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. Author manuscript; available in PMC: 2022 May 19.
Published in final edited form as: J Am Chem Soc. 2021 Apr 15;143(19):7368–7379. doi: 10.1021/jacs.1c00131

Figure 3.

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.