PR n poly‐dipeptides encoded by the repeat expansion in C9orf72 block nuclear import and export

Expansion of the (GGGGCC) n hexanucleotide repeat within the first intron of the C9orf72 gene is the mutation that leads to the most prevalent heritable form of amyotrophic lateral sclerosis (ALS). The expanded repeat is aberrantly transcribed from both sense and anti‐sense strands relative to the C9orf72 gene, and both transcripts are translated in an ATG‐independent manner to yield five distinct poly‐dipeptides. Expression of either the glycine:arginine (GR n ) or proline:arginine (PR n ) poly‐dipeptide in Drosophila leads to neurodegneration of the eye, and when introduced into culturing medium, synthetic forms of both peptides lead to the death of human cells. We recently identified PR n poly‐dipeptide interacting partners in vivo using an unbiased proteomics approach, demonstrating that the nuclear pore complex (NPC) as a major binding target. The biological significance of the PR n peptide‐NPC interaction became apparent when we observed major defects in both mature mRNA export to the cytoplasm and protein import into the nucleus after cells were treated with PR n . The functional nucleocytoplasmic transport defect caused by PR n peptide was due to binding of the peptide to the central channel of the NPC as visualized by super‐resolution microscopy. The NPC central channel is comprised of phenylalanine‐glycine (FG) domain nucleoporins, and these proteins are essential for the NPC's active transport and passive size‐exclusion permeability barrier functions. Purified FG domains of Nup54 and Nup98, both identified in our proteomics study as PR n targets, polymerized into structurally labile, cross‐β sheet fibers under physiological conditions . Several lines of evidence suggested that the polymerized state of FG domains is relevant to intact NPC. First, PR n peptides only bound to polymerized Nup54 and Nup98 and not to the soluble versions of either protein, potentially representing how PR n binds to the FG‐rich central channel. Second, the aliphatic alcohol 1,6‐hexanediol (HD) selectively disrupted the permeability barrier of the NPC, while 2,5‐hexanediol had no effect. Similarly, 1,6‐HD can effectively solubilize Nup FG polymers in vitro, while 2,5‐HD had no effect on polymer stability. Finally, PR n binding to the NPC abrogated the disruption of the permeability barrier by 1,6‐HD. Similarly, PR n also protected the Nup FG polymers from solubilization by 1,6‐HD . Our study elucidated a major mechanism by which the C9orf72 repeat expansion associated PR n poly‐dipeptide inhibit the transport of macromolecules in and out of the nucleus, leading to a major disruption of cellular physiology. Our results support a model in which the FG domains of the NPC exist in equilibrium between the polymerized and unpolymerized states. By binding to polymerized FG domains and stabilizing them, the PR n peptide shifts the equilibrium toward the polymerized state, with consequent blockage of nuclear transport. Aliphatic alcohol 1,6‐hexanediol has the opposite effect, shifting the equilibrium toward the unstructured state of FG domains and disrupting the permeability barrier. The PR n peptide and aliphatic alcohols represent extreme conditions, but subtler changes in the equilibrium between structured and unstructured FG domains may regulate nuclear pore function in living cells.