FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD

Abstract

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

Keywords: ALS; C9orf72; D. melanogaster; FUS; RAN translation; RNA chaperone; neuroscience; repeat expansion disease.

Figure 1.. Screening for RNA-binding proteins (RBPs) that suppress G₄C₂ repeat-induced toxicity in C9-ALS/FTD flies.

(A) Light microscopic images of the eyes in flies expressing both (G₄C₂)_{42 or 89} and the indicated RBPs using the GMR-Gal4 driver. Coexpression of FUS, IGF2BP1, or hnRNPA2B suppressed eye degeneration in both (G₄C₂)₄₂ and (G₄C₂)₈₉ flies, indicated by ‘Suppression (strong).’ Coexpression of hnRNPR, SAFB2, SF3B3, hnRNPA1, or hnRNPL suppressed eye degeneration in either (G₄C₂)₄₂ or (G₄C₂)₈₉ flies, indicated by ‘Suppression (weak)’ (see also Figure 1—source data 2). Scale bar: 100 μm. (B) Quantification of eye size in (G₄C₂)₈₉ flies coexpressing the indicated RBPs (n = 5). (C, D) Quantification of eye pigmentation in (G₄C₂)₈₉ flies (C) or (G₄C₂)₄₂ flies (D) coexpressing the indicated RBPs (n = 5). (E) Expression levels of (G₄C₂)₈₉ RNA in flies expressing both (G₄C₂)₈₉ and the indicated RBPs using the GMR-Gal4 driver (five independent experiments, n = 25 flies per genotype). The (G₄C₂)₈₉(H) fly line expresses (G₄C₂)₈₉ RNA at a high level (see also Figure 1—figure supplement 1). In (B–E), data are presented as the mean ± SEM; p<0.0001, as assessed by one-way ANOVA; n.s., not significant, ^*p<0.05, ^**p<0.01, and ^***p<0.001, as assessed by Tukey’s post hoc analysis. The detailed statistical information is summarized in Figure 1—source data 3.

Figure 1—figure supplement 1.. Characterization of C9-ALS/FTD flies.

(A) (G₄C₂)_n constructs used in this study. These constructs do not include an ATG start codon downstream of the UAS sequence, and were expressed in a tissue-specific manner using the GAL4-UAS system. (B) Light microscopic images of the eyes in flies expressing (G₄C₂)_n using the GMR-Gal4 driver. Scale bar: 100 μm. (C) Expression levels of (G₄C₂)_n RNAs in flies expressing (G₄C₂)_n using the GMR-Gal4 driver. Strong eye degeneration with decreased eye size and loss of pigmentation was observed in (G₄C₂)_{42 or 89} flies, but not in (G₄C₂)₉ flies. Eye degeneration was confirmed in (G₄C₂)₄₂ and two (G₄C₂)₈₉ independent fly lines. Degree of eye degeneration in two (G₄C₂)₈₉ fly lines was expression-level dependent [(L) vs. (H) in (G₄C₂)₈₉] (three independent experiments, n = 15 flies per each genotype). Expression of G₄C₂ repeat RNA of the sense transcripts but not that of the antisense transcripts was confirmed. (D) Climbing ability at 1 d of age in flies expressing (G₄C₂)_n using the elav-Gal4 driver. Flies expressing (G₄C₂)₈₉(H) in neurons showed lethality. Decreasing climbing ability was observed in (G₄C₂)_{42 or 89} flies compared with (G₄C₂)₉ flies (five independent experiments, n = 100 flies per each genotype). (E) Fluorescence in situ hybridization (FISH) analyses of G₄C₂ repeat RNA in the salivary glands of fly larvae with two copies of GMR-Gal4 and (G₄C₂)_{9 or 89} (red: G₄C₂ RNA; yellow: G₂C₄ RNA; blue [DAPI]: nuclei). RNA foci formation (arrowheads) of the sense transcripts but not that of the antisense transcripts was confirmed. RNA foci were observed in (G₄C₂)₈₉ flies, but not in (G₄C₂)₉ flies. Scale bars: 100 μm (low magnification) or 20 μm (high magnification). (F) Immunohistochemical analyses of dipeptide repeat proteins (DPRs) stained with anti-DPR antibodies in the eye imaginal discs of fly larvae with two copies of GMR-Gal4 and (G₄C₂)_{9 or 89} (magenta: poly(GR); orange: poly(GA); green: poly(GP); blue [DAPI]: nuclei). Expression and cytoplasmic aggregation (arrowheads) of three DPRs in (G₄C₂)₈₉ flies, but not in (G₄C₂)₉ flies, were confirmed. Scale bars: 50 μm (low magnification), 10 μm (middle magnification), and 5 μm (high magnification). In (B–F), L: low-expression line; H: high-expression line. In (C, D), data are presented as the mean ± SEM; p<0.0001, as assessed by one-way ANOVA; n.s., not significant, and ^***p<0.001, as assessed by Tukey’s post hoc analysis. The detailed statistical information is summarized in Figure 1—figure supplement 1—source data 2.

