PhD degree in Molecular Medicine (curriculum in Molecular Oncology) European School of Molecular Medicine (SEMM), University of Milan and University of Naples “Federico II” Settore disciplinare: bio/10 Characterization of the E3 ligase dHecw, a novel member of the Drosophila melanogaster Nedd4 family Fajner Valentina Fondazione IFOM, Milan Matricola n. R10751 Supervisor: Dr. Polo Simona Fondazione IFOM, Milan Anno accademico 2017-2018 TABLE OF CONTENTS LIST OF ABBREVIATIONS ..................................................................................................................................... 6 FIGURE INDEX ............................................................................................................................................................ 8 TABLE INDEX ........................................................................................................................................................... 10 ABSTRACT ................................................................................................................................................................. 11 INTRODUCTION ...................................................................................................................................................... 13 1. The multifunctional role of Ubiquitin .............................................................................................. 13 1.1 E3 ligases: catalysts and matchmakers of the Ubiquitin cascade ........................................ 16 1.1.1 RING ligases .............................................................................................................................................................................. 17 1.1.2 HECT E3 ligases ...................................................................................................................................................................... 18 1.1.3 RBR ligases ................................................................................................................................................................................. 19 2. NEDD4 family ................................................................................................................................................ 19 2.1 Domain description ...................................................................................................................................... 19 2.2 Regulation of HECT activity ...................................................................................................................... 22 2.3 Function in physiology and disease ..................................................................................................... 24 2.3.1 HECW1 and HECW2 ........................................................................................................................................................... 27 2.3.2 HECW1 ......................................................................................................................................................................................... 28 2.3.4 HECW2 ......................................................................................................................................................................................... 29 2.4 Model organism to study the NEDD4 family ................................................................................... 31 Drosophila melanogaster model organism ...................................................................................................................... 31 Drosophila melanogaster Nedd4 family ............................................................................................................................. 33 3. mRNA localization and local translation ........................................................................................ 34 3.1 RNA granules .................................................................................................................................................... 36 3.1.1 mRNA regulation in ribonucleoparticles .............................................................................................................. 37 3.1.2 RNP cytoplasmic transport and de-repression following localization ............................................ 39 1 3.2 Studying RNA localization in Drosophila ovaries ......................................................................... 41 3.2.1 Drosophila ovaries ................................................................................................................................................................. 41 3.2.1 RNA localization in Drosophila oocyte .................................................................................................................... 43 3.2.2 mRNA regulation in the germline .............................................................................................................................. 45 3.2.3 dFmrp contributes to mRNA regulation in the ovaries .............................................................................. 46 3.2.4 Ubiquitination in RNPs regulation. ........................................................................................................................... 47 4. Autophagy ....................................................................................................................................................... 47 4.1 The autophagic pathway ........................................................................................................................... 48 4.2 Ubiquitin in Autophagy .............................................................................................................................. 49 4.2.1 The NEDD4 family and Autophagy ........................................................................................................................... 50 4.3 RNA and Autophagy ..................................................................................................................................... 50 4.3.1 Autophagic clearance of RNPs ..................................................................................................................................... 51 4.3.2 Pathological implications of altered RNP homeostasis .............................................................................. 51 AIM OF THE THESIS .............................................................................................................................................. 54 MATERIAL AND METHODS .............................................................................................................................. 55 1. Cell culture ...................................................................................................................................................... 55 1. Gene knock down by RNA interference in S2 cells .................................................................... 55 2.1 Synthesis of double stranded RNA ....................................................................................................... 55 2.2 dsRNA mediated knocked down in S2 cells ..................................................................................... 58 2. Drosophila S2 Cells immunostaining ................................................................................................ 58 3. Fly strains ........................................................................................................................................................ 59 4. UAS-RFP dHecw line generation ......................................................................................................... 59 5. Targeted overexpression and knock down in Drosophila ................................................... 61 6. CRISPR Cas9 mutant generation ......................................................................................................... 62 2 7.1 CRISPR Cas9 mutant screening ......................................................................................................... 64 7. Drosophila behavior assay ..................................................................................................................... 66 8.1 Lifespan Analysis ........................................................................................................................................... 66 8.2 Climbing Assay ................................................................................................................................................ 66 8. Nucleic acid assays ..................................................................................................................................... 67 9.1 Genomic DNA extraction ............................................................................................................................ 67 9.2 RNA extraction and reverse transcription ....................................................................................... 67 9. Protein assays ............................................................................................................................................... 67 10.1 dHecw antibody production ................................................................................................................. 67 10.2 Protein extraction and quantification ............................................................................................. 68 10.3 Immunoblot analysis