Repeat-associated non-AUG-initiated translation of expanded CGG repeats (CGG RAN) from the FMR1 5′-leader produces toxic proteins that contribute to neurodegeneration in fragile X-associated tremor/ataxia syndrome. Here we describe how unexpanded CGG repeats and their translation play conserved roles in regulating fragile X protein (FMRP) synthesis. In neurons, CGG RAN acts as an inhibitory upstream open reading frame to suppress basal FMRP production. Activation of mGluR5 receptors enhances FMRP synthesis. This enhancement requires both the CGG repeat and CGG RAN initiation sites. Using non-cleaving antisense oligonucleotides (ASOs), we selectively blocked CGG RAN. This ASO blockade enhanced endogenous FMRP expression in human neurons. In human and rodent neurons, CGG RAN-blocking ASOs suppressed repeat toxicity and prolonged survival. These findings delineate a native function for CGG repeats and RAN translation in regulating basal and activity-dependent FMRP synthesis, and they demonstrate the therapeutic potential of modulating CGG RAN translation in fragile X-associated disorders.
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We thank P.K.T. and M.A.S.’s laboratory members and A. Goldstrohm for helpful discussions, commentary and technical advice. C. Pearson provided TC43-97 fibroblasts originally generated by M. Turker44. Compound 1A was a gift from M. Disney35. This work was funded by the NIH (grant nos. R01NS099280 and R01NS086810), and philanthropic donations to P.K.T. C.M.R was supported by the National Institute of Neurological Disorders and Stroke (NIN.DS, grant no. F31 NS090883-03). M.G.K was supported by grant no. F32NS089124 and is now supported by grant no. K99GM.126064. J.M.H. was funded by grant no. T32NS007222 and a post-doctoral fellowship from FRAXA. S.J.B. and B.N.F. were supported by the NIH (grant nos. R01-NS097542 and 1P30AG053760). S.E.W. was supported by the NIH (grant no. T-32-NS076401). M.I. and G.B. were supported by the NINDS (grant no. 5P30NS05507708). M.R.G. was supported by the NIH (grant no. T32NS007222). M.A.S. was supported by the Michigan Discovery Fund. F.R. and P.J.-N. were supported by Ionis Pharmaceuticals, who also funded reagent generation. The iPSC work was supported by the University of Michigan Human Stem Cell and Genome Editing Core.
P.K.T. served as a consultant with Denali Therapeutics and has licensed technology through the University of Michigan to Denali. P.K.T., C.M.R., F.R. and P.J. hold a patent on ASOs targeting CGG RAN. F.R. and P.J. are paid employees of Ionis Pharmaceuticals. All other authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
A, Left: in vitro translated (CGG)n FMRP-nluc reporter mRNAs harboring different CGG repeat sizes. ‡‡‡: FMRP-nanoluciferase-3X FLAG protein. ‡: N-terminal extension of FMRP from RAN initiation at the ACG codon in the (polyarginine) 0-frame. ‡ is detected at up to 18 repeats, but is attenuated at normal repeat sizes as previously described . ‡‡: N-terminal extension of FMRP from initiation in the 0RAN, downstream of the repeat. The ‡‡ product is only detectable in vitro, and is not detectable in cells. Right: Luciferase activity of in vitro translated FMRP-nLuc reporters (n=3; 0 vs 18: p=0.000000000000033; 18 vs 28: p=000001109; 28 vs 45: p=0.00234; 28 vs 57: p=0.0000518; 28 vs 69: p=0.0004997; 28 vs 100: p=0.001371). B, Luciferase activity from FMRP reporter in vitro after replacement of (CGG)25 repeat with unstructured (GAA)25 repeat (n=3; p=0.4940). C, Schematic of RAN translation reporters. D, Left: luciferase activity showing relative levels of FMRP, +1 RAN, and 0-frame RAN reporters at 25 and 100 repeats in HEK293 cells (n=3; CGG25: FMRP vs +1 RAN p=0.00002612, FMRP vs 0-frame RAN p=0.00001338; CGG100: FMRP vs +1 RAN p=0.0009, FMRP vs 0-frame RAN p=0.0003). E, Top: Immunoblot of (CGG)n FMRP-nLuc reporters in HEK293T cells with indicated mutations. Bottom: luciferase activity from reporters translated in vitro (RRL). +1-AUG represents insertion of AUG in place of +1 ACG RAN