Fig. 5
RAN translation of G4C2 repeats is down-regulated by a short upstream open reading frame (uORF). (a) Retention of intron 1 in C9ORF72 repeat-containing transcripts creates an uORF located 226 nucleotides upstream of the start CUG codon. This uORF may inhibit or enhance G4C2 RAN translation. (b) To interrogate the regulation of RAN translation by this uORF, RNAs harboring the 5′ full-length sequence including C9ORF72 exon 1A (#1) and a AUG > CCG mutation in the uORF start codon (#2) were compared to RAN translation from RNA without the uORF (#4). Black boxes represent exons 1a and 1b and the gray box represents the uORF overlapping exon 1a and intron 1. (c) Translation in RRL system was performed in presence of [35S]-methionine and capped RNA (#1, #2, or #4) followed by detection of [35S]-methionine proteins by autoradiography. (d) Poly-GA, poly-GP, and poly-GR were detected by immunoblots using antibodies against HA-tag, His-tag, and FLAG-tag, respectively
