Fig 2: RuvBL overexpression reduces transcription of C9orf72 sense DNA.(A) HeLa cells transfected with empty vector control (ev), FLAG-RuvBL1, or HA-RuvBL2 were co-transfected with empty vector or with 45 uninterrupted sense GGGGCC repeats (45xG4C2). RuvBL overexpression was confirmed via immunoblot with GAPDH indicating equal loading of samples. (B, C, D) The levels of transcription of the sense repeats (B), GAPDH (C), and endogenous C9orf72 (D) was quantified by RT-qPCR using 18S as a housekeeping gene (mean ± SEM, N = 4 independent experiments; one-way ANOVA with Dunnett’s post-test: ns, non-significant, ****P < 0.0001).

Fig 3: RuvBL overexpression slows the rate of dipeptide repeat (DPR) production and interacts with the translational machinery.(A) HeLa cells transfected with empty vector control (Ctrl), FLAG-RuvBL1, or HA-RuvBL2 were co-transfected with empty vector or with 45 uninterrupted sense GGGGCC repeats (45xG4C2) before treating with cycloheximide (CHX) for the indicated time to block further protein translation. RuvBL overexpression was confirmed via immunoblot with GAPDH indicating equal loading of samples, and cyclin D demonstrating efficacy of the CHX treatment. (B) Levels of repeat-associated non-AUG translated poly(GP) DPRs were determined via MSD-ELISA allowing for the monitoring of protein turnover. (C) HeLa cells transfected with empty vector control (ev), FLAG-RuvBL1, or HA-RuvBL2 were co-transfected with empty vector or with V5-tagged 45 uninterrupted sense GGGGCC repeats (V5-45xG4C2). Proteins were harvested at the indicated times post transfection to follow rate of production. (D) Levels of repeat-associated non-AUG translated poly(GP) DPRs were determined via MSD-ELISA and are presented relative to the empty control transfected 0 h sample. (E) The level of poly(GP) DPRs at 8 h post transfection with empty vector control, FLAG-RuvBL1, or HA-RuvBL2. Poly(GP) DPRs are presented relative to the empty vector control (mean ± SEM, N = 3 independent experiments; one-way ANOVA with Tukey post-test: *P < 0.05, **P < 0.01, ***P < 0.001). Lysates from HeLa cells transfected with empty vector control or FLAG-tagged RPL10A were subjected to immunoprecipitation with anti-FLAG antibodies. (F, G) Immune pellets were probed for RuvBL1 (F) and RuvBL2 (G) on immunoblot.

Fig 4: Knockdown of RuvBL does not affect dipeptide repeat protein levels in vitro.HeLa cells treated with control siRNA (siCtrl), RuvBL-targeting siRNA (siRuvBL1), or RuvBL2-targeting siRNA (siRuvBL2) were transfected with empty vector control or 45 uninterrupted sense GGGGCC repeats (V5-45xG4C2). (A, B) RuvBL1 levels (A) and RuvBL2 levels (B) were determined via immuoblot with GAPDH used to indicate equal loading. RuvBL1 and RuvBL2 levels were normalised to GAPDH (mean ± SEM; one-way ANOVA with Tukey post-test: ***P ≤ 0.001, ****P ≤ 0.0001; N = 4 independent experiments). (C) Levels of repeat-associated non-AUG translated poly(GP) dipeptide repeats were determined via MSD-ELISA (one-way ANOVA with Tukey post-test: **P ≤ 0.01, ns, non-significant, N = 4 independent experiments).

Fig 5: C9ALS/FTD patient cells have reduced levels of RuvBL proteins.(A, B, C) RuvBL1 (left immunoblot) and RuvBL2 (right immunoblot) protein levels from 3 C9orf72-ALS/FTD patient iNPC lines and their age and sex-matched controls were determined by immunoblot. Levels of RuvBL1 (left graph) and RuvBL2 (right graph) were normalised to GAPDH and are shown relative to the age and sex-matched control (mean ± SEM; unpaired t test: *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns, non-significant; N = 3 independent experiments). (D, E, F) Expression of RuvBL1 (upper graphs) and RuvBL2 (lower graphs) transcripts were quantified by RT-qPCR using 18S as a housekeeping gene (mean ± SEM, N = 3 independent experiments; unpaired t test: **P ≤ 0.005, ***P < 0.001, ****P < 0.0001).