Abstract
Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid–liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-β2 (Kapβ2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapβ2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapβ2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapβ2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapβ2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapβ2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.
Highlights
Mutations in the Fused in Sarcoma (FUS) RNA-binding protein have been linked to the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS), as they cause approximately 5% of the cases for familial amyotropic lateral sclerosis (ALS) and 1% of sporadic A LS1
The complex crystallized in the same space group of P21212 with similar crystallographic parameters as the Kapβ2·Wild type (WT) FUSPY-NLS12
The Pro[525] to leucine (P525L) mutation changes the structure of the proline-tyrosine nuclear localization signal (PY-NLS) main chain from residues 522–525, which diverged from the peptide main chain of the WT PY-NLS and moved slightly farther away from Kapβ[2] (Fig. 1D,E)
Summary
Mutations in the Fused in Sarcoma (FUS) RNA-binding protein have been linked to the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS), as they cause approximately 5% of the cases for familial ALS and 1% of sporadic A LS1. Mutations related to the PY-NLS of FUS (FUSPY-NLS) cause the FUS variants to localize to different extents to the cytoplasm and cause different degrees of disease severity in patients. A small fraction FUS(P525L) localizes to the nucleus even though the protein is substantially mislocalized to the cytoplasm. It is unclear how the P525L missense mutation affects interactions of the PY-NLS with Kapβ[2]. It is not known how FUS(R495X) is transported into the nucleus when it is missing the PY-NLS. KD determined by error-surface projection in the global analysis of the duplicates or triplicates of each experimental set
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