Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X)

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.

The RNA-binding protein FUS is chaperoned and imported into the nucleus by a network of import receptors

“…Our results now show that additional NTRs can perform similar functions. Of note, a subset of alternative NTRs has also been reported to interact with FUS during the review process of our article ( 52 ). Apart from TNPO1/2, TNPO3 (also known as transportin-SR2, Trn-SR2, Trn-3) and its splice variant transportin-SR ( 53 , 54 , 55 , 56 , 57 ) have been implicated in the nuclear import of several RBPs by binding to their arginine–serine (RS)-rich regions, both in a phosphorylation-dependent and phosphorylation-independent manner ( 29 , 57 ).…”

Wild-type FUS corrects ALS-like disease induced by cytoplasmic mutant FUS through autoregulation

“…Unexpectedly, a significant fraction of the mouse FUS protein was found in the nucleus of adult Fus ∆NLS/∆NLS mice with hFUS transgene, albeit the endogenous FUS protein is completely truncated of the NLS. This is a priori surprising as the NLS is the major domain responsible for interaction with nuclear import receptors such as karyopherin ß2 [ 24 , 25 ]. There are at least two possible explanations to this observation.…”

“…Second, nuclear import of FUS might be possible through NLS-independent mechanisms. Indeed, recent work has shown that ∆NLS mutants of FUS could still interact through RGG domains with karyopherin ß2 [ 24 , 25 ], as well as other nuclear import receptors [ 25 ] leading to significant nuclear import [ 24 ] .…”

(Dis)Solving the problem of aberrant protein states

“…In some cases, protein cargo bear disease-linked mutations in the NLS, as with FUS P525L , in which the proline residue of the PY-NLS is mutated, or in FUS R495X , in which the PY-NLS is largely deleted ( Guo et al, 2019 ). These mutant forms of FUS are associated with very aggressive forms of early-onset ALS, and are more refractory to Kapβ2 activity ( Gonzalez et al, 2021 ; Guo et al, 2018 ). In sporadic ALS and other neurodegenerative diseases for which there is no clear genetic component, NIR levels may decline due to stress and aging or otherwise become less effective, as defects in nucleocytoplasmic transport are a key feature of many neurodegenerative diseases ( Guo et al, 2019 ; Kim and Taylor, 2017 ).…”