C9orf72 arginine-rich dipeptide repeat proteins disrupt importin β-mediated nuclear import

Disruption of nucleocytoplasmic transport (NCT), including mislocalization of the importin β cargo, TDP-43, is a hallmark of amyotrophic lateral sclerosis (ALS), including ALS caused by a hexanucleotide repeat expansion in C9orf72. However, the mechanism(s) remain unclear. Importin β and its cargo adaptors have been shown to co-precipitate with the C9orf72-arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to NCT protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with nuclear transport receptors in the vicinity of the nuclear envelope.


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A GGGGCC hexanucleotide repeat expansion (HRE) in C9orf72 is the most common 29 known cause of amyotrophic lateral sclerosis (ALS) and is also a major cause of frontotemporal

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Using this method, we performed live imaging of nuclear import of Rango, a direct 125 importin β cargo whose Ran-, importin β-, and energy-dependent nuclear translocation is 126 conferred by the IBB domain (Kalab et al., 2006). We verified that Rango import in

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Consistent with the expected lower efficiency of tripartite nuclear import complex assembly, R-

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The mechanisms of cargo recognition for importin b differ significantly even from its 145 structurally closest relative TNPO1 (KPNB2), whose cargos are marked by the PY-NLS motif 146 . However, since the sequence of the PY-NLS also contains basic residues, we 147 tested the effect of R-DPRs on the nuclear import of YFP-M9-CFP (hereafter referred to as M9), 148 a TNPO1 substrate based on the prototypic PY-NLS sequence of hnRNPA1 (Siomi and  To further validate the direct interaction between R-DPRs and importin b, we performed 156 the bead halo assay. This equilibrium-based binding assay is capable of identifying both low-157 and high-affinity interactions between 'bait' proteins immobilized to beads, and fluorescent 'prey' 158 in the surrounding buffer, which forms a fluorescent halo on the bead surface (Patel et al., 2007; 159 Patel and Rexach, 2008). First, we examined the propensity for all five DPRs to interact with 160 biotinylated importin β, immobilized on the surface of neutravidin beads (Figure 2A). Controls 161 included bare beads and beads coated with biotinylated BSA. As a positive control, we 162 observed that the Rango sensor exclusively bound to full-length importin β-coated beads, and 163 not the control beads. Fluorescent dextran, the negative control, did not form a halo in any 164 conditions. AF488-labeled PR10 and GR10 (200 nM) both showed a modest degree of non-165 specific binding to all controls which was equivalent to the binding seen to bare beads.

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However, there was an approximately two-fold more intense halo around importin β-coated  To test if the disruption of nuclear import resulted from changes in the passive exclusion 177 limit of NPCs, we tested the effects of R-DPRs on the passive influx of small cargoes. Passive 178 diffusion of GFP and small fluorescent dextrans into nuclei of permeabilized HeLa cells was 9 imaged at 10-second intervals for 5 minutes, and nuclear fluorescence quantified over time. All 180 experiments were done in the context of energy and cell lysate, identical to the active transport 181 conditions, so as not to miss putative effects that may depend on simultaneous active transport 182 (i.e., recruitment of importins and DPRs to the NPC). Under these conditions, we observed the

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The rate of passive transport is thought to be governed by the FG-Nup barrier in

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Importantly, based on our passive transport studies, these interactions do not confer an 204 impedence to transport as previously suggested, but rather a modest increase in NPC   2). We compared supernatant versus pellet fractions for all five DPRs compared to control 225 lysates in which no DPRs were added, to assess the degree to which proteins were being 226 sequestered and depleted from the soluble fraction. We saw enrichment in the pellet for importin      RNAse-sensitive ( Figure 5A). However, this did not appear to be attributable to significant

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To further test if DPR-induced aggregate formation is relevant to the mechanism of 269 nuclear import blockade, we performed a series of assays in which the supernatant was 270 separated from the insoluble pellet prior to initiating transport (diagrammed in figure 5B). We 271 reasoned that if aggregates sequester key transport factors, the remaining supernatant would 272 be insufficient to drive nuclear import. However, if the aggregates contain inhibitor(s) of nuclear 273 import or are themselves inhibitory, depleting them could rescue transport impairment. The 274 results were markedly different for GR versus PR ( Figure 5C). For GR10, removing the 275 insoluble pellet restored nuclear import to normal, confirming that the inhibitory factor was 276 present in (or was) the aggregates. In contrast, nuclear import remained perturbed in the 277 supernatants of the PR10 aggregates, although it was restored by the addition of RNA.

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Next, we monitored the location of the R-DPRs with respect to the aggregates by adding 279 AF488-labeled DPRs to the transport reactions. By confocal microscopy, we observed that the 280 transport disruption correlated with the presence of DPRs in the vicinity of the nuclear envelope 281 ( Figure 5D). AF488-GR10 fully sedimented into the pellet, leaving no visible GR10 in the 282 13 supernatant, where transport proceeded normally. In contrast, a subset of AF488-PR10 283 remained in the supernatant and was present at the nuclear envelope, paralleling the persistent 284 inhibition of nuclear import by the PR10 supernatants. RNA dispersed AF488-R-DPRs from the 285 permeabilized cell nuclei in all conditions, restoring nuclear import. These results suggest that 286 the import inhibition depends on GR or PR acting directly, rather than through putative 287 intermediary factor(s), to inhibit nuclear import. The strikingly divergent segregation of GR10 vs.

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PR10 between the soluble and insoluble phase indicated that, while both share importin β as 289 their target, the mechanisms and locations of their intracellular actions could differ significantly.

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The critical steps of importin β-mediated nuclear import take place at NPCs via 291 interactions with FG-Nups. To test whether interaction between R-DPRs and the NPC is 292 sufficient to confer the block to import, we ran two parallel sets of import reactions (diagrammed 293 in figure 5E). In the "lysate preincubation" paradigm, as for previous active import assays, R-

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DPRs were added to lysates used to supply transport factors, preincubated for 1 hour, and then 295 added to permeabilized cells along with Rango and energy to initiate the transport reaction. In 296 the "nuclei preincubation" set, we first exposed the permeabilized cell nuclei to R-DPRs (in the 297 presence of lysate and energy, but no fluorescent cargo). After 1 hour, the DPR-lysate mix was 298 removed from the nuclei, and fresh transport lysate, energy, and cargo added to initiate 299 transport (without R-DPRs). We hypothesized that, if the DPRs inhibited Rango import by 300 associating with and perturbing the NPC, we should see reduced import rate in the "nuclei 301 preincubation" group. However, transport proceeded normally ( Figure 5F). These results 302 support a model in which the R-DPRs inhibit nuclear import by directly interfering with factor(s) 303 present in the soluble phase of the NCT machinery ( Figure 5G), which is consistent with the 304 biochemical evidence for importin β as one of their direct targets.

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Importin β is composed of 19 tandem HEAT repeats, coiled into a superhelix with 338 exposed N-terminal RanGTP-binding domain and C-terminal importin a-binding domain 339 (Cingolani et al., 1999). We predicted that R-DPRs might mimic the arginine-and lysine-rich IBB

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we tested the effect of adding total cellular RNA to the transport reaction, and observed dose-388 dependent rescue. Total protein aggregates were not strongly reduced by the RNA, however 389 significantly less AF488-labeled R-DPRs were observed in the vicinity of the nuclear envelope.

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Our electrophoretic mobility shift assay shows that a broad range of cellular RNAs can bind to 391 R-DPRs directly, and previous evidence in a purified system showed that synthetic RNAs can

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The authors declare no competing interests.