initiation codon. RAN initiation sites in the (CGG)n FMRP-nLuc reporters were mutated to preclude initiation in the 0-frame (0-AAA), the +1 reading frame (+1-AAA), or both (0/+1-AAA) (n=3; CGG25: WT vs 0-AAA p=0.0001, WT vs +1-AAA p=0.1977, WT vs 0/1-AAA p=0.0001; CGG100: WT vs 0-AAA p=0.0001, WT vs +1-AAA p=0.4437, WT vs 0/1-AAA p=0.0001). F, RT-qPCR to nLuc mRNA from SH-SY5Y cells expressing the indicated FMRP reporters with 100 repeats (n=3; p=0.4940). G, Flag immunocytochemistry for (CGG)100 FMRP-nLuc reporters in rat neurons co-expressing mCherry (red) to fill the cell. H, Quantification of Flag signal for the WT (CGG)100 reporter (n=23) and for the 0/+1-AAA (CGG)100 reporter (n=21), where “n” is the mean CTCF signal from 5 neurons (p=0.0258). Panel A: One-way ANOVA with multiple comparisons. Panels D, E: One-way ANOVA with multiple comparisons, within repeat groups. Panel B, F, H: two sided Student’s t-test. n.s.=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Graphs are mean ± S.E.M. Source data
A, Left: Venus with and without (∆AUG) an AUG initiation codon serve as a positive and negative translation control. Right: Venus fluorescent proteins with the AUG deleted were inserted after the 5′ leader of FMR1, in the +1 reading frame to serve as a reporter for +1CGG RAN. B, Live imaging of mature rat hippocampal neurons expressing indicated Venus reporters (green) and mCherry (red). C, Quantification of Venus reporter signals with 32 repeats (n=11) or 90 repeats (n=14) (p=0.0002). D, Single molecule imaging of CGG RAN translation in distal neuronal processes expressing indicated +1CGG RAN-Venus reporters after photo bleaching. Green dots represent individual translation events. E, Quantification of CGG RAN events in processes (n=3; p=0.2355). Panels C,E: Two sided Student’s t-test. n.s.=not significant, ***p<0.001. Graphs are mean ± S.E.M.
A, Mouse embryonic fibroblasts were transfected with +1 (CGG)100 RAN-nLuc reporters or mock transfected were probed for nLuc RNA by Hybridization Chain Reaction (HCR) and co-stained for FLAG. The specificity of the probes for nLuc is illustrated by the presence of signal (red) in transfected cells only. B, Left: HCR of nLuc mRNA combined with ICC to co-transfected mApple verifies dendritic export of reporter RNAs. Right: Quantification of dendritic nLuc reporter mRNA; AUG-nLuc (n=16), 0 repeat (n=16), 20 repeats (n=18), 90 repeats (n=20) (AUG vs 0: p=0.1447; AUG vs 20: p=0.0373; AUG vs 90: p=0.0044). One-way ANOVA with correction for multiple comparisons. Box extends to 25th/75th percentiles with a line at mean and whiskers indicate 95% CI.
A, Schematic of the AUG (+1) CGGn FMRP-nLuc-3′ UTR reporter, which drives translation in the +1 RAN reading frame and reports for FMRP. B, relative nLuc values in neurons transfected with CGG25 or CGG100 FMRP-nLuc-3′ UTR reporters with or without a +1 AUG mutant 5′ leader region (n=3; CGG20: p=0.0002; CGG100: p=0.0036). C, Relative nLuc values in neurons transfected with CGG25 FMRP-nLuc-3′ UTR reporters: WT (n=12), +1 AUG/Mock (n=12), +1 AUG/DHPG (n=11) (WT/Mock vs +1AUG/mock: p=0.005; WT/Mock vs +1AUG/DHPG: p=0.4504). D, Rat hippocampal neurons were transfected with a nLuc construct for the C9Orf72 G4C2 hexanucleotide repeat in the Glycine/Alanine reading frame (n=6; p=0.8642). E, Hippocampal neurons were treated with 1A at 20 hours post-transfection. Quantification of nLuc expression following 3 hours of 1A treatment is represented for each indicated reporter (n=3; AUG/Vehicle vs AUG/1A: p=0.8038; RAN/Vehicle vs RAN/1A: p=0.0437; FMRP/Vehicle vs FMRP/1A: p=0.5728). F, Rat hippocampal neurons were treated with ISRIB 6 hours before treatment with vehicle or DHPG (Mock/Mock: n=18, Mock/DHPG n=18, ISRIB/Mock n=17, ISRIB/DHPG n=18; Mock/Mock vs Mock/DHPG: p=0.0342, Mock/DHPG vs ISRIB/DHPG: p=0.0678, ISRIB/Mock vs ISRIB/DHPG: p=0.0058). Panel B, C, F: One-way ANOVA with multiple comparisons. Panel D, E: Two sided Student’s t-test. n.s.=not significant, *p<0.05, **p<0.01, ***p<0.001. Graph is mean ± S.E.M.
