Fig. 1.

RAN translation of C9ORF72 hexanucleotide repeats can initiate with and without 5′-cap. a Schematic of monicistronic dual-luciferase reporters for RAN translation and canonical AUG translation. b HeLa Flp-In cells were induced to express translation reporters by doxycycline for 24 h. Relative RAN translation products from Frame-GA and Frame-GP were compared to no-repeat control. NLuc signals were normalized to FLuc in each sample. c Immunoprecipitation using MYC antibody from cells expressing Neg-NLuc or C9R-NLuc, followed by immunoblotting with GA or GP antibody. d Schematic of bicistronic reporters for cap-independent RAN translation and AUG translation. e Relative RAN translation products from Frame-GA and Frame-GP were compared to no-repeat control. NLuc signals were normalized to FLuc in each sample. f Immunoprecipitation using MYC antibody from bicistronic reporter cells followed by immunoblotting with GA or GP antibody. g Schematic timeline of siRNA transfection and induction of luciferase reporters in HeLa Flp-In cells. h Reporter cells were transfected with non-targeting siRNA or siRNA against cap-binding protein eIF4E. Immunoblotting of eIF4E showed the knockdown efficiency. GAPDH was blotted as internal control. i Expression of AUG-FLuc translation (left) and C9R-NLuc RAN translation (right) reporters in presence of eIF4E siRNA compared to non-targeting siRNA control. *P < 0.05, **P < 0.005, ***P < 10^–6, two-tailed t-test. j Reporter cells were treated with mTOR inhibitor Torin 1 for 24 h. Immunoblotting of phospho-4E-BP1 using antibodies recognizing different phosphorylation sites. β-actin was blotted as internal control. k Expression changes of AUG-FLuc and C9R-NLuc in bicistronic reporter cells under mTOR pathway inhibition by Torin 1 treatment. **P < 0.005, ***P < 0.0005, two-tailed t-test. l The relative translation levels of C9R-NLuc (Frame-GA and Frame-GP) with and without a functional 5′-cap. HeLa cells were transfected with in vitro transcribed monocistronic C9R-NLuc and Neg-NLuc RNA with either 5′-m⁷G cap or ApppG cap analog. The NLuc luciferase was normalized to RNA level in each condition, and each sample was compared with 5′-m⁷G capped C9R-NLuc in frame-GA (set as 100). Data are mean ± SEM from three biological replicates