Nuclear localization sequence of FUS and induction of stress granules by ALS mutants

Abstract

Mutations in FUS have been reported to cause a subset of familial amyotrophic lateral sclerosis (ALS) cases. Wild-type FUS is mostly localized in the nuclei of neurons, but the ALS mutants are partly mislocalized in the cytoplasm and can form inclusions. Little is known about the regulation of FUS subcellular localization or how the ALS mutations alter FUS function. Here we demonstrate that the C-terminal 32 amino acid residues of FUS constitute an effective nuclear localization sequence (NLS) as it targeted beta-galactosidase (LacZ, 116 kDa) to the nucleus. Deletion of or the ALS point mutations within the NLS caused cytoplasmic mislocalization of FUS. Moreover, we identified the poly-A binding protein (PABP1), a stress granule marker, as an interacting partner of FUS. PABP1 formed large cytoplasmic foci that co-localized with the mutant FUS inclusions. No such foci, which resemble stress granules, were observed in the presence of wild-type FUS. In addition, processing bodies, which are functionally related to stress granules, were adjacent to but not co-localized with the mutant FUS inclusions. Our results suggest that the ALS mutations in the C-terminal NLS of FUS can impair FUS nuclear localization and induce cytoplasmic mislocalization, inclusion formation, and potential perturbation of RNA metabolism.

Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

“…4 and Table 1). Loss of Kapβ2 binding in these mutants correlates with the degree of cytoplasmic mislocalization in cells (2,(6)(7)(8)(9). Fig.…”

“…Such high affinities may be consistent with the initiation of disease later in life. High affinities for Kapβ2 may allow near-normal nuclear function and localization for some FUS mutants or partial mislocalization for others, which are tolerated until the aging cells encounter additional hits such as further impairment of the nuclear transport machinery or other cellular stress that exacerbate FUS mislocalization or trigger aggregation of mislocalized FUS (2,5,7,9). Furthermore, even though ALS mutants bind Kapβ2 rather tightly, effects of decreased affinities compared with those of wild-type FUS may be amplified in cells due to competition with other high-affinity Kapβ2 cargos and with nonspecific competitors (16,17,30).…”

“…More recently, the signal was shown to direct Kapβ2-mediated nuclear import in cells (2) and to be heavily mutated in ∼5% of familial amyotrophic lateral sclerosis (ALS) (3)(4)(5), a progressive and fatal neurodegenerative disorder. ALS mutations in the PY-NLS disrupted nuclear import of FUS, causing its mislocalization and aggregation in the cytoplasm, as evidenced by cytoplasmic FUS inclusions in motor neurons of ALS patients (2,(6)(7)(8)(9). The PY-NLS of FUS is also mutated in another neurodegenerative disease, frontotemporal lobar dementia (FTLD), which also is characterized by cytoplasmic mislocalization and aggregation of FUS (10,11).…”

Self-assembled FUS binds active chromatin and regulates gene transcription

“…Such observations suggest two potential disease-causing mechanisms: loss of FUS normal function in the nucleus and gain of toxic function in the cytoplasm. It remains to be determined which mechanism plays a more critical role in ALS etiology and the two mechanisms are not necessarily exclusive of each other.Cytoplasmic FUS inclusions resemble stress granules, indicated by colocalization of FUS with different stress granule components (11,12). Stress granules are temporary cellular structures containing RNAs and proteins from suspended translation apparatus (14).…”

Highly Efficient Targeted Mutagenesis in Mice Using TALENs

“…Mutations disrupting the C-terminal nuclear localization sequence (NLS) of FUS have been identified in ALS patients (Kwiatkowski et al 2009), whereas a hexanucleotide repeat expansion in the first intron of the C9orf72 gene was found in patients representing ALS, FTLD, or both diseases (DeJesus-Hernandez et al 2011;Renton et al 2011). In the Fus gene dominant mutations within the NLS disrupt the nuclear import of FUS and lead to its cytoplasmic deposition in the brain and spinal cord of patients (Bosco et al 2010;Gal et al 2011;Ito et al 2011;Kino et al 2011;Dormann and Haass 2013). This defect is a key to pathogenesis since mutations that severely impair nuclear import, such as the P525L replacement, lead to an early onset and rapid progression of the disease.…”