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W4384698769 abstract "Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Data availability References Peer review Author response Article and author information Metrics 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. eLife assessment This important study demonstrates that the human FUS protein, which is implicated in ALS and related conditions, interacts with RNAs containing GGGGCC repeats and can regulate their translation by altering three-dimensional structures caused by these repeats. The study is carefully executed and the data provide convincing evidence for its major claims. This work will likely be of interest to researchers studying RNA binding proteins, and to those working on ALS and related diseases. https://doi.org/10.7554/eLife.84338.3.sa0 About eLife assessments Introduction Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are incurable neurodegenerative diseases with overlapping genetic and neuropathological features. Abnormal expansions of the GGGGCC (G4C2) repeat sequence in the noncoding region of the C9orf72 gene have been found to be the most common genetic mutation responsible for ALS/FTD (DeJesus-Hernandez et al., 2011; Gijselinck et al., 2012; Renton et al., 2011). Three major pathomechanisms are thought to be involved in the pathogenesis of C9orf72-linked ALS/FTD (C9-ALS/FTD): first, expansion of the G4C2 repeats results in decreased expression of the C9orf72 gene, leading to its haploinsufficiency (Boivin et al., 2020; DeJesus-Hernandez et al., 2011; Gijselinck et al., 2012; Shi et al., 2018; Waite et al., 2014; Zhu et al., 2020). Second, the transcribed G4C2 repeat-containing RNA accumulates as RNA foci in the affected tissues, sequestering various RNA-binding proteins (RBPs) and altering their function (Conlon et al., 2016; Cooper-Knock et al., 2014; Donnelly et al., 2013; Haeusler et al., 2014; Lee et al., 2013; Mori et al., 2013a). Third, this G4C2 repeat RNA is also translated into dipeptide repeat (DPR) proteins, despite the lack of an AUG initiation codon, by noncanonical repeat-associated non-AUG (RAN) translation (Ash et al., 2013; Gendron et al., 2013; Mori et al., 2013b; Mori et al., 2013c; Zu et al., 2011; Zu et al., 2013). Since RAN translation occurs in all reading frames and the expanded G4C2 repeat sequence is bidirectionally transcribed, five distinct DPRs, that is, poly(glycine-arginine) [poly(GR)], poly(glycine-alanine) [poly(GA)], poly(glycine-proline) [poly(GP)], poly(proline-arginine) [poly(PR)], and poly(proline-alanine) [poly(PA)], are produced and observed in patients’ brains (Ash et al., 2013; Gendron et al., 2013; Mori et al., 2013b; Mori et al., 2013c; Zu et al., 2013) and cerebrospinal fluid (Gendron et al., 2017; Krishnan et al., 2022; Lehmer et al., 2017; Su et al., 2014). DPRs have been shown to exert toxic effects in multiple C9-ALS/FTD models, such as cultured cells, flies, and mice (Choi et al., 2019; Jovičić et al., 2015; May et al., 2014; Mizielinska et al., 2014; Rudich et al., 2017; Wen et al., 2014; Zhang et al., 2016; Zhang et al., 2018). Importantly, the toxicity of DPRs was confirmed in DPR-only flies, which express DPRs translated from non-G4C2 repeat RNAs with alternative codons and show neurodegeneration, whereas RNA-only flies expressing G4C2 repeat RNAs with stop codon interruptions, which eliminate DPRs production, did not show any obvious degenerative phenotypes (Mizielinska et al., 2014). In addition, increased DPR production, but not RNA foci, was reported to correlate with G4C2 repeat-induced toxicity in a C9-ALS/FTD Drosophila model (Tran et al., 2015). Taken together, these studies have strongly suggested that DPRs play a central role in the pathogenesis of C9-ALS/FTD. Indeed, DPRs have been reported to disrupt various biological pathways, such as nucleocytoplasmic transport (Hutten et al., 2020; Jovičić et al., 2015; Zhang et al., 2016) and membraneless organelle dynamics (Kwon et al., 2014; Lee et al., 2016; Lin et al., 2016). Therefore, elucidating the regulatory mechanism of RAN translation is a significant challenge toward developing potential therapies for C9-ALS/FTD. Since the discovery of RAN translation in 2011 (Zu et al., 2011), many studies to date have focused on its molecular mechanisms, that is, whether it has functional overlap with canonical AUG-dependent translation. Previous studies on C9-ALS/FTD using monocistronic