Substrate for C9ORF72 intron translation. (A) Lysates from HeLa Flp-In cells stably expressing the reporter plasmid were ultracentrifuged through 0.5 M and 1 M sucrose to pellet polysomes. Parallel experiments were performed when lysates were treated with EDTA, which dissociated polysomes. (B) Spliced RNA was measured from exon 1 to exon 2 and was far more abundant in our input compared to unspliced RNA (pair 3), relative to hygromycin (hyg) RNA expressed from the same plasmid. (C) RNA from the pellets as well as input was subjected to RT-qPCR with the primers in intron 1 and exon 1 (pair 1), within intron 1 (pair 2), and intron 1 and exon 2 (pair 3). (D) The ratios of the RT-qPCR products using primer pair 1 (exon 1 and intron 1) to primer pair 2 (both in intron 1) in the polysome pellet and total input RNA is shown. (E) Lysates from iPSCs C9#1 (also referred to as C9 26#6) were ultracentrifuged through 0.5 M and 1 M sucrose to pellet polysomes. RNA from the pellets was subjected to RT-qPCR with the primers in intron 1 and exon 1 (pair 1), within intron 1 (pair 2), and intron 1 and exon 2 (pair 3). Parallel experiments were performed when lysates were treated with EDTA, which dissociated polysomes. (F) The ratios of the RT-qPCR products using primer pair 1 (exon 1 and intron 1) to primer pair 2 (both in intron 1) in the polysome pellet and total input RNA are shown. All experiments were performed in biological triplicate. (*) P < 0.01, (***) P < 0.001 (Student t-test).
