Extended Data Fig. 9: Cryptic stop codon analysis.
From: The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center
a, Transcript (top line) and polypeptide (bottom line) sequence of the reporter designed to test translation termination at cryptic stop codons. The reporter contains an N-terminal 8xHis tag (blue), a modified calmodulin sequence (light orange), the test codon position (dark orange; stop1), a modified sequence encoding the autonomously folding villin headpiece (VHP) domain followed by the cytosolic domain of Sec61β (light green), and the UAA stop codon (black; stop2). The reporter sequence was modified to remove all codons, except for one UGG codon (pink), that start with a U and contain a purine (A/G) in the second and/or third positions. Additional cryptic codons that start with a C and contain purines in the second and third positions (purple) were tested by mutations (see panel c). b, SRI-41315 induces translation termination at specific cryptic stop codons. Quantification of the ratio of stop1 to stop2 products from reporters containing UUA (green) or UAU (orange) in the test codon position synthesized in vitro with increasing concentrations of SRI-41315. The normalized average (line) and individual ratios (dots) from three independent experiments are shown. c, Mutagenesis of additional cryptic stop codons. Assays as in Fig. 4a with UGA (lane 1) or UGG (lanes 2–5) in the test codon position and additional mutation of CAG and CAA codons (Qmut; purple) upstream of the stop1 position and/or of the UGG codon (Wmut; pink) downstream of the stop1 position as indicated in panel a. Radiolabeled reporter products generated without (left) or with (right) 100 µM SRI-41315 are shown. Note: changes in products upon mutation of these codons occur specifically with SRI-41315, representative of 3 replicates with similar results. Purple dots denote products abolished by mutating the CAG/CAA codons; pink dot denotes the lower band of a doublet abolished by mutating the UGG codon. Yellow dot denotes a band with increased intensity after mutation of the CAG/CAA codons.