reporters containing a G4C2 repeat sequence revealed cap-dependent translation initiation from the upstream near-cognate CUG initiation codon, requiring the cap-binding eukaryotic translation factor 4F complex (Green et al., 2017; Tabet et al., 2018). On the other hand, studies using bicistronic reporters with a G4C2 repeat sequence in the second cistron also produced DPRs by RAN translation in all reading frames, suggesting cap-independent translation initiation within the G4C2 repeat sequence (Cheng et al., 2018; Sonobe et al., 2018). This is reminiscent of internal ribosomal entry site translation initiation, which is another type of noncanonical cap-independent translation in which specific factors are directly recruited to the highly structured mRNA for initiation (Kwan and Thompson, 2019). While such initiation mechanisms of RAN translation have been explored to date, specific roles of the repeat sequence on RAN translation remain enigmatic. Considering a repeat length dependency of RAN translation (Mori et al., 2013c; Zu et al., 2011; Zu et al., 2013), the repeat sequence itself would also be essential for the initiation or elongation steps of RAN translation. Based on our previous findings of the protective role of TDP-43 on UGGAA repeat-induced toxicity in spinocerebellar ataxia type 31 (SCA31) models (Ishiguro et al., 2017), we hypothesized that RBPs specifically binding to repeat sequences of template RNA play a role in RAN translation. Using Drosophila models of C9-ALS/FTD, we here demonstrate the regulatory roles of the ALS/FTD-linked RBP FUS on RAN translation from G4C2 repeat RNA, which lead to the significant modulation of neurodegeneration. We found that FUS suppresses RNA foci formation and DPR production, resulting in the suppression of repeat-induced degeneration. This suppressive effect on degeneration was abolished by mutations in the RNA-recognition motif (RRM) of FUS. In contrast, knockdown of endogenous caz, a Drosophila homologue of FUS, enhanced DPR aggregation and RNA foci formation, resulting in the enhancement of repeat-induced degeneration. Moreover, FUS was found to directly bind to G4C2 repeat RNA and modify its G-quadruplex structure as an RNA chaperone, resulting in the suppression of RAN translation in vitro. In addition, other G-quadruplex-targeting RBPs also suppressed RAN translation and G4C2 repeat-induced toxicity in our C9-ALS/FTD flies. These results strongly indicate that FUS regulates RAN translation and suppresses DPR toxicity through modulating the G-quadruplex structure of G4C2 repeat RNA. Our findings shed light on the regulatory mechanisms of RAN translation by G-quadruplex-targeting RBPs and propose novel therapeutic strategies for repeat expansion diseases by regulating RAN translation. Results Screening for RBPs that suppress G4C2 repeat-induced toxicity in C9-ALS/FTD flies We established Drosophila models of C9-ALS/FTD that express pathogenic length 42 or 89 G4C2 repeats [(G4C2)42, or (G4C2)89 flies, respectively] and confirmed that expanded G4C2 repeat sequences induce eye degeneration and motor dysfunction accompanied with the formation of RNA foci and the production of three types of DPRs (Figure 1—figure supplement 1), consistent with previous studies (Freibaum et al., 2015; Goodman et al., 2019; Mizielinska et al., 2014; Xu et al., 2013). We also established Drosophila expressing normal length 9 G4C2 repeats as a control [(G4C2)9 flies] and found that they did not show eye degeneration, motor dysfunction, RNA foci formation, or DPR aggregation (Figure 1—figure supplement 1). We selected 18 RBPs that have been reported to bind to G4C2 repeat RNA (Mori et al., 2013a), as well as TDP-43, an ALS/FTD-linked RBP that does not bind to G4C2 repeat RNA (Xu et al., 2013; Figure 1—source data 1), and examined their roles in neurodegeneration in our C9-ALS/FTD fly models. We found that coexpression of FUS, IGF2BP1, or hnRNPA2B1 strongly suppressed the eye degeneration in both flies expressing (G4C2)42 or 89, which show decreased eye size and loss of pigmentation (Figure 1A–D and Figure 1—source data 2). Coexpression of five RBPs, namely, hnRNPR, SAFB2, SF3B3, hnRNPA1, and hnRNPL, also partially suppressed the eye degeneration, whereas coexpression of the other six RBPs had no effect, and two RBPs enhanced the phenotypes (Figure 1A–D and Figure 1—source data 2). In addition, coexpression of TDP-43 had no effect on the eye degeneration