 Phosphorylated NELFE on S115 binds directly to 14-3-3 .............. 44 2.4 NELF phosphorylation regulates transcriptional elongation ................. 46 2.4.1 NELFE is required for cell survival after UV-light exposure ........... 46 2.4.2 p38 MAPK promotes dissociation of the NELF complex from chromatin ........................................................................................................... 47 2.4.3 NELFE release is accompanied by transcriptional elongation ....... 48 Discussion ................................................................................................................ 51 3.1 The p38-MK2/3 pathway is activated independently of canonical DNA damage signaling .................................................................................................. 51 3.1.1 ATR-CHEK1 pathway in DDR ........................................................ 51 3.1.2 The role of p38 MAPK in DDR ....................................................... 52 3.1.3 MK2/3 act redundantly downstream of p38 MAPK after UV-light exposure ............................................................................................................ 53 3.2 14-3-3 proteins are general p38-MK2/3 phosphorylation readers ........ 53 3.3 Regulation of transcription after UV-light exposure .............................. 54 3.3.1 The role of the p38-MK2/3 pathway in transcriptional elongation .. 54 3.3.2 The role of the NELF complex in the regulation of transcriptional elongation after UV-light exposure .................................................................... 55 3.3.3 NELF role in tumorigenesis ............................................................ 57 3.4 The role of the p38-MK2/3 pathway in RNA splicing, stability, and translation ............................................................................................................. 58 Conclusions ................................................................................................ 58 Materials and methods ............................................................................................. 59 4.1 List of solutions and buffers .................................................................. 59 4.1.1. Buffers and solutions .................................................................... 59 4.1.2. Enzymes, reagents, and commercially available kits .................... 60 4.2 Cell culture ........................................................................................... 62 4.2.1 Transfection of cells ....................................................................... 62 4.2.2 Genotoxic treatment of cells .......................................................... 62 4.2.3 Colony formation assay ................................................................. 62 4.2.4 Cell viability .................................................................................... 63 4.2.5 Immunofluorescence and confocal microscopy ............................. 63 4.3 Molecular biology ................................................................................. 63 4.3.1 Gateway cloning ............................................................................ 63 4.3.2 Site-directed mutagenesis ............................................................. 63 4.4 Biochemistry ......................................................................................... 64 4.4.1 SDS-PAGE and western blotting ................................................... 64 4.4.2 Total cell lysis ................................................................................ 64 4.4.3 Cellular fractionation ...................................................................... 64 4.4.4 Pull-downs using GFP-Trap agarose or StrepTactin sepharose .... 65 4.4.5 Co-immunoprecipitation ................................................................. 65 4.4.6 Purification of GST-14-3-3 and GST-pull downs ............................ 65 4.4.7 Peptide pull downs ......................................................................... 65 4.4.8 Structure determination .................................................................. 66 4.5 Mass spectrometry-based proteomics .................................................. 66 4.5.1 Cell lysis for phosphoproteomics ................................................... 66 4.5.2 In-solution digestion ....................................................................... 66 4.5.3 Phosphopeptide enrichment .......................................................... 67 4.5.4 Micro tip-based strong cation exchange chromatography (Micro-SCX) .......................................................................................................................... 67 4.5.5 In-gel digestion .............................................................................. 67 4.4.6 Desalting and concentration of peptides ........................................ 68 4.5.7 MS analysis ................................................................................... 68 4.5.8 MS peptide identification ................................................................ 68 4.5.9 Phosphorylation site occupancy analysis....................................... 69 4.5.10 In vitro kinase assay .................................................................... 69 4.5.11 Computational