Figure 1—figure supplement 2.. Coexpression of FUS suppresses G₄C₂ repeat-induced toxicity in flies expressing (G₄C₂)₈₉.

(A) Light microscopic images of the eyes in flies expressing both (G₄C₂)_{42 or 89} and FUS using the GMR-Gal4 driver. FUS-2 and FUS-3 are different strains from that in Figure 1. Scale bar: 100 μm. (B) Quantification of the eye size in (G₄C₂)₈₉ flies of the indicated genotypes (n = 10). (C, D) Quantification of eye pigmentation in (G₄C₂)₈₉ flies (C) or (G₄C₂)₄₂ flies (D) of the indicated genotypes (n = 10). In (B–D), data are presented as the mean ± SEM; p<0.0001, as assessed by one-way ANOVA; ^***p<0.001, as assessed by Tukey’s post hoc analysis. The detailed statistical information is summarized in Figure 1—figure supplement 2—source data 1.

Figure 2.. FUS suppresses G₄C₂ repeat-induced toxicity via its RNA-binding activity.

(A) Light microscopic images of the eyes in flies expressing both (G₄C₂)₈₉ and either FUS or FUS-RRMmut using the GMR-Gal4 driver. Scale bar: 100 μm. (B) Quantification of eye size in the flies of the indicated genotypes (n = 10). (C) Quantification of eye pigmentation in the flies of the indicated genotypes (n = 10). (D) Egg-to-adult viability in flies expressing both (G₄C₂)₄₂ and either FUS or FUS-RRMmut using the GMR-Gal4 driver (>500 flies per genotype). (E) Climbing ability in flies expressing both (G₄C₂)₄₂ and FUS using the elav-GeneSwitch driver (five independent experiments, n = 100 flies per each genotype). In (B–E), data are presented as the mean ± SEM. In (B, C), p<0.0001, as assessed by one-way ANOVA; n.s., not significant, and ^***p<0.001, as assessed by Tukey’s post hoc analysis. In (D), n.s., not significant and ^***p<0.001, as assessed by Tukey’s multiple-comparison test using wholly significant difference. In (E), n.s., not significant, ^*p<0.05, ^**p<0.01, and ^***p<0.001, as assessed by two-way repeated-measures ANOVA with Tukey’s post hoc analysis. The detailed statistical information is summarized in Figure 2—source data 1.

Figure 2—figure supplement 1.. Western blot analysis showing expression levels of FUS and FUS-RRMmut proteins.

(A) Western blot analysis of the FUS and FUS-RRMmut proteins in the heads of adult flies expressing EGFP, FUS, or FUS-RRMmut using the GMR-Gal4 driver, with an anti-FUS antibody. The arrowhead indicates bands from the FUS and FUS-RRMmut proteins, whereas the asterisk indicates bands resulting from nonspecific antibody binding. (B) Quantification of the FUS and FUS-RRMmut proteins from the western blot analysis in (A) (n = 3). In (B), data are presented as the mean ± SEM; n.s., not significant, as assessed by the unpaired t-test. The detailed statistical information is summarized in Figure 2—figure supplement 1—source data 1.