in (G4C2)42 flies and resulted in lethality in (G4C2)89 flies, likely due to the toxicity of TDP-43 expression itself (Figure 1A and D and Figure 1—source data 2). The variation in the effects of these G4C2 repeat-binding RBPs on G4C2 repeat-induced toxicity may be due to their different binding affinities to G4C2 repeat RNA and the different toxicity of overexpressed RBPs themselves. We then analyzed the expression levels of G4C2 repeat RNA in flies coexpressing (G4C2)89 and three RBPs that strongly suppressed eye degeneration. We found that coexpression of IGF2BP1 or hnRNPA2B1 significantly decreased G4C2 repeat RNA levels, whereas they were not altered upon coexpression of FUS (Figure 1E). Although the suppressive effects of IGF2BP1 and hnRNPA2B1 could simply be explained by the decreased levels of G4C2 repeat RNA, the molecular mechanisms by which FUS suppresses G4C2 repeat-induced toxicity remain to be clarified. The suppressive effects of FUS on G4C2 repeat-induced toxicity were confirmed using multiple FUS fly lines, showing the significant suppression of decreased eye size and loss of pigmentation in (G4C2)42 or 89 flies coexpressing FUS (Figure 1—figure supplement 2). Therefore, we decided to further focus on FUS, which is another ALS/FTD-linked RBP, and investigated its mechanism of the suppression of G4C2 repeat-induced toxicity. Figure 1 with 2 supplements see all Download asset Open asset 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—source data 1 RNA-binding proteins and their cDNA accession numbers screened in the genetic analyses in Figure 1. https://cdn.elifesciences.org/articles/84338/elife-84338-fig1-data1-v1.xlsx Download elife-84338-fig1-data1-v1.xlsx Figure 1—source data 2 Summary of the genetic analyses in Figure 1. https://cdn.elifesciences.org/articles/84338/elife-84338-fig1-data2-v1.xlsx Download elife-84338-fig1-data2-v1.xlsx Figure 1—source data 3 Statistical data related to Figure 1B–E. https://cdn.elifesciences.org/articles/84338/elife-84338-fig1-data3-v1.xlsx Download elife-84338-fig1-data3-v1.xlsx FUS suppresses G4C2 repeat-induced toxicity via its RNA-binding activity We next investigated whether the suppressive effects of FUS on G4C2 repeat-induced toxicity are mediated by its binding to G4C2 repeat RNA, using flies expressing FUS with mutations in the RRM (FUS-RRMmut), which have been reported to eliminate its RNA-binding activity (Daigle et al., 2013). Western blot analysis confirmed that the FUS-RRMmut fly line expresses almost an equivalent level of the FUS proteins to the FUS fly line (Figure 2—figure supplement 1). We found that coexpression of FUS-RRMmut did not restore the eye degeneration in flies expressing (G4C2)89, suggesting that the RNA-binding activity of FUS is essential for its suppressive effects on G4C2 repeat-induced toxicity (Figure 2A–C). We also evaluated the reduced egg-to-adult viability of (G4C2)42 flies and confirmed that this phenotype was rescued by coexpression of FUS, but not by coexpression of FUS-RRMmut (Figure 2D). Expression of G4C2 repeat RNA in the nervous system of flies after eclosion using the elav-GeneSwitch driver induces motor dysfunction, and coexpression of FUS significantly alleviated this motor dysfunction (Figure 2E), indicating that FUS suppresses the neuronal phenotypes of flies expressing G4C2 repeat RNA. It is notable that the motor dysfunction caused by the expression of FUS alone was also alleviated by coexpression of (G4C2)42 (Figure 2E), indicating that the G4C2 repeat RNA conversely suppresses FUS toxicity. This result is consistent with our previous observations in SCA31 flies that UGGAA repeat RNA reduced the aggregation and toxicity of TDP-43 (Ishiguro et al., 2017). Moreover, recent studies demonstrated that RNA buffers the phase separation of TDP-43 and FUS, resulting in the suppression of their aggregation (Maharana et al., 2018; Mann et al., 2019). These findings hence suggest that balancing the crosstalk between repeat RNAs and RBPs neutralizes the toxicities of each other. Figure 2 with 1 supplement see all Download asset Open asset 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—source data 1 Statistical data related to Figure 2B–E. https://cdn.elifesciences.org/articles/84338/elife-84338-fig2-data1-v1.xlsx Download elife-84338-fig2-data1-v1.xlsx FUS suppresses RNA foci formation and RAN translation from G4C2 repeat RNA We next analyzed the effects of FUS expression