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Identification of the Nuclear Export Pathways of TDP‐43 and FUS

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Dissertation der Graduate School of Systemic Neurosciences der Ludwig‐Maximilians‐Universität München Identification of the nuclear export pathways of TDP‐43 and FUS Ph.D. (Doctor of Philosophy) Thesis HELENA EDERLE Born in Kisel, Russia 21st December 2017 Dissertation der Graduate School of Systemic Neurosciences der Ludwig‐Maximilians‐Universität München Identification of the nuclear export pathways of TDP‐43 and FUS Ph.D. (Doctor of Philosophy) Thesis HELENA EDERLE Born in Kisel, Russia 21st December 2017 This Ph.D. thesis was conducted and written under the supervision of Dorothee Dormann in the BioMedical Center (BMC) of the Ludwig Maximilians University Munich, Germany in the time from the 1th September 2014 to the 21st December 2017. 1st appraiser and supervisor: Dorothee Dormann, Ph.D. Ludwig Maximilians University (LMU) Munich BMC – BioMedical Center, Institute for Cell Biology (Anatomy III) Grosshaderner Str. 9 82152 Planegg‐Martinsried Germany 2nd appraiser: Prof. Dieter Edbauer, M.D. DZNE – German Center for Neurodegenerative Diseases Feodor‐Lynen Str. 17 81377 Munich Germany 3rd appraiser: Prof. Paul Walther, Dr. sc. nat. (ETH) Head of the Electron Microscopy Core Facility University of Ulm Albert‐Einstein‐Allee 11 89081 Ulm Germany The oral defense of the Ph.D. thesis took place on Wednesday, the 25th April 2018. Dedicated to ANGELA and JOHANNES WINKLER ABBREVIATION INDEX ALS Amyotrophic lateral sclerosis C‐Terminus Carboxy‐terminus C9orf72 Chromosome 9 open reading frame 72 CRM1 Chromosome region maintenance 1 FTD Frontotemporal dementia FUS Fused in sarcoma hnRNP Heterogenous nuclear ribonucleoprotein hnRNP‐C Heterogenous nuclear ribonucleoprotein C1/C2 lncRNA Long non‐coding RNA mRNA Messenger RNA mRNP Messenger ribonucleoprotein miRNA MicroRNA N‐Terminus Amino‐Terminus NES Nuclear export signal NLS Nuclear localization signal NPC Nuclear pore complex NUP Nulceoporin PY‐NLS Proline – tyroline nuclear localization signal RBP RNA‐binding protein RGG Arginine (A) – glycine (G) – glycine (G) RNP Ribonucleoprotein RRM RNA‐recognition motif TDP‐43 TAR DNA‐binding protein of 43 kDa UTR Untranslated region XPO1 Exportin‐1 * List of most common abbreviations used in the manuscript 5 INDEX MAIN PROJECT ............................................................................................................................................ 8 Identification of the nuclear export pathways of TDP‐43 and FUS ........................................................... 8 ABSTRACT ............................................................................................................................................... 8 I. INTRODUCTION ................................................................................................................................... 9 1. Amyotrophic lateral sclerosis and frontotemporal dementia: TDP‐43 and FUS in neurodegenerative diseases ............................................................................................................. 9 2. Nuclear import defects in amyotrophic lateral sclerosis and frontotemporal dementia .......... 10 3. TDP‐43 and FUS: of functions and malfunctions ........................................................................ 12 3.1. Functional domains of TDP‐43 and FUS .............................................................................. 12 3.2. From pre‐mRNA to microRNA: functions of TDP‐43 and FUS in the nucleus ..................... 13 3.2.1. Nuclear functions of TDP‐43 ........................................................................................ 13 3.2.2. Nuclear functions of FUS .............................................................................................. 14 3.3. mRNA stability, trafficking and translation: functions of TDP‐43 and FUS in the cytoplasm ..................................................................................................................................................... 16 3.3.1. Cytoplasmic functions of TDP‐43 ................................................................................. 16 3.3.2. Cytoplasmic functions of FUS ....................................................................................... 17 4. Nuclear export of TDP‐43 and FUS: of evidence and ideas ........................................................ 19 4.1. Evidence for nuclear export of TDP‐43 and FUS ................................................................. 19 4.2. Potential export routes: how TDP‐43 and FUS could exit the nucleus ............................... 19 4.2.1. Receptor‐ or mRNA‐driven export ............................................................................... 19 4.2.2. Export of TDP‐43: receptor‐mediated or alongside with mRNA? ............................... 20 4.2.3. Export of FUS: alongside with mRNA or a single receptor? ......................................... 21 4.2.4. An alternative possibility: passive diffusion as an export route for TDP‐43 and FUS . 22 5. Ph.D. project goals ....................................................................................................................... 23 II. RESULTS ............................................................................................................................................ 24 1. Analysis of nuclear export of TDP‐43 and FUS in the interspecies heterokaryon assay ............ 24 2. Predicted Exportin‐1/CRM1‐dependet nuclear export signals of TDP‐43 and FUS are not functional ......................................................................................................................................... 28 3. Nuclear export of TDP‐43 and FUS is independent of Exportin‐1/CRM1 ................................... 30 4. Nuclear export of TDP‐43 and FUS is independent of RNA binding or the mRNA export pathway ......................................................................................................................................................... 33 5. Alternative export factors in nuclear export of TDP‐43 and FUS ............................................... 35 6. Potential domains involved in nuclear export of TDP‐43 and FUS ............................................. 37 7. Enlargement of TDP‐43 and FUS impairs their nuclear egress ................................................... 40 8. Nuclear egress of TDP‐43 is promoted upon decreased transcription or RNA‐binding of TDP‐43 ......................................................................................................................................................... 45 III. DISCUSSION ..................................................................................................................................... 49 1. Transport defects in ALS and FTD ............................................................................................... 49 2. Export via Exportin‐1/CRM1 – a misconception for TDP‐43 and FUS ........................................ 50 3. Of alternative routes and factors: TDP‐43 and FUS seem to leave the nucleus via passive diffusion ........................................................................................................................................... 52 SIDE PROJECT ............................................................................................................................................ 56 6 Identification of additional import factors regulating nuclear localization of TDP‐43 and FUS ............. 56 I. BACKGROUND & PRELIMINARY RESULTS ......................................................................................... 56 II. OUTLOOK .......................................................................................................................................... 61 EXPERIMENTAL PROCEDURES .................................................................................................................. 63 1. Automated‐imaging screen and analysis ......................................................................................... 63 2. Cell culture, transfection and drug treatment ................................................................................ 63 3. Cell fractionation and analysis ......................................................................................................... 64 4. Cloning and cDNA constructs .......................................................................................................... 65 5. Fluorescence Loss In Photobleaching (FLIP) and analysis ............................................................... 66 6. Hormone‐inducible nuclear transport assay ................................................................................... 66 7. Image acquisition and analysis ........................................................................................................ 67 8. Immunocytochemistry ..................................................................................................................... 67 9. Interspecies heterokaryon assay ..................................................................................................... 67 10. NEX‐TRAP assay .............................................................................................................................. 68 11. Poly(A)+RNA in situ hybridization .................................................................................................. 68 12. Preparation of cell lysates and immunoblotting ..........................................................................
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