Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics

Abstract

Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

Keywords: FUS; LLPS; amyotrophic lateral sclerosis; frontotemporal lobar degeneration; hnRNP; intrinsically disordered protein; low complexity domain; phase transition; prion-like domain; protein aggregation.

Graphical abstract

Figure 1

Arginine-Rich Motifs Are Enriched in Cellular Liquid-like Compartments and Can Phase Separate In Vitro (A) Proteins in ribonucleoprotein complexes (Castello et al., 2012, Topisirovic et al., 2009), liquid compartments (Andersen et al., 2005, Jain et al., 2016, Liu et al., 2010), or proteins with prion-like domains (Kato et al., 2012, March et al., 2016) are enriched for both tri-RGG/di-RGG (Thandapani et al., 2013) and R motifs (Mitrea et al., 2016). Binomial test. (B) Balloon plot indicating sequence redundancy of RGG boxes. (C) Synthetic RGG boxes and arginine-rich C9orf72 DPRs phase separate following the addition of a molecular crowder. Measured at room temperature (RT), mean ± SD is depicted. The extent of phase separation is correlated with arginine content (Spearman). (D) Examples of RGG and DPR phase separation. Pictures taken at 4°C. Scale bar represents 10 μm. See also Figure S1.

Figure 2

PR₂₀ Peptide Undergoes Liquid-Liquid Phase Separation (A) A solution of 1 mM PR₂₀ with 20% PEG is clear at room temperature, but phase separates after cooling. (B) Demixed PR₂₀ droplets dissolve following a temperature increase. (C) PR₂₀ droplets are highly circular suggesting surface tension minimization (250 μM PR₂₀, 30% PEG, RT). (D) Large PR droplets deform when shear stress is applied (arrows), but take up a circular shape after stress relief as indicated by an aspect ratio approximating one (250 μM PR₂₀, 30% PEG, RT). (E) Volume is conserved following the fusion of two PR₂₀ droplets (250 μM PR₂₀, 30% PEG, RT). Scale bars represent 10 μm.

Figure 3

Molecular Determinants of PR₂₀ Phase Separation (A) PR₂₀ LLPS is dependent on molecular crowding. (B) PR LLPS is length dependent. (C) Addition of 1,6-hexanediol does not affect PR₂₀ LLPS. (D) Addition of NaCl inhibits PR₂₀ LLPS. (E and E’) PR-PEG LLPS is dependent on inorganic counteranions and correlated to anionic charge. (F and F’) RNA dose dependently induces PR₂₀ LLPS in the absence of molecular crowder. (G and G’) Addition of PR₂₀ clusters poly-tyrosine polymers in the absence of molecular crowder. Depict schemes of molecular interactions and phase contrast images of solutions (E’, F’, and G’). Mean ± SD, n = 3 (A–G). All measurements at RT. Scale bars represent 20 μm. See also Figures S1 and S2.

Figure 4

PR-RNA Droplets Are Dynamic Liquid Compartments (A) Fluorescently tagged PR₂₀ and polyU RNA colocalize in phase separated droplets. Scale represents 10 μm. (B) Spontaneous fusion behavior of PR₂₀-polyU RNA droplets over time, as expressed by changes in hydrodynamic radius over time as measured by DLS. Successive additions of polyU RNA indicated by red arrows. Final RNA concentration (green). The radius of polyU is 22nm (green line). (C) Cryo-TEM of two PR₂₀-polyU droplets (green) wetting carbon support (brown). No obvious structures can be observed within the droplets. (D) Full droplet bleach of PR₂₀ fluorescence indicates exchange between droplet and solution. Scale represents 5 μm. (E) Within-droplet bleach on old versus young droplets indicates that PR₂₀-polyU droplets do not mature or age. Mean ± SD, n = 12 droplets. See also Figure S2.

Figure 5

PR₃₀ Initiates Phase Separation of RNA Granule Components and Metastable Proteins In Vitro (A) Addition of PR₃₀ to cleared cell lysate induces LLPS with the formation of an insoluble fraction. Background from pre-cleared lysate not treated with PR was effectively zero. Weak interactions can be stabilized by mild PFA crosslinking. (B) The PR₃₀ interactome (n = 874) is enriched for GO biological processes centered on RNA and protein metabolism. Benjamini-Hochberg. (C and D) The PR₃₀ interactome significantly overlaps with cellular liquid compartments (Andersen et al., 2005, Jain et al., 2016) (C) and metastable proteins (Kato et al., 2012, Olzscha et al., 2011) (D). Binomial test. (E and F) The PR₃₀ interactome is enriched for RNA binding domains (E) and arginine-rich motifs (Mitrea et al., 2016) (F). Binomial test. (G and H) The PR₃₀ interactome is enriched for disordered (G) and supersaturated proteins (Ciryam et al., 2013) (H). Boxplots, whiskers indicate range. Mann-Whitney. See also Figures S3–S5 and Table S1.

Figure 6

PR₁₀₀ Expression Alters Stress Granule Dynamics in Cells (A) Expression of PR₁₀₀ in HeLa cells induces cytoplasmic SGs assembly positive for PR. (B) PR₁₀₀ is more efficient at inducing SGs than PA₁₀₀. (C) Arsenite and puromycin increase number of SGs in PR₁₀₀ transfected cells, whereas cyclohexamide decreases them. (D) Non-phosphorylatable form (AA) of eIF2α prevents PR₁₀₀-induced SG assembly in MEFs. (E) G3BP1/2 knockout prevents PR₁₀₀ induced SG assembly in U2OS cells. (F) PR₁₀₀-induced SGs have a mildly reduced FRAP recovery for G3BP1 compared to arsenite-induced ones. Repeated-measures ANOVA. Mean ± SEM, n = 17 SGs. (G) SG enrichment of several RNA binding proteins is altered in PR₁₀₀-induced SGs compared to arsenite-induced ones. Boxplots, whiskers indicate range. Mann-Whitney. n > 60 SGs, from three independent experiments. Fischer’s exact test. n ≥ 100 cells, from three independent experiments (A–E). See also Figure S6.

Figure 7

PR₃₀ Affects Phase Transitions of PrLDs (A) FUS LC droplets spontaneously fuse over time into larger droplets, as characterized by an increase in droplet size. This spontaneous fusion behavior is prevented by addition of PR₃₀, but not GP₃₀. Boxplots, whiskers indicate range. Kruskal-Wallis. n = (318–3,349) droplets. 50 mM MES buffer, 200 μM NaCl. (B) The FUS LC domain (amino acids [aa] 1–163) poorly phase separates, but addition of PR₃₀ increases phase separation dose dependently. (C) Kinetic analysis of ThT fluorescence over a 12-hr period for different FUS LC-PR₃₀ combinations. Addition of PR increases ThT fluorescence. Of note, PR₃₀ by itself did not yield any increase in ThT fluorescence over time (data not shown). Mean ± SD (B and C). See also Figure S7.