A Helicase Unwinds Hexanucleotide Repeat RNA G-Quadruplexes and Facilitates Repeat-Associated Non-AUG Translation

Abstract

The expansion of a hexanucleotide repeat GGGGCC (G4C2) in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 expansion leads to repeat-associated non-AUG (RAN) translation and the production of toxic dipeptide repeat (DPR) proteins, but the mechanisms of RAN translation remain enigmatic. Here, we report that the RNA helicase DHX36 is a robust positive regulator of C9orf72 RAN translation. DHX36 has a high affinity for the G4C2 repeat RNA, preferentially binds to the repeat RNA's G-quadruplex conformation, and efficiently unwinds the G4C2 G-quadruplex structures. Native DHX36 interacts with the G4C2 repeat RNA and is essential for effective RAN translation in the cell. In induced pluripotent stem cells and differentiated motor neurons derived from C9orf72-linked ALS patients, reducing DHX36 significantly decreased the levels of endogenous DPR proteins. DHX36 is also aberrantly upregulated in tissues of C9orf72-linked ALS patients. These results indicate that DHX36 facilitates C9orf72 RAN translation by resolving repeat RNA G-quadruplex structures and may be a potential target for therapeutic intervention.

Figure 1.

DHX36 protein shows high affinity for G-quadruplex structures formed by GGGGCC RNA repeats. (A), (C), (E), (G) EMSA experiments were performed by annealing 20 nM of four Cy5-labeled RNA probes, r(GGGGCC)₈, r(GGGGCC)₄, r(GTGTCC)₄, and r(CCCCGG)₄, gradually from 95 °C to 25 °C and then incubating them with DHX36 protein in increasing concentrations (0, 2.5, 5, 10, 20, 40, and 80 nM) in the presence of 100 mM KCl or LiCl. The G-quadruplexes (GQ) that specifically appeared on the gels in the presence of K⁺ are indicated by arrows. (B), (D), (F), (H) Quantification was performed to generate the binding curves of DHX36 protein to various RNA probes in the presence of K⁺ or Li⁺ (N = 3), as indicated. Data are given as means ± SD of three independent experiments.

Figure 2.

DHX36 efficiently unwinds G-quadruplexes formed by GGGGCC RNA repeats. (A) Schematic representation of the G-quadruplex-hemin RNAzyme system. (B) Absorption spectra of r(GGGGCC)₁₀, r(CCCCGG)₁₀, or the buffer control in the colorimetric reactions. [RNA]=1 μM, [Hemin]=2.5 μM, [ABTS]=2.5 μM, and [H₂O₂]=0.4 mM. (C) DHX36 or BSA proteins were added to the r(GGGGCC)₁₀ RNAzyme system at increasing concentrations (0, 100, 200, 400, or 800 nM) before triggering the colorimetric reaction. Maximum absorbance at 420 nm for each group was used to compare the RNAzyme activities. Data are given as means ± SD of four independent experiments. **P <0.01. (D) Schematic of smFRET using RNA r(GGGGCC)₄-U10. (E) FRET histograms before and after addition of the DHX36 protein. FRET values were collected from >4000 molecules in 20 different fields of view. (F) Two sets of representative smFRET traces upon addition of DHX36 (arrows). The smFRET trace shows fluorescence intensities observed for Cy3 (green) and Cy5 (red) and the calculated FRET efficiency (blue) when 10 nM DHX36 protein was added. (G) The smFRET trace taken after DHX36 was added to the G4C2-U10 substrate. (H) FRET histograms taken 3, 10, and 20 minutes after 1 mM ATP was added to the DHX36 and G4C2-U10 substrate mix. (I) Three sets of representative smFRET traces after ATP was added.

Figure 3.

DHX36 binding to C9orf72 repeat RNA is required for efficient RAN translation in cells. (A) Schematic of the inducible luciferase-based C9orf72 RAN translation reporter system in HeLa Flp-In cells. The wide and narrow rectangles represent exons and introns, respectively. (B) RNA FISH with a (CCCCGG)₄-Cy3 probe demonstrated repeat RNA foci in the cells expressing the (GGGGCC)₇₀. (C) DHX36 knockdown significantly decreased C9orf72 RAN translation efficiency, measured as the ratio of Nano luciferase signal to firefly luciferase signal, in the HeLa Flp-In cells (left, N = 3). The knockdown of DHX36 was confirmed by immunoblot analysis of the cells expressing (GGGGCC)₇₀ (right). (D) RNA immunoprecipitation experiments in HeLa Flp-In cells with or without (GGGGCC)₇₀ demonstrated the enrichment of (GGGGCC)₇₀-containing transcripts (Nano Luc RNAs) but not the internal control firefly Luc transcripts upon pulldown of DHX36 (N = 3). (E) Immunoblotting confirmed the pulldown of DHX36 in the RNA immunoprecipitation experiments in HeLa Flp-In cells with and without (GGGGCC)₇₀. Data are given as means ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 4.

DHX36 is required for efficient translation elongation through GGGGCC repeat RNAs. (A) Schematic of the reporter constructs used to monitor the effects of (GGGGCC)n repeats on translation elongation. (B) The effects of (GGGGCC)n repeats on transcription were compared by RT-PCR analyses of EGFP RNA levels in HEK293 cells expressing reporter constructs containing various GGGGCC repeats and non-G-quadruplex-forming control sequences (non-GQ-mers) of the same sizes. The level of EGFP RNA was normalized to that of NeoR RNA, which is independently expressed on the same reporter construct. (C) Immunoblot analysis of EGFP from HEK293 cells expressing the reporter and control constructs. β-actin was used as the loading control. (D) Immunoblot analysis of EGFP from HEK293 cells expressing the reporter and control constructs after the cells were treated with shRNAs against DHX36 or non-targeting control shRNAs. Data are given as means ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 5.

Reduction in poly-GP by knockdown of DHX36 in patient-derived iPSCs and iMNs and upregulation of DHX36 in the spinal cords of C9orf72-linked ALS patients. (A) C9orf72-linked ALS patient-derived iPSCs were analyzed for poly-GP levels by ELISA, with or without the stable knockdown of DHX36. Data are given as means ± SD of four replicates from two independent experiments. **P < 0.01, ***P < 0.001. (B) C9orf72-linked ALS patient-derived iMNs were analyzed for poly-GP levels by ELISA, with or without the stable knockdown of DHX36. Data are given as means ± SD of four replicates from two independent experiments. ***P < 0.001, ****P < 0.0001. (C) DHX36 protein expression levels are higher in the spinal cord tissues from C9orf72-linked ALS patients than in the controls. Data are given as means ± SD of samples from five patients or five controls. *P = 0.0335.