on RNA foci and DPR production in flies expressing G4C2 repeat RNA. We performed RNA fluorescence in situ hybridization (FISH) of the salivary glands of fly larvae expressing (G4C2)89 and found that coexpression of FUS significantly decreased the number of nuclei containing RNA foci in (G4C2)89 flies, whereas it was not altered by coexpression of FUS-RRMmut (Figure 3A and B). We confirmed that the expression levels of G4C2 repeat RNA in (G4C2)89 flies were not altered by coexpression of FUS or FUS-RRMmut (Figure 3C). These results were in good agreement with our previous study on SCA31 showing the suppressive effects of FUS and other RBPs on RNA foci formation of UGGAA repeat RNA through altering RNA structures and preventing aggregation of misfolded repeat RNA as RNA chaperones (Ishiguro et al., 2017), raising the possibility that FUS has RNA-chaperoning activity also for G4C2 repeat RNA. Immunohistochemistry of the eye imaginal discs of fly larvae expressing (G4C2)89 revealed that coexpression of FUS significantly decreased the number of DPR aggregates in (G4C2)89 flies, whereas coexpression of FUS-RRMmut did not (Figure 3D and E). Quantitative analyses of poly(GP) by immunoassay also demonstrated that poly(GP) levels were greatly decreased in (G4C2)89 flies upon coexpression of FUS, but not FUS-RRMmut (Figure 3F), indicating that FUS suppresses RAN translation from the G4C2 repeat RNA to reduce DPR production. Considering that the 5′ upstream sequence of the G4C2 repeat in the C9orf72 gene is reported to affect RAN translation activity (Green et al., 2017; Tabet et al., 2018), we used flies expressing the G4C2 repeat sequence with the upstream intronic sequence of the C9orf72 gene, namely, LDS-(G4C2)44GR-GFP (Goodman et al., 2019). Since this construct has a 3′-green fluorescent protein (GFP) tag in the GR reading frame downstream of the G4C2 repeat sequence, the GR-GFP fusion protein is produced by RAN translation (Figure 3—figure supplement 1). We confirmed that coexpression of FUS significantly decreased the expression level of GR-GFP, whereas coexpression of FUS-RRMmut had no effect (Figure 3G–I). Figure 3 with 2 supplements see all Download asset Open asset 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)44GR-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—source data 1 Statistical data related to Figure 3B, C, E, F, H and I. https://cdn.elifesciences.org/articles/84338/elife-84338-fig3-data1-v1.xlsx Download elife-84338-fig3-data1-v1.xlsx Figure 3—source data 2 Source data related to Figure 3G. https://cdn.elifesciences.org/articles/84338/elife-84338-fig3-data2-v1.zip Download elife-84338-fig3-data2-v1.zip We further excluded the possibility that FUS directly interacts with DPRs, rather than with G4C2 repeat RNA, to decrease DPR levels and exert its suppressive effects. Using DPR-only flies expressing DPRs translated from non-G4C2 RNAs with alternative codons (Mizielinska et al., 2014), we confirmed that FUS did not suppress the eye degeneration in DPR-only flies expressing poly(GR), but rather enhanced their phenotypes, likely due to the additive effects of FUS toxicity (Figure 3—figure supplement 2). Together with the finding that FUS decreases not only DPR expression but also RNA foci formation (Figure 3A and B), these results collectively indicate that FUS indeed interacts with G4C2 repeat RNA and regulates RAN translation from G4C2 repeat RNA in Drosophila models of C9-ALS/FTD. Reduction of endogenous caz expression enhances G4C2 repeat-induced toxicity, RNA foci formation, and DPR aggregation To elucidate the physiological role of FUS on RAN translation, we also investigated the role of endogenous caz, a Drosophila homologue of FUS, on G4C2 repeat-induced toxicity in flies expressing G4C2 repeat RNAs. Coexpression of caz as well as FUS suppressed eye degeneration in flies expressing (G4C2)42 or 89 (Figure 4—figure supplement 1). These data suggest that caz is a functional homologue of FUS. In contrast, knockdown of caz by RNA interference or its hemizygous deletion modestly but significantly enhanced the eye degeneration in (G4C2)89 flies (Figure 4A–D), indicating that reduced caz expression enhances G4C2 repeat-induced toxicity. We next analyzed the effects of caz knockdown on RNA foci formation and DPR production in flies expressing (G4C2)89. FISH analysis of the salivary glands revealed that knockdown of caz significantly increased the number of