Figure 3.. FUS suppresses RNA foci formation and RAN translation from G₄C₂ repeat RNA.

(A) Fluorescence in situ hybridization (FISH) analyses of G₄C₂ repeat RNA in the salivary glands of fly larvae expressing both (G₄C₂)₈₉ and either FUS or FUS-RRMmut using two copies of the GMR-Gal4 driver (red: G₄C₂ RNA; blue [DAPI]: nuclei). Arrowheads indicate RNA foci. Scale bar: 20 μm. (B) Quantification of the number of nuclei containing RNA foci from the FISH analyses in (A) (n = 10). (C) Expression levels of (G₄C₂)₈₉ RNA in fly larvae expressing both (G₄C₂)₈₉ and either FUS or FUS-RRMmut using the GMR-Gal4 driver (10 independent experiments, n = 50 flies per each genotype). (D) Immunohistochemical analyses of dipeptide repeat proteins (DPRs) stained with anti-DPR antibodies in the eye imaginal discs of fly larvae expressing both (G₄C₂)₈₉ and either FUS or FUS-RRMmut using two copies of the GMR-Gal4 driver (magenta: poly(GR); orange: poly(GA); green: poly(GP)). Arrowheads indicate cytoplasmic aggregates. Scale bars: 20 μm (low magnification) or 5 μm (high magnification). (E) Quantification of the number of DPR aggregates from the immunohistochemical analyses in (D) (n = 14 or 15 [GR], or 10 [GA or GP]). (F) Immunoassay to determine poly(GP) levels in flies expressing both (G₄C₂)₈₉ and either FUS or FUS-RRMmut using the GMR-Gal4 driver (three independent experiments, n = 30 flies per each genotype). (G) Western blot analysis of the heads of adult flies expressing both LDS-(G₄C₂)₄₄^GR-GFP and any of DsRed, FUS or FUS-RRMmut using the GMR-Gal4 driver, using either an anti-GFP (upper panel) or anti-GR antibody (middle panel). (H, I) Quantification of GR-GFP protein levels from the western blot analysis in (G) (nine independent experiments, n = 90 flies per each genotype). In (B, C, E, F, H, I), data are presented as the mean ± SEM. In (B, E, F), p<0.0001, as assessed by one-way ANOVA; n.s., not significant, ^*p<0.05, ^**p<0.01, and ^***p<0.001, as assessed by Tukey’s post hoc analysis. In (C), p=0.452, as assessed by one-way ANOVA; n.s., not significant, as assessed by Tukey’s post hoc analysis. In (H), p=0.0148, as assessed by one-way ANOVA; n.s., not significant and ^*p<0.05, as assessed by Tukey’s post hoc analysis. In (I), p=0.0072, as assessed by one-way ANOVA; n.s., not significant and ^*p<0.05, as assessed by Tukey’s post hoc analysis. The detailed statistical information is summarized in Figure 3—source data 1.

Figure 3—figure supplement 1.. Schema of the LDS-(G₄C₂)₄₄^GR-GFP construct.

Schema of the LDS-(G₄C₂)₄₄^GR-GFP construct containing the (G₄C₂)₄₄ sequence and 114 nucleotides of the 5′-flanking region of intron 1 of the human C9orf72 G₄C₂ repeat sequence. A GFP tag in the GR frame was introduced downstream of the (G₄C₂)₄₄ repeat sequence.

Figure 3—figure supplement 2.. Overexpression of FUS does not suppress eye degeneration in dipeptide repeat protein (DPR)-only flies expressing DPRs translated from non-G₄C₂ RNAs.

Light microscopic images of the eyes in DPR-only flies coexpressing either the poly(GR) or poly(GA) protein, and either FUS or FUS-RRMmut using the GMR-Gal4 driver. Overexpression of FUS did not suppress the eye degeneration in flies expressing either (GR)₃₆ or (GR)₁₀₀. Overexpression of FUS also caused mild eye degeneration in flies expressing EGFP, (GA)₃₆, or (GA)₁₀₀, likely due to FUS toxicity.

