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-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 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 Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.
Highlights
Nuclear import receptors (NIRs) transport RNA-binding proteins (RBPs) and modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs)
We observed the release of maltose-binding protein (MBP):fused in sarcoma (FUS)-NLS from Kapβ[2] in the presence of PR poly-dipeptides (Fig. 5c), suggesting that PR poly-dipeptides compete with NLS of FUS for Kapβ[2]
We showed that PR/GR poly-dipeptides inhibited the Kapβ[2] function as a modifier of RBPs phase transitions, whereas PA/GP/ GA poly-dipeptides did not, and that PR poly-dipeptides target the NLS-binding site of Kapβ[2], which contains a negatively charged region
Summary
Nuclear import receptors (NIRs) transport RNA-binding proteins (RBPs) and modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf[72] interact with NIRs and cause nucleocytoplasmic transport deficit. The molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Nuclear import receptor (NIR) karyopherin-β2 (Kapβ2) controls the nucleocytoplasmic distribution of RBPs and acts as a phase modifier to regulate self-association of FUS by recognizing proline–tyrosine nuclear localization signal (NLS)[7,8,9,10]. One mechanism of the disruption of NCT is that PR poly-dipeptides bind and stabilize phenylalanine:glycine-rich domains of nuclear pore proteins[3], controversial results have been reported[23]. Arginine-rich poly-dipeptides have been found to interact with NIRs and cause NCT deficit[24,25], the molecular basis for the way in which arginine-rich poly-dipeptides affect NIRs function as phase modifiers remains elusive
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