title: Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila.
abstract: Inclusions containing Fused in Sarcoma (FUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline-tyrosine nuclear localization sequence (PY-NLS) of FUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of FUS is mediated by transportin, which binds the PY-NLS and the last arginine/glycine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of FUS in an in vivo model, we generated different transgenic Drosophila lines expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, we analysed the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming FUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, we screened the Drosophila PRMT proteins (DART1-9) and found that knockdown of Dart1 led to a reduction in methylation of hFUS P525L and aggravated its phenotype. These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS autoregulation are conserved between humans and Drosophila. In addition to the well-known neurodegenerative effects of FUS loss-of function, our data suggest toxic potential of overexpressed FUS in the nucleus and of insoluble FUS.
authors.affiliation: German Center for Neurodegenerative Diseases (DZNE) Tübingen, Germany; Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, Tübingen, Germany. Electronic address: sandra.jaeckel@dzne.de.
authors.forename: Sandra
authors.initials: S
authors.lastname: Jäckel
authors.affiliation: Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, Tübingen, Germany.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Summerer
authors.affiliation: Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, Tübingen, Germany.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Thömmes
authors.affiliation: Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany.
authors.forename: Xia
authors.initials: X
authors.lastname: Pan
authors.affiliation: Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany.
authors.forename: Aaron
authors.initials: A
authors.lastname: Voigt
authors.affiliation: Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany.
authors.forename: Jörg B
authors.initials: JB
authors.lastname: Schulz
authors.affiliation: Hertie Institute for Clinical Brain Research, Synaptic Plasticity Group, Tübingen Germany.
authors.forename: Tobias M
authors.initials: TM
authors.lastname: Rasse
authors.affiliation: Adolf Butenandt Institute, Metabolic Biochemistry, Ludwig Maximilians University, Munich, Germany.
authors.forename: Dorothee
authors.initials: D
authors.lastname: Dormann
authors.affiliation: Adolf Butenandt Institute, Metabolic Biochemistry, Ludwig Maximilians University, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE) Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Haass
authors.affiliation: German Center for Neurodegenerative Diseases (DZNE) Tübingen, Germany; Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, Tübingen, Germany. Electronic address: philipp.kahle@uni-tuebingen.de.
authors.forename: Philipp J
authors.initials: PJ
authors.lastname: Kahle
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D030801:Animals, Genetically Modified; D019175:DNA Methylation / Q000502:physiology; D004195:Disease Models, Animal; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D005123:Eye / Q000378:metabolism / Q000473:pathology; D055785:Gene Knockdown Techniques; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D009046:Motor Neurons / Q000378:metabolism / Q000473:pathology; D009069:Movement Disorders / Q000473:pathology / Q000503:physiopathology; D009154:Mutation; D019636:Neurodegenerative Diseases / Q000473:pathology / Q000503:physiopathology; D034622:RNA Interference; D034702:RNA-Binding Protein FUS / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D035362:Transcription Factor TFIID / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; C000599550:FUS protein, human; D028884:Karyopherins; D034702:RNA-Binding Protein FUS; D016601:RNA-Binding Proteins; C000592699:Tnpo protein, Drosophila; D035362:Transcription Factor TFIID; C094754:caz protein, Drosophila; C487898:Art1 protein, Drosophila; D008780:Methyltransferases
keywords: ALS; Caz; Drosophila; FTLD; FUS; PRMT; Transportin