title: RNA-binding ability of FUS regulates neurodegeneration, cytoplasmic mislocalization and incorporation into stress granules associated with FUS carrying ALS-linked mutations.
abstract: Amyotrophic lateral sclerosis (ALS) is an uncommon neurodegenerative disease caused by degeneration of upper and lower motor neurons. Several genes, including SOD1, TDP-43, FUS, Ubiquilin 2, C9orf72 and Profilin 1, have been linked with the sporadic and familiar forms of ALS. FUS is a DNA/RNA-binding protein (RBP) that forms cytoplasmic inclusions in ALS and frontotemporal lobular degeneration (FTLD) patients' brains and spinal cords. However, it is unknown whether the RNA-binding ability of FUS is required for causing ALS pathogenesis. Here, we exploited a Drosophila model of ALS and neuronal cell lines to elucidate the role of the RNA-binding ability of FUS in regulating FUS-mediated toxicity, cytoplasmic mislocalization and incorporation into stress granules (SGs). To determine the role of the RNA-binding ability of FUS in ALS, we mutated FUS RNA-binding sites (F305L, F341L, F359L, F368L) and generated RNA-binding-incompetent FUS mutants with and without ALS-causing mutations (R518K or R521C). We found that mutating the aforementioned four phenylalanine (F) amino acids to leucines (L) (4F-L) eliminates FUS RNA binding. We observed that these RNA-binding mutations block neurodegenerative phenotypes seen in the fly brains, eyes and motor neurons compared with the expression of RNA-binding-competent FUS carrying ALS-causing mutations. Interestingly, RNA-binding-deficient FUS strongly localized to the nucleus of Drosophila motor neurons and mammalian neuronal cells, whereas FUS carrying ALS-linked mutations was distributed to the nucleus and cytoplasm. Importantly, we determined that incorporation of mutant FUS into the SG compartment is dependent on the RNA-binding ability of FUS. In summary, we demonstrate that the RNA-binding ability of FUS is essential for the neurodegenerative phenotype in vivo of mutant FUS (either through direct contact with RNA or through interactions with other RBPs).
authors.affiliation: Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA, USA.
authors.forename: J Gavin
authors.initials: JG
authors.lastname: Daigle
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Lanson
authors.forename: Rebecca B
authors.initials: RB
authors.lastname: Smith
authors.forename: Ian
authors.initials: I
authors.lastname: Casci
authors.forename: Astha
authors.initials: A
authors.lastname: Maltare
authors.forename: John
authors.initials: J
authors.lastname: Monaghan
authors.forename: Charles D
authors.initials: CD
authors.lastname: Nichols
authors.forename: Dmitri
authors.initials: D
authors.lastname: Kryndushkin
authors.forename: Frank
authors.initials: F
authors.lastname: Shewmaker
authors.forename: Udai Bhan
authors.initials: UB
authors.lastname: Pandey
mesh_terms: D020816:Amino Acid Motifs; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D009046:Motor Neurons / Q000378:metabolism; D020125:Mutation, Missense; D021381:Protein Transport; D034702:RNA-Binding Protein FUS / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; C439908:FUS protein, Drosophila; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D034702:RNA-Binding Protein FUS