Research Collection Doctoral Thesis Interactions of the alternative splicing factor RBFOX with non- coding RNAs Author(s): Stoltz, Moritz Publication Date: 2015 Permanent Link: https://doi.org/10.3929/ethz-a-010496253 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO. 22872 Interactions of the alternative splicing factor RBFOX with non- coding RNAs A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) Presented by Moritz Stoltz Master of Science in Chemistry, University of Basel born on 24.07.1983 citizen of Marburg, Germany accepted on the recommendation of Prof. Dr. Jonathan Hall Prof. Dr. Gisbert Schneider 2015 2 Don't speak to me of anarchy or peace of calm revolt, man We're in a play of slow decay orchestrated by Boltzmann It's entropy, it's not a human issue Entropy, it's matter of course Entropy, energy at all levels Entropy, from it you cannot divorce And your pathetic moans of suffrage tend to lose all significance Extinction, degradation; the natural outcomes of our ordered lives Power, motivation; temporary fixtures for which we strive Something in our synapses assures us we're okay But in our disequilibrium we simply cannot stay From “Entropy” by Bad Religion, written by Greg Graffin 3 4 Content Summary ................................................................................................................................................. 7 Zusammenfassung ................................................................................................................................... 9 Acknowledgments ................................................................................................................................. 11 1: Interactions of FOX proteins with microRNAs................................................................................... 15 1: Introduction ................................................................................................................................... 15 1.1 Surface Plasmon Resonance spectroscopy (SPR) ........................................................................ 15 1.1.1..: History of the development of SPR ................................................................................... 15 1.1.2.: Theoretical background of SPR .......................................................................................... 15 1.1.3.: Experimental set-up of the SPR method ............................................................................ 18 1.1.4.: Fitting experimental data from SPR experiments .............................................................. 20 1.2.: RNA Synthesis ............................................................................................................................ 24 1.2.1.: Oligonucleotide deprotection. ........................................................................................... 25 1.2.2.: Activation and coupling of the phosphoramidites ............................................................. 26 1.2.3.: Capping of the unreacted reagents. ................................................................................... 27 1.2.4 Oxidation .............................................................................................................................. 28 1.2.5.: Workup and purification of oligonucleotides .................................................................... 29 1.3.: MicroRNA biogenesis ................................................................................................................ 30 1.3.1 Nuclear processing ............................................................................................................... 30 1.3.3 Cytoplasmic processing ........................................................................................................ 32 1.3.4 The RNA induced silencing complex (RISC) .......................................................................... 32 1.3.5.: Additional regulatory elements.......................................................................................... 35 1.3.6.: MiRNA in human disease ................................................................................................... 36 1.4.: RBFOX proteins .......................................................................................................................... 37 1.4.1.: Structure of the RBFOX/RNA complex ............................................................................... 38 1.4.2.: RBFOX in alternative splicing .............................................................................................. 43 1.4.3.: Variation in the recognition of RNA by RBFOX proteins .................................................... 45 1.4.4.: MicroRNA regulation of RBFOX-3....................................................................................... 46 1.5.: Detection of new RNA binding motifs ....................................................................................... 47 1.5.1. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) ................................. 47 1.5.2 Cross-linking immunoprecipitation (CLIP) based methods .................................................. 50 Aim of the project ................................................................................................................................. 54 2. Results and Discussion ...................................................................................................................... 55 2.1.: ELISA Assay of RBFOX against pre-miRNAs ............................................................................... 55 2.2: Synthesis of two RNA libraries ................................................................................................... 59 2.3: Immobilization of the RBFOX RRM on a biosensor .................................................................... 60 5 2.4.: Screen for new RNA binding motifs of RBFOX RRM .................................................................. 63 2.5.: Single base variants of the consensus RBFOX binding element ................................................ 70 2.5.1.:. Use of dimethyl-cytidine (dMC) and monomethyl-cytidine (mMC) to gain insights into RBP/RNA recognition. ................................................................................................................... 74 2.6. Interaction of RBFOX with precursor miRNAs ............................................................................ 77 2.6.1. SPR studies of RBFOX/miRNA interactions ......................................................................... 77 2.6.2.: Influence of RNA structure upon binding to RBFOX RRM ................................................. 83 2.6.4.: The influence of the sugar pucker of the ribose on the binding affinity against RBFOX ... 86 2.7.: Effects of the alternative splicing factor RBFOX-2 on the biogenesis of pre-miR-20b .............. 89 3.: Summary and Outlook...................................................................................................................... 92 4.: Materials and Methods .................................................................................................................... 94 4.1.: List of used Chemicals ............................................................................................................... 94 4.2.: List of used Equipment .............................................................................................................. 95 4.3.: Methods .................................................................................................................................... 96 5.: Supplementary tables and figures ................................................................................................. 104 5.1.: List of Abbreviations ................................................................................................................ 104 5.2.: Tables ...................................................................................................................................... 106 5.3.: Figures ..................................................................................................................................... 139 6.: Manuscripts .................................................................................................................................... 147 6.1.: Development of a RNA negative control ................................................................................. 147 6.2.: Rapid high-yield cell-free expression of quantitatively biotinylated proteins (Draft) ............ 155 4.4.: List of tables ............................................................................................................................ 176 4.5.: List of tables ............................................................................................................................ 180 5.: References ...................................................................................................................................... 182 CURRICULUM VITAE: MORITZ STOLTZ ............................................................................................... 199 6 Summary RNA binding proteins (RBPs) play roles in the post-transcriptional regulation of RNA metabolism including