Figure 4.. Reduction of endogenous caz expression enhances G₄C₂ repeat-induced toxicity, RNA foci formation, and dipeptide repeat protein (DPR) aggregation.

(A) Light microscopic images of the eyes in flies expressing (G₄C₂)₈₉ using the GMR-Gal4 driver, with knockdown of caz. Scale bar: 100 μm. (B) Quantification of eye size in flies of the indicated genotypes shown in (A) (n = 10). (C) Light microscopic images of the eyes in flies expressing (G₄C₂)₈₉ using the GMR-Gal4 driver, with a hemizygous deletion of caz. Scale bar: 100 μm. (D) Quantification of eye size in the flies of the indicated genotypes shown in (C) (n = 10). (E) Fluorescence in situ hybridization (FISH) analyses of G₄C₂ repeat RNA in the salivary glands of fly larvae expressing (G₄C₂)₈₉ using the GMR-Gal4 driver, with knockdown of caz (red: G₄C₂ RNA; blue [DAPI]: nuclei). Arrowheads indicate RNA foci. Scale bar: 20 μm. (F) Quantification of the number of nuclei containing RNA foci from the FISH analyses in (E) (n = 10). (G) Expression levels of (G₄C₂)₈₉ RNA in fly larvae expressing (G₄C₂)₈₉ using the GMR-Gal4 driver, with knockdown of caz (four independent experiments, n = 20 flies per each genotype). (H) Immunohistochemical analyses of DPRs stained with anti-DPR antibodies in the eye imaginal discs of fly larvae expressing (G₄C₂)₈₉ using two copies of the GMR-Gal4 driver, with the knockdown of caz. (magenta: poly(GR); orange: poly(GA); green: poly(GP)). Arrowheads indicate cytoplasmic aggregates. Scale bars: 20 μm (low magnification) or 5 μm (high magnification). (I) Quantification of the number of DPR aggregates from the immunohistochemical analyses in (H) (n = 10). In (B, D, F, G, I), data are presented as the mean ± SEM. In (B, D), p<0.0001, as assessed by one-way ANOVA; ^***p<0.001, as assessed by Tukey’s post hoc analysis. In (F, G, I), n.s., not significant, ^*p<0.05, ^**p< 0.01, and ^***p<0.001, as assessed by the unpaired t-test. The detailed statistical information is summarized in Figure 4—source data 1.

Figure 4—figure supplement 1.. Endogenous caz is a functional homologue of FUS for the suppression of G₄C₂ repeat-induced toxicity.

(A) Light microscopic images of the eyes in flies expressing both (G₄C₂)_{42 or 89} and either caz (FLAG-caz) or FUS-4 (FLAG-FUS) using the GMR-Gal4 driver. FUS-4 is a different strain from those used in Figure 1 and Figure 1—figure supplement 2. Scale bar: 100 μm. (B) Quantification of the eye size in (G₄C₂)₈₉ flies of the indicated genotypes (n = 10). (C, D) Quantification of eye pigmentation in (G₄C₂)₈₉ flies (C) or (G₄C₂)₄₂ flies (D) of the indicated genotypes (n = 10). In (B–D), data are presented as the mean ± SEM; p<0.0001, as assessed by one-way ANOVA; n.s., not significant, ^*p<0.05, ^**p<0.01, and ^***p<0.001, as assessed by Tukey’s post hoc analysis. The detailed statistical information is summarized in Figure 4—figure supplement 1—source data 1.

Figure 5.. FUS directly binds to and modulates the G-quadruplex structure of G₄C₂ repeat RNA, resulting in the suppression of RAN translation in vitro.

