RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response

Abstract

Repeat-associated non-AUG (RAN) translation allows for unconventional initiation at disease-causing repeat expansions. As RAN translation contributes to pathogenesis in multiple neurodegenerative disorders, determining its mechanistic underpinnings may inform therapeutic development. Here we analyze RAN translation at G₄C₂ repeat expansions that cause C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9RAN) and at CGG repeats that cause fragile X-associated tremor/ataxia syndrome. We find that C9RAN translation initiates through a cap- and eIF4A-dependent mechanism that utilizes a CUG start codon. C9RAN and CGG RAN are both selectively enhanced by integrated stress response (ISR) activation. ISR-enhanced RAN translation requires an eIF2α phosphorylation-dependent alteration in start codon fidelity. In parallel, both CGG and G₄C₂ repeats trigger phosphorylated-eIF2α-dependent stress granule formation and global translational suppression. These findings support a model whereby repeat expansions elicit cellular stress conditions that favor RAN translation of toxic proteins, creating a potential feed-forward loop that contributes to neurodegeneration.

Fig. 1

C9RAN translation-specific reporters reveal differential expression across reading frames. a Schematic of reporters used in this study. C9RAN translation reporters were designed by placing the C9orf72 intron 1 sequence, including 70 G₄C₂ repeats, upstream of a start codon mutant NanoLucifearse (NLuc) and a C-terminal 3xFLAG-tag, in separate reading frames relative to the repeat. b Anti-FLAG western blot of control and C9RAN translation reporters expressed in rabbit reticulocyte lysate (RRL). GAPDH is used as a loading control. To prevent over-exposure, the AUG-NLuc control reaction was diluted 1:5 in sample buffer (indicated by ^#). c–e Relative expression from C9RAN NLuc reporters (c) normalized to GGG-NLuc in RRL (n = 15), or normalized to GA-NLuc in (d) HEK293 cells (n = 18), and (e) primary rat hippocampal neurons (n = 9). Graph in (c) represents mean ± SD. Graphs in d and e represent mean ± SEM. Two-tailed Student’s t test with Bonferroni and Welch’s correction, ^∗∗ p < 0.01; ^∗∗∗∗ p < 0.0001. GA glycine–alanine, GP glycine–proline, GR glycine–arginine, PSP precision protease cleavage site

Fig. 2

C9RAN is cap- and eIF4A-dependent and can initiate at a near-cognate start codon. a Schematic of 5′-cap C9RAN reporter mRNAs and near-cognate start codon mutations. b Expression of m⁷G-capped and A-capped control and C9RAN reporters in RRL, n = 6. c Expression of control and C9RAN reporters in RRL when excess free m⁷G (250 μM) or equimolar A-cap was added to inhibit eIF4E in trans, n = 6. d Expression of reporter mRNAs when eIF4A, the canonical helicase required for ribosome scanning during initiation, is inhibited with 4 μM hippuristanol (hipp), n = 6. e Mutational analysis of near-cognate start codons upstream of the repeat in the GA frame as depicted in a, n = 6. Graphs represent mean ± SEM. Two-tailed Student’s t test with Bonferroni and Welch’s correction, ^∗ p < 0.05; ^∗∗∗ p < 0.001; ^∗∗∗∗ p < 0.0001. GA glycine–alanine, GP glycine–proline, GR glycine–arginine, CrPV cricket paralysis virus

Fig. 3

RAN translation is selectively activated by the integrated stress response. a Schematic of the integrated stress response pathway. b Western blot analysis of the ER stress pathway and C9RAN reporter levels in HEK293 cells after treatment with 2 μM TG. GAPDH was used as a loading control. c Expression of control and C9RAN NLuc reporters and co-transfected FLuc in HEK293 cells treated with 2 μM TG, n = 9. d Schematic of the previously published +1 and +2 CGG RAN translation NLuc reporters. e, f Expression of control and CGG RAN translation reporters and co-transfected FLuc in HEK293T cells treated with 2 μM TG analyzed by (e) luciferase activity, n = 9, and (f) anti-FLAG western blot. Tubulin was used as a loading control. g Fluorescence intensity of mApple and co-transfected GFP (left) or (G₄C₂)×66-GFP (right) in primary rat cortical neurons, imaged with automated fluorescent microscopy 3 days after treatment with 0.5, 1, or 2 μM TG, n > 30. Graphs represent mean ± SEM. Two-tailed Student’s t test with Bonferroni and Welch’s correction, ^∗∗ p < 0.01; ^∗∗∗ p < 0.001; ^∗∗∗∗ p < 0.0001. PERK endoplasmic reticulum ER-resident kinase, HRI heme-regulated inhibitor kinase, SA sodium arsenite, TG thapsigargin, TM tunicamycin

