Mechanism of C9ORF72 intron translation. (A) A hairpin (−50 kcal/mol) or an identical size control sequence CAA(21) that forms no hairpin was inserted two bases downstream from the cap in the reporter construct. The plasmids were transfected into HEK cells followed by determination of nano and firefly luciferase activities and RNA levels. (B) The constructs shown in Figure 1D were transfected into HEK cells followed by HHT treatment as well as 4EGI, a small molecule inhibitor of the eIF4E–eIF4G interaction. 4EGI was applied to cells at 100 µM for 3 h. Ribosome profiling was then performed as in Figure 1E. (C) A HeLa Flp-In cell line expressing the construct depicted in Figure 1D was transduced with lentiviruses expressing a nonspecific sequence, negative siRNA (nsi) or shRNAs for DAP5, eIF3d, or eIF4E. Nano and firefly luciferase activities and RNAs were determined. These proteins as well as vinculin were then western blotted and quantified. (D) C9ORF72 and GAPDH RNA immunoprecipitation was performed with DAP5 antibody or nonspecific IgG on HeLa Flp-In cells. RNA values were normalized to DAP5, which was set at 1. All experiments were performed in biological triplicate. (*) P < 0.01, (***) P < 0.001 (one-way ANOVA). (E) A model for C9ORF72 GGGGCC repeat-containing RNA translation. Ribosome profiling shows initiating ribosomes on 3 CUG codons upstream in the intron of G4C2(n). Two of the CUG codons are in-frame with poly(GA) and one in-frame with poly(GP). A uORF initiating at an AUG codon at the end of exon 1 is a negative regulator of translation. Recruitment or positioning of ribosomes on the uORF AUG may be facilitated by the eIF4G-like protein DAP5. Ribosome stalling may occur on the G4C2(n) hairpins formed by the repeat expansion. Created with BioRender.com.
