Fig. 3
G4C2 RAN translation of all reading frames initiates at the same near-cognate CUG start codon in RRL. (a) Scheme of the pre-initiation complex loaded at the 5′cap and the 40S ribosomal subunit ready to scan toward the start codon. RAN translation occurs in absence of an AUG codon. (b) Schemes of the transcripts #4 to #11 showing mutations in the 5′ flanking sequence of (G4C2)66 used in (c-f). Construct #4 contains the native sequence of 113 nucleotides upstream of the G4C2 repeat. Construct #5 has a CUG > CCG mutation (blue nucleotide) in a near-cognate start codon located in a perfect Kozak sequence 24 nucleotides upstream of the repeat. Construct #6 has CUG > CCG and GAG > GGG (blue nucleotides) mutations in two potential start codons located 24 and 13 nucleotides upstream of the repeat, respectively. Construct #7 contains a GAG > GGG mutation in a potential near-cognate start codon located 13 nucleotides upstream of the repeat. Constructs #8 and #9 harbor a deletion leaving 33 nucleotides (including CUG and GAG codons) and eight nucleotides (deleting both CUG and GAG codons) upstream of the repeat, respectively. Construct #10 has a CUG > AUG mutation in the near-cognate start codon. Construct #11 has GCUCUGG > UCUCUGC mutations in the Kozak sequence. (c–f) Translation was performed in presence of [35S]-methionine using each RNA variant separately (#4 to #11). (c and e) [35S]-methionine RAN translation products were detected by autoradiography. (d and f) Poly-GA, poly-GP, and poly-GR were detected by immunoblot using antibodies against HA-tag, His-tag, and FLAG-tag, respectively. Asterisk indicates unspecific proteins translated in the RRL system