A, Schematic of other tested non-cleaving RAN blocking ASOs. Colored bars overlap the corresponding FMR1 5′ leader sequence and start sites; 0 frame ACG (orange), +1 frame ACG (+1RAN ASO-1, purple(18nt) or blue(16nt)) and +1 frame GUG (+1RAN ASO-2, maroon). B, Effect of +1RAN ASO-16 nucleotide on endogenous FMRP expression (0nM vs 25nM: p=0.0021; 0nM vs 75nM: p=0.0404; 0nM vs 100nM: p=0.2021). C, Effect of +0 RAN ASO (18nt) on endogenous FMRP expression (0nM vs 25nM: p=0.9999; 0nM vs 75nM: p=0.9997; 0nM vs 100nM: p=0.9580). D, Effect of Control ASO on endogenous FMRP expression (0nM vs 25nM: p=0.8183; 0nM vs 75nM: p=0.1780; 0nM vs 100nM: p=0.8486). E, Effect of combinatorial treatment with (+1RAN ASO-1 and +1RAN ASO-2 on endogenous FMRP expression at indicated doses (0nM vs 50nM: p=0.6231; 0nM vs 75nM: p=0.0127; 0nM vs 100nM: p=0.0171). F, Impact of +1RAN ASO-1 transfection into patient derived fibroblasts (0nM vs 50nM: p=0.2795; 0nM vs 75nM: p=0.0336; 0nM vs 100nM: p=0.5035). G, Representative immunoblot of FMRP expression after treatment with +1RAN ASO-1 (technical replicates of main Fig. 4a) in transfected control iPSCs. For all experiments, n=3, replicated in 3 independent experiments. For all graphs: One-way ANOVA with a Fisher’s LSD test for dose dependency. n.s.=not significant, *p<0.05, **p<0.01. Graphs are mean ± S.E.M. Source data
A, Immunocytochemistry against FMRpolyG on Control ((CGG)23), FXTAS ((CGG)100-117), and FXS ((CGG)931-940, fully methylated) patient derived lymphoblasts. B, Rater-blinded quantification of FMRpolyG staining expressed as a ratio to pre-immune serum at the same concentration (μg/mL) on the same cells. Values are expressed relative to the FXS line, which does not express the FMR1 transcript (Control n=50, FXTAS n=133, FXS n=102; p=0.0000000000005). C, Immunocytochemistry to FMRpolyG (red) in mature control human neurons (TUJ1-positive (green)) treated with +1RAN ASO-1 or Control ASO treatment. D, Quantification of FMRpolyG signal with +1RAN ASO-1 (n=90) or Control ASO (n=69) treatment, where “n” is the mean CTCF signal from 5 neurons (p=0.0485). Panel B: Kruskal Wallis test with post-hoc two sided Mann Whitney U tests. Panel D: Two sided unpaired Student t-test. *p<0.05, ****p<0.0001. Box extends to 25th/75th percentiles with a line at mean and whiskers indicate 95% CI. Marked dots are only shown for values outside the 95% CI.
