Toxic PR_n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

The toxic proline:arginine (PR_n) poly-dipeptide encoded by the (GGGGCC)_n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PR_n poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-β polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PR_n poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PR_n-mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PR_n poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PR_n poly-dipeptide toxicity in the context of a prominent form of ALS.