LIQUID-LIQUID PHASE SEPARATION: MOLECULAR MECHANISMS AND INFLUENCE ON THE mRNA DECAPPING MACHINERY DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von STEFAN SCHÜTZ aus QUEDLINBURG im Jahr 2019 Das Promotionsgesuch wurde eingereicht am: 14. Juni 2019 Die Arbeit wurde angeleitet von: PROF. DR. REMCO SPRANGERS Unterschrift: Stefan Schütz - 2 - - 3 - List of Publications and Manuscripts Published 1. Schütz, S.; Nöldeke, E. R.; Sprangers, R. A Synergistic Network of Interactions Promotes the Formation of in Vitro Processing Bodies and Protects mRNA against Decapping. Nucleic Acids Res. 2017, 45 (11), 6911–6922. https://doi.org/10.1093/nar/gkx353. 2. Damman R; Schütz, S; Luo, Y; Weingarth, M; Sprangers, R; Baldus, M. Atomic-level insight into the maturated state of mRNA processing bodies by combining solid and solution-state NMR spectroscopy. Nat Commun. 2019, 10 (1), 4536. https://doi.org/10.1038/s41467-019- 12402-3. Accepted 3. Schütz, S.; Sprangers, R. Methyl TROSY spectroscopy: A versatile NMR approach to study challenging biological systems. Prog. Nucl. Magn. Reson. Spectrosc., 2019, in press, https://doi.org/10.1016/j.pnmrs.2019.09.004 In preparation 4. Schütz, S; Sprangers, R. Deciphering the contributions of molecular interactions that lead to liquid-liquid phase separation of the conserved DEAD-box protein Dhh1. The contributions of the individual authors to the listed publications and manuscripts are indicated at the beginning of the respective chapters. - 4 - TABLE OF CONTENT SUMMARY ........................................................................................................................................... - 8 - SELECTED ABBREVIATIONS .................................................................................................................. - 9 - CHAPTER 1 General Introduction ................................................................................................. - 10 - 1.1 A SHORT VIEW ON THE LIFE OF AN mRNA ........................................................................ - 10 - 1.2 mRNA DEGRADATION IN EUKARYOTES ............................................................................. - 12 - 1.2.1 3´-5´ decay ................................................................................................................. - 14 - 1.2.2 5´-3´ decay and the mRNA degradation machinery .................................................. - 14 - 1.3 LIQUID-LIQUID PHASE SEPARATION .................................................................................. - 15 - 1.4 NMR SPECTROSCOPY ......................................................................................................... - 18 - 1.4.1 The TROSY experiment .............................................................................................. - 18 - 1.4.2 Methyl TROSY ............................................................................................................ - 19 - 1.4.3 Methyl labeling .......................................................................................................... - 20 - 1.4.4 Methyl resonance assignment .................................................................................. - 22 - 1.4.5 CSP experiments ........................................................................................................ - 27 - 1.4.6 Methionine scanning ................................................................................................. - 29 - 1.5 AIMS OF THIS THESIS ......................................................................................................... - 32 - CHAPTER 2 A synergistic network of interactions promotes the formation of in vitro processing bodies and protects mRNA against decapping ....................................... - 33 - 2.1 INTRODUCTION ................................................................................................................. - 33 - 2.2 MATERIALS AND METHODS .............................................................................................. - 35 - 2.2.1 Protein expression and purification .......................................................................... - 35 - 2.2.2 Protein fluorescence labeling .................................................................................... - 37 - 2.2.3 RNA in vitro transcription, purification and capping ................................................. - 37 - 2.2.4 Liquid-liquid phase separation experiments ............................................................. - 39 - 2.2.5 Microscopy ................................................................................................................ - 39 - 2.2.6 NMR ........................................................................................................................... - 39 - 2.2.7 Decapping assays ....................................................................................................... - 40 - 2.2.8 HPLC analysis ............................................................................................................. - 40 - 2.2.9 Analysis of degradation data ..................................................................................... - 40 - 2.2.10 RNase A protection assays ........................................................................................ - 40 - 2.3 RESULTS ............................................................................................................................. - 41 - 2.3.1 High throughput approach for LLPS determination .................................................. - 41 - 2.3.2 Pdc1 stimulates LLPS ................................................................................................. - 43 - - 5 - 2.3.3 RNA strongly stimulates LLPS .................................................................................... - 44 - 2.3.4 RNA is protected against degradation by LLPS .......................................................... - 46 - 2.3.5 LLPS reduces the catalytic activity of Dcp2 ............................................................... - 46 - 2.3.6 The IDR in Edc3 specifically interacts with RNA ........................................................ - 48 - 2.3.7 Interactions of the Edc3 IDR and the RNA-helicase Dhh1 are conserved from yeast to human .......................................................................................................... - 51 - 2.3.8 Maturation of processing bodies .............................................................................. - 51 - 2.4 DISCUSSION ....................................................................................................................... - 54 - CHAPTER 3 Atomic level insight into the maturated state of mRNA processing bodies by combining solid- and solution-state NMR spectroscopy ...................................... - 57 - 3.1 INTRODUCTION ................................................................................................................. - 57 - 3.2 MATERIALS AND METHODS .............................................................................................. - 59 - 3.2.1 Protein expression and purification .......................................................................... - 59 - 3.2.2 RNA in vitro transcription and purification ............................................................... - 61 - 3.2.3 Liquid-liquid phase separation assays ....................................................................... - 62 - 3.2.4 Solution-state NMR experiments .............................................................................. - 62 - 3.2.5 Solid-state NMR experiments .................................................................................... - 62 - 3.2.6 Molecular Dynamics Simulations .............................................................................. - 63 - 3.3 RESULTS ............................................................................................................................. - 63 - 3.3.1 The LSm domain of Edc3 is mobile in the matured state .......................................... - 63 - 3.3.2 The YjeF_N domain forms a rigid core in the matured state .................................... - 65 - 3.3.3 Interactions between the IDR and the Yjef_N domain are important for phase separation of Edc3 ..................................................................................................... - 67 - 3.3.4 Details of the interactions between the IDR and the Yjef_N domain ....................... - 69 - 3.3.5 RNA interacts with the IDR and thereby increases rigidity ....................................... - 72 - 3.4 DISCUSSION ....................................................................................................................... - 74 - CHAPTER 4 Deciphering the contributions of molecular interactions that lead to liquid-liquid phase separation of the conserved DEAD-box protein Dhh1 ............... - 76 - 4.1 INTRODUCTION ................................................................................................................. - 76 - 4.2 MATERIALS AND METHODS .............................................................................................. - 80 - 4.2.1 Protein expression and purification .......................................................................... - 80 - 4.2.2 Complex reconstitution ............................................................................................