(A) Analysis of the binding of His-tagged FUS proteins to biotinylated (G₄C₂)₄ RNA by the filter binding assay. The nitrocellulose membrane (left) traps RNA-bound FUS proteins, whereas unbound RNAs are recovered on the nylon membrane (right), and then the RNAs trapped on each of the membranes was probed with streptavidin-horseradish peroxidase (HRP). Biotinylated (AAAAAA)₄ and (UUAGGG)₄ were used as negative and positive controls, respectively. (B–D) CD spectra of (G₄C₂)₄ RNA incubated with or without FUS in the presence of 150 mM KCl (B), NaCl (C), or LiCl (D). The CD spectrum of FUS alone was subtracted from that of (G₄C₂)₄ RNA incubated with FUS. The original data are shown in Figure 5—figure supplement 2B–2D. (E) Schema of the template RNA containing the (G₄C₂)₈₀ sequence and 113 nucleotides of the 5′-flanking region of intron 1 of the human C9orf72 G₄C₂ repeat sequence. A Myc tag in the GA frame was introduced downstream of the (G₄C₂)₈₀ repeat sequence. (F) Western blot analysis of samples from in vitro translation using rabbit reticulocyte lysate in the presence or absence of increasing concentrations of FUS or FUS-RRMmut. The GA-Myc fusion protein was detected by western blotting using the anti-Myc antibody. (G) Quantification of the GA-Myc fusion protein in (F) (n = 3). In (G), data are presented as the mean ± SEM; ^*p<0.05, ^**p<0.01, and ^***p<0.001, as assessed by the unpaired t-test. The detailed statistical information is summarized in Figure 5—source data 1.

Figure 5—figure supplement 1.. FUS colocalizes with G₄C₂ RNA foci.

Combined fluorescence in situ hybridization (FISH) and immunohistochemical analyses of G₄C₂ repeat RNA and FUS in the salivary glands of flies expressing both (G₄C₂)₈₉ and FUS using two copies of the GMR-Gal4 driver. Arrowheads indicate colocalization of FUS with RNA foci. Scale bar: 10 μm (low magnification) and 5 μm (high magnification).

Figure 5—figure supplement 2.. FUS modulates the G-quadruplex structure of G₄C₂ repeat RNA.

(A–C) CD spectra of (G₄C₂)₄ RNA incubated with or without FUS in the presence of 150 mM KCl (A), NaCl (B), or LiCl (C). CD spectra of (G₄C₂)₄ RNA alone (black), FUS alone (green), sum of (G₄C₂)₄ RNA and FUS (blue), and the spectra of their coincubation (magenta) are shown. FUS interacts with (G₄C₂)₄ RNA under KCl or NaCl buffer conditions. (D) Imino proton NMR spectra of (G₄C₂)₄ RNA incubated with increasing amounts of FUS in the presence of 150 mM KCl.

Figure 6.. Identification of G-quadruplex-targeting RNA-binding proteins (RBPs) that suppress G₄C₂ repeat-induced toxicity in C9-ALS/FTD flies.

(A) Light microscopic images of eyes in flies expressing both (G₄C₂)₈₉ and the indicated G-quadruplex-targeting RBPs using the GMR-Gal4 driver. Scale bar: 100 μm. (B) Quantification of eye size in the flies of the indicated genotypes (n = 10). (C) Quantification of eye pigmentation in the flies of the indicated genotypes (n = 10). (D) Expression levels of (G₄C₂)₈₉ RNA in flies expressing both (G₄C₂)₈₉ and the indicated G-quadruplex-targeting RBPs using the GMR-Gal4 driver (five independent experiments, n = 25 flies per each genotype). (E) Immunohistochemical analyses of poly(GA) stained with anti-GA antibody in the eye imaginal discs of fly larvae expressing both (G₄C₂)₈₉ and the indicated G-quadruplex-targeting RBPs using two copies of the GMR-Gal4 driver (orange: poly(GA)). Arrowheads indicate cytoplasmic aggregates. Scale bars: 20 μm (low magnification) or 5 μm (high magnification). (F) Quantification of the number of poly(GA) aggregates from the immunohistochemical analyses in (E) (n = 10). In (B, C, D, F), data are presented as the mean ± SEM; p<0.0001, as assessed by one-way ANOVA; n.s., not significant, ^*p<0.05 and ^***p<0.001, as assessed by Tukey’s post hoc analysis. The detailed statistical information is summarized in Figure 6—source data 2.