A, Immunocytochemistry for mGluR5 in control human iPSC-derived neurons. B, mGluR mRNA expression as quantified by RT-PCR in Control iPSCs vs. neurons at day 49 of differentiation (n=2). C, Impact of 5 minutes of 50μΜ DHPG on calcium transients in human iPSC derived neurons. Graph represents number of neurons with active calcium transients compared to total number of neurons tracked over two independent neuronal cultures (n=2). D, Time course of DHPG effect on FMRP levels in human neurons (0 min: n=20, 5 min: n=35, 30 min: n=34, 60 min: n=46; 0 vs 5 min: p=0.2972, 0 vs 30 min: p=0.0158, 0 vs 60 min: p=0.0072). E, Left: Immunoblot from Control human neurons treated with +1RAN ASO-1 with or without DHPG treatment. Right: Quantification of FMRP expression in human neurons treated with DHPG after pretreatment with +1RAN ASO-1 relative to Control ASO treated neurons (n=3; p=0.0110). F, Representative western of FMRP expression after DHPG in control human neurons pretreated with the indicated ASOs and then treated with vehicle or DHPG. G, PKR has a 30-nucleotide CGG repeat in its 5′ leader. Endogenous expression of PKR was assessed after vehicle or DHPG treatment in iPSC-derived control human neurons. Left: representative immunoblot to PKR after indicated treatments. Right: Quantification of PKR expression by immunoblot in response to DHPG treatment (n=4; p=0.9696). Panel B, C, E, G: Two sided unpaired student t-test. Panel D: One-way ANOVA with multiple comparisons and post-hoc LSD. *p<0.05, **p<0.01. Graphs are mean ± S.E.M (±SD for B and C). Source data
A, Detection of three pluripotency markers—OCT-3/4, Nanog, and SSEA4—in the TC43-97 iPSCs confirms successful reprogramming of the fibroblast line. B, Cytogenetic analysis of cells in metaphase revealed an apparently normal male karyotype of TC43-97 iPSCs. C, Methylation sensitive qPCR of FMR1 promoter demonstrates lack of DNA methylation in TC43-97 iPSCs. Methylation levels were calculated relative to the FX hESC condition (n=3). D, Quantification of immunoblots to FMRP in the TC43-97 iPSCs relative to Control (n=3; p=0.0088). Panel D: Two sided Student’s t-test, **p<0.01. Graphs are mean ± S.E.M.
A, RT-PCR from indicated iPSC lines 24 hours after transfection with indicated ASOs: Control/Control ASO (n=4), Control/+1RAN ASO-1 (n=4), TC43-97/Control ASO (n=7), TC43-97/+1RAN ASO-1 (n=6, p=0.0522). B, Representative western blot showing relative FMRP expression in both Control and TC43-97 neurons treated with increasing doses of +1RAN ASO-1. Each lane is quantified relative to GAPDH and as a percent of the untreated control neurons. C, Soma (DAPI, blue) and processes (Tuj1, red) of differentiated neurons are TUJ1 positive in both control and TC43-97 neurons. D, Representative FMRP immunoblot from TC43-97 neurons treated with DHPG and indicated ASOs. E, Quantification of FMRP fluorescence by immunocytochemistry in human neurons treated as indicated, normalized to untreated Control ASO neurons quantified in parallel. Average fluorescence was binned for every 5 neurons consecutively analyzed to represent an individual data point. (Control ASO/Mock: n=7, Control ASO/DHPG: n=5, +1RAN ASO-1/Mock: n=6, +1RAN ASO-1/DHPG: n=5; Control ASO/Mock vs +1RAN ASO-1/Mock: p=0.0163). F, Survival analysis of TC43-97 iPSC derived neurons treated with 150nM +1RAN ASO-1 (n=272) or Control ASO (n=310), independent experiment and neuronal derivation #2 (p=0.000148). G, Survival analysis of TC43-97 iPSC derived neurons treated with 150nM +1RAN ASO-1 (n=220) or Control ASO (n=190), independent experiment and neuronal derivation #3 (p=0.027). Survival is plotted as cumulative risk of death. Panel C: Two sided Student T-test. Panel E: Two-way ANOVA with post-hoc correction for multiple comparisons. Panels F, G: Cox proportional hazard analysis. n.s.= not significant. * p<0.05. ***p<0.001. Graph is mean ± S.E.M. Box extends to 25th/75th percentiles with a line at mean and whiskers indicate 95% CI. Source data
CGG RAN regulates FMRP synthesis by limiting access of initiation complexes to the AUG initiation codon of FMRP in a repeat-dependent manner. II: mGluR activation bypasses CGG RAN, which allows for enhanced synthesis of FMRP. III: In the absence of CGG RAN or CGG repeat, steady-state FMRP synthesis increases but is decoupled from mGluR activation. IV: Non-cleaving RAN ASOs prevent CGG RAN initiation. This increases steady-state FMRP production, decreases FMRpolyG production and enhances neuronal survival.
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Rodriguez, C.M., Wright, S.E., Kearse, M.G. et al. A native function for RAN translation and CGG repeats in regulating fragile X protein synthesis. Nat Neurosci 23, 386–397 (2020). https://doi.org/10.1038/s41593-020-0590-1