Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats

Abstract

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1, and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter cell lines and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation - suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation elongation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

Figure 1.. NEMF, LTN1, and ANKZF1 act as genetic modifiers of RAN translation from G4C2 and CGG repeats.

(A) Schematic of mRNA and protein surveillance pathways(63).(B) Schematic of luciferase reporters used to assess RAN translation product abundance. (C) Targeted screen of factors involved in mRNA and protein surveillance pathways. The relative expression of NLuc to cell titer was compared to a non-targeting siRNA control (NTC). Data represent mean with error bars ± SEM of n = 6 cross at least 2 independent experiments ns = not significant; *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, multiple unpaired t-test after Welch’s correction.

Figure 2.. Depletion of NEMF, LTN1, and ANKZF1 enhances G4C2 C9 RAN translation in a repeat length-dependent manner across all reading frames.

(A) Schematic AUG-driven NLuc-3xFLAG and C9 RAN G4C2 repeat length and reading frame reporters. (B-E) Luciferase assays after NEMF, LTN1, both NEMF and LTN1 or ANKZF1 depletion. All graphs show mean with error bars ± SD. Each N is shown as an open circle (n=6–9/group). Asterisks above each bar are comparisons of expression between NTC and gene(s) knockdown. ns = not significant; *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, as determined with two-way ANOVA with Sidak’s multiple comparison test. Asterisks placed inside each bar are comparisons between AUG-driven no-repeat control and different repeat lengths of the GA frame of cells treated with gene(s) knockdown. ns = not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, represent unpaired t-tests with Welch’s correction for multiple comparisons.

Figure 3.. Detection of partially made products translated from GC-rich transcripts.

(A) Schematic of AUG-V5-(G4C2)₆₉-NLuc-3xFLAG in GA frame and AUG-V5-+1(CGG)₁₀₁-NLuc-3xFLAG in polyG frame. (B) FLAG and V5 IP workflow to enrich for incomplete products generated from GC-rich transcripts. 3xFL: 3xFLAG (C-D) V5 antibody IP reveals short/stalled products (line next to the blot) that are generated from G4C2 (panel C) or CGG repeats (panel D) that do not contain the carboxyl-terminal FLAG tag. FT: Flow-through.

Figure 4.. Depletion of NEMF, LTN1, and ANKZF1 enhances the generation of both full-length and partially made GA and polyG products from GC-rich transcripts.

(A-D) Immunoblots of HEK293 cells transfected with in vitro synthesized G4C2 (GA frame) and CGG repeat RNA reporters (polyG frame) in the presence and absence of NEMF + LTN1, and ANKZF1. Samples were processed as in Figure 3B. Blots represent two biological replicates. Lines next to the blots indicate short/stalled product products.

Figure 5.. Enhancement of polyG production with NEMF, LTN1, and ANKZF1 depletion requires the CGG repeat RNA structure.

(A-B) Prediction of the optimal RNA secondary structure and calculation of the minimum free energy in CGG₁₀₁ and GGN₁₀₃ repeats. Only the repeat region from each repeat was used to predict the RNA secondary structure and calculate the minimum free energy. The results were computed by RNAfold 2.5.1. (C)Schematics of AUG-V5-NLuc-3xFLAG, AUG-V5-+1(CGG)₁₀₁-NLuc-3xFLAG, and AUG-V5-(GGN)₁₀₃-NLuc-3xFLAG transcripts. (D) Knockdown of NEMF, LTN1, and ANKZF1 in HEK293 with no-repeat control, polyG from GGN repeats, and CGG repeats RNA transfection. Data represent means with error bars ±SD of n = 12, ns = not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 by Tukey’s multiple comparisons tests. The statistic result placed in the legend is group comparisons by two-way ANOVA Sidak’s multiple comparisons tests.

Figure 6.. Depletion of NEMF enhances repeat-associated toxicity in a fly model of C9 ALS/FTD and DPR accumulation in human neurons.

(A) Representative images of Drosophila eyes expressing (G4C2)28 repeats under the GMR-GAL4 driver in the presence or absence of NEMF at 25°C [BDSC36955 #1 and BDSC25214 #2]. Rough eye phenotypes quantified using a nominal scoring system are shown as violin plots on the right. Individual flies are represented by single data points. n= 30–33/genotype. (B) (G4C2)28 repeats expressed with a GMR-GAL4 driver at 29°C show decreased eye width that is enhanced by the depletion of NEMF, as quantified on the right. Graphs represent the mean with error bars ±SD, n = 21–24. For A and B, *P < 0.05; ****P ≤ 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. (C) Schematic workflow for studies with C9ALS patient-derived iNeurons (iN). (D-F) RNA Expression of NEMF, LTN1, and ANKZF1 transcripts from C9ALS and isogenic control iN lysates. Data represent means with error bars ±SD. n = 3–6/gene, ns = not significant; *P < 0.05 by Student’s t-test. (G-I) Quantification of GP by MSD assay from C9ALS and isogenic control iNs treated with lenti-empty vector or lenti-shRNA of NEMF, LTN1, or ANKZF1. Data represent mean ±SD; n = 3–6, ns = not significant; **P ≤ 0.01; ***P ≤ 0.001 by two-way ANOVA with Sidak’s multiple comparison test.

Figure 7.. Overexpression of NEMF, LTN1, and ANKZF1 decreases RAN translation from G4C2 and CGG repeats.

(A-B) Relative expression of AUG-driven no repeats, (G4C2)70 repeats in the GA frame, and (CGG)100 repeats in the polyG frame when overexpressing empty vector (EV) versus hNEMF or hLTN1. (C) Relative expression of AUG-driven no-repeat control and (G4C2)70 repeats in the GA, GP, and GR frames when overexpressing empty vector (EV) versus hANKZF1. (D) Relative expression of AUG-driven no-repeat control and (CGG)100 repeats in the polyG frame when overexpressing empty vector (EV) versus hANKZF1. Data represent means with error bars ±SD of n = 9–12, ns = not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 by two-way ANOVA with Sidak’s multiple comparison test. (E-G) Relative GP response of C9 and isogenic control iN treated with lenti-empty vector or lenti-hNEMF, hLTN1, and hANKZF1. The level of GP was measured by MSD. Data represent means with error bars ±SD of n = 3–5, ns = not significant; *P < 0.05; ****P ≤ 0.0001 by two-way ANOVA with Sidak’s multiple comparison test.