Ribosome profiling of C9ORF72. (A) iPSCs derived from three patients with expanded G4C2 repeats (C9) and three controls (cntrl) were treated with cycloheximide (CHX) or homoharringtonine (HHT) and then processed for ribosome profiling of the C9ORF72 RNA. Total read counts are provided in Supplemental Table 1. (B) An area of intron 1 upstream of G4C2 repeats was expanded to reveal two clusters of ribosome footprints. Also shown is an expanded area of intron 1 from a C9 patient following lactimidomycin D (LTM) treatment, which has an effect similar to HHT and ribosome profiling. (C) iPSC-derived neurons from a C9ORF72 patient and a healthy control were treated with HHT followed by ribosome profiling. The relevant area of intron 1 is shown. (D) A reporter construct containing a portion of C9ORF72 exon 1, C9ORF72 intron 1, G4C2(70), nano luciferase, a 3′ portion of C9ORF72 intron 1, the 5′ end of C9ORF72 exon 2, and firefly luciferase was expressed in HEK293T cells for ribosome profiling. (E) HEK cells were transfected with the plasmid noted above, or an identical one in which G4C2(70) was removed, followed by HHT treatment and ribosome profiling. Three ribosome footprints were detected on RNA derived from both plasmids, and are designated 1 (at 222), 2 (at 269), and 3 (at 281). The dip in the signal on the G4C2(70) RNA is due to a point mutation of an A for a G after 18 repeats in the reporter, which does not affect the reading frame. (F) Mapping of start sites with codon resolution shows that footprint 1 contains a putative CUG start site and is in-frame with poly(GP). Footprint 2 contains a putative CUG start site and is in-frame with poly(GA); however, a stop codon is immediately adjacent to this CUG codon. Footprint 3 contains a putative CUG start site in-frame with poly(GA).
