ABSTRACT
Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.
Subject(s)
Active Transport, Cell Nucleus/genetics , C9orf72 Protein/chemistry , Peptides/chemistry , beta Karyopherins/chemistry , Binding Sites , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Cloning, Molecular , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HeLa Cells , Humans , Models, Molecular , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Peptides/genetics , Peptides/metabolism , Phase Transition , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , beta Karyopherins/antagonists & inhibitors , beta Karyopherins/genetics , beta Karyopherins/metabolismABSTRACT
Liquid-liquid phase separation (LLPS) of proteins and nucleic acids to form membraneless cellular compartments is considered to be involved in various biological functions. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vivo and in vitro. Here, we investigated the effects of pressure and temperature on the LLPS of FUS by high-pressure microscopy and high-pressure UV/vis spectroscopy. The phase-separated condensate of FUS was obliterated with increasing pressure but was observed again at a higher pressure. We generated a pressure-temperature phase diagram that describes the phase separation of FUS and provides a general understanding of the thermodynamic properties of self-assembly and phase separation of proteins. FUS has two types of condensed phases, observed at low pressure (LP-LLPS) and high pressure (HP-LLPS). The HP-LLPS state was more condensed and exhibited lower susceptibility to dissolution by 1,6-hexanediol and karyopherin-ß2 than the LP-LLPS state. Moreover, molecular dynamic simulations revealed that electrostatic interactions were destabilized, whereas cation-π, π-π, and hydrophobic interactions were stabilized in HP-LLPS. When cation-π, π-π, and hydrophobic interactions were transiently stabilized in the cellular environment, the phase transition to HP-LLPS occurred; this might be correlated to the aberrant enrichment of cytoplasmic ribonucleoprotein granules, leading to amyotrophic lateral sclerosis.
Subject(s)
Amyotrophic Lateral Sclerosis , RNA-Binding Protein FUS/chemistry , Humans , Protein Domains , TemperatureABSTRACT
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-ß2 (Kapß2), 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 Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 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 Kapß2 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.