Fig. 4

RAN translation is resistant to eIF2α phosphorylation. a Expression of control and C9RAN NLuc reporters and co-transfected FLuc in HEK293 cells treated with 40 μM Sal003, n = 6. b Expression of control and CGG RAN NLuc reporters and co-transfected FLuc in HEK293T cells treated with 20 μM Sal003, n = 9. c Western blot analysis of control, GA70 RAN and CGG RAN NLuc reporters in HEK293T cells treated with 20 μM Sal003. d Expression of control and C9RAN NLuc reporters and co-transfected FLuc in HEK293 cells transfected with either WT or S51D (phosphomimetic) eIF2α, n = 6. e Expression of control and CGG RAN NLuc reporters and co-transfected FLuc in HEK293T cells transfected with either WT or S51D (phosphomimetic) eIF2α, n = 9–15. f Expression of control, CGG, and C9RAN NLuc reporters normalized to co-transfected FLuc in WT eIF2α-S51 (S/S) and non-phosphorylatable homozygous eIF2α-S51 A/A mutant mouse embryonic fibroblasts (MEFs) following treatment with 1 μg mL⁻¹ tunicamycin (TM), n = 6–9. Graphs represent mean ± SEM. Two-tailed Student’s t test with Bonferroni and Welch’s correction, ^∗∗ p < 0.01; ^∗∗∗ p < 0.001; ^∗∗∗∗ p < 0.0001

Fig. 5

Near-cognate codons are sufficient to allow for stress-induced translation. a Top: schematic of C9RAN reporter with AUG codon inserted upstream of repeat. Bottom: expression of control, C9RAN NLuc, and AUG-driven C9 reporters and co-transfected FLuc in HEK293 cells treated with 2 μM TG, n = 6–9. b Top: schematic of CGG RAN reporter with a near-cognate codon mutated to AUG. Bottom: expression of control, CGG RAN NLuc, and AUG-driven CGG reporters and co-transfected FLuc in HEK293T cells treated with 2 μM TG, n = 9. c Top: schematic showing location of near-cognate codon substitutions made in AUG-NLuc. Bottom: expression of control and near-cognate codon NLuc reporters and co-transfected FLuc in HEK293T cells treated with 2 μM TG, n = 9. d Expression of control and near-cognate codon NLuc reporters and co-transfected FLuc in HEK293T cells treated with 20 μM Sal003 for 5 h, n = 12. Graphs represent mean ± SEM. Two-tailed Student’s t test with Bonferroni and Welch’s correction, ^∗∗ p < 0.01; ^∗∗∗ p < 0.001; ^∗∗∗∗ p < 0.0001

Fig. 6

CGG and G₄C₂ repeat expansions induce phosphorylated-eIF2α-dependent stress granules. a Top left: immunofluorescent images of HEK239 cells treated with vehicle or 0.5M SA. Bottom: immunofluorescent images of HEK239 cells expressing control, (G₄C₂)×70, or CGG×100 reporters, scale bar = 100 µm. Top right: quantification of the proportion of FLAG-positive cells with FMRP-positive stress granules (SGs) for each genotype, n > 70. b Western blot and quantification of puromycin incorporation in cells transfected with control AUG-NLuc or CGG×100 reporter, or treated with 2 µM TG as a positive control. GAPDH is used as a loading control. Graph represents mean ± SEM. c mApple fluorescent intensity in primary rat cortical neurons co-transfected with GFP or CGG×100-GFP, longitudinally imaged with automated fluorescent microscopy for 10 days following transfection, n > 68. Graph represents mean ± 95% confidence interval. d Left: immunofluorescent images of WT eIF2α- S51 S/S and eIF2α- S51 A/A MEFs expressing control, (G₄C₂)×70, or CGG×100 reporters, scale bar = 100 µm. Right: quantification of the proportion of FLAG-positive cells with FMRP-positive SGs for each genotype, n > 40. FLAG marks reporter expressing cells, FMRP mark SGs. For a and d, Fisher’s exact test, ^∗∗∗ p < 0.001; ^∗∗∗∗ p < 0.0001. For b and c, two-tailed Student’s t test with Bonferroni and Welch’s correction, ^∗ p < 0.05; ^∗∗ p < 0.01; ^∗∗∗∗ p < 0.0001

Fig. 7

Working model for how a feed-forward loop activates RAN translation and cellular stress pathways. Repeat expansions trigger RAN translation. RAN proteins or the repeat RNAs themselves then elicit stress granules and suppress global protein synthesis in a phosphorylated-eIF2α-dependent manner. Activation of the integrated stress response (ISR) and phosphorylation of eIF2α, either by the repeat RNAs or RAN proteins directly or through exogenous cellular stress, can further trigger stress granule formation and suppress global translation while selectively enhancing RAN translation. This creates a feed-forward loop that can contribute to neuronal dysfunction and death