nuclei containing RNA foci in (G4C2)89 flies (Figure 4E and F). We also confirmed that the expression levels of G4C2 repeat RNA in (G4C2)89 flies were not altered by the knockdown of caz (Figure 4G). Immunohistochemical analysis showed that knockdown of caz significantly increased the number of DPR aggregates in (G4C2)89 flies (Figure 4H and I). These results indicate that the reduction of caz expression enhances RNA foci formation and DPR aggregation, compatible with the results of FUS coexpression in flies expressing (G4C2)89 (Figure 3), and that FUS functions as an endogenous regulator of RAN translation. Figure 4 with 1 supplement see all Download asset Open asset 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—source data 1 Statistical data related to Figure 4B, D, F, G and I. https://cdn.elifesciences.org/articles/84338/elife-84338-fig4-data1-v1.xlsx Download elife-84338-fig4-data1-v1.xlsx FUS directly binds to and modulates the G-quadruplex structure of G4C2 repeat RNA, resulting in the suppression of RAN translation in vitro We next confirmed the direct interaction of FUS with G4C2 repeat RNA by the filter binding assay. We found that His-tagged FUS binds to the (G4C2)4 RNA in a dose-dependent manner, but not to the control (AAAAAA)4 RNA (Figure 5A), and His-tagged FUS-RRMmut had almost no binding affinity to the (G4C2)4 RNA, consistent with a previous study (Mori et al., 2013a). We also confirmed the interaction of FUS with the G4C2 repeat RNA in our C9-ALS/FTD flies by showing the colocalization of FUS with the RNA foci (Figure 5—figure supplement 1), consistent with a recent study using C9-ALS/FTD patient fibroblasts (Bajc Česnik et al., 2019). Since G4C2 repeat RNA was reported to form both G-quadruplex and hairpin structures (Fratta et al., 2012; Haeusler et al., 2014; Reddy et al., 2013; Su et al., 2014), we next characterized the interactions of FUS with G4C2 repeat RNA having different structures. G4C2 repeat RNA is known to form G-quadruplex structures in the presence of K+, whereas they form hairpin structures in the presence of Na+ (Su et al., 2014). Surface plasmon resonance (SPR) analyses demonstrated that FUS preferentially binds to (G4C2)4 RNA with the G-quadruplex structure in KCl buffer (Table 1, dissociation constant (KD) = 1.5 × 10–8 M) and weakly to (G4C2)4 RNA with the hairpin structure in NaCl buffer (Table 1, KD = 1.3 × 10–7 μM). We also confirmed that FUS has poor binding affinity to (G4C2)4 RNA in LiCl buffer (Table 1, KD = 1.4 × 10–5 μM), which destabilizes the G-quadruplex structure (Hardin et al., 1992), and was an almost similar level to its binding affinity to the negative control (A4C2)4 RNA (not shown). These results suggest the preferential binding of FUS to G4C2 repeat RNA with the G-quadruplex structure, which is consistent with a previous report showing preferential binding of FUS to G-quadruplex structured Sc1 and DNMT RNAs (Ozdilek et al., 2017). Considering that higher-order structures, including G-quadruplex and hairpin structures, are reported to be involved in RAN translation (Mori et al., 2021; Simone et al., 2018; Wang et al., 2019; Zu et al., 2011), we next investigated the effects of FUS on the structure of G4C2 repeat RNA. The circular dichroism (CD) spectrum of (G4C2)4 RNA in KCl buffer was found to exhibit a positive peak at approximately 260 nm and a negative peak at 240 nm (Figure 5B, black line), consistent with previous reports (Fratta et al., 2012; Haeusler et al., 2014; Reddy et al., 2013; Su et al., 2014). Interestingly, upon the addition of FUS, these two peaks were notably shifted to longer wavelengths with substantial CD spectrum changes, indicating a significant structural alteration in (G4C2)4 RNA (Figure 5B, red line). We confirmed that the CD spectrum of FUS alone in the wavelength range of 240–300 nm was almost negligible (Figure 5—figure supplement 2A, green line), indicating that this change in CD spectrum is attributed to structural changes in the (G4C2)4 RNA. We also observed CD spect" @default.
W4384698769 created "2023-07-20" @default.
W4384698769 date "2023-07-18" @default.
W4384698769 modified "2023-09-26" @default.
W4384698769 title "Reviewer #3 (Public Review):: FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD" @default.
W4384698769 doi "https://doi.org/10.7554/elife.84338.3.sa3" @default.
W4384698769 hasPublicationYear "2023" @default.
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