Fig. 5: Rolling circle translation of endogenous circRNAs.

Source data are provided as a Source Data file for panels (c–g). a The higher-energy collisional dissociation MS/MS spectrum of the peptide across the back-splice junctions of the human circPSAP (MMMHMEEILVYLEK), circPFAS (LLEVGPRNL), and circABHD12 (LPRILSVK). The annotated b- and y-ions are marked in red and green, respectively. b The rcORF translation reporters. The coding region of the endogenous rcORF was inserted into a back-splicing reporter, with an in-frame V5 epitope for detection. c Three rcORF reporters were transfected into 293 cells, the cells were collected at 48 h after transfection and analyzed by western blotting and RT-PCR. The blue arrows represent the predicted MW of the single cycle of translation product from cPSAP (22.4kD), cPFAS (15.6kD), or cABHD12 (10.5kD). d The rcORF translation reporters were transfected into 293T cells and then treated with 10 µM MG132 for 2 h, or 10 µM chloroquine for 4 h before cell collection. The bar graph represents the quantification of protein levels relative to GAPDH, which were also normalized to the RNA (N.S. not significant). e The circPFAS contains two IRES-like hexamers (AATTCA and AAGAAG), which were mutated into neutral sequences (mut1 and mut2). The downstream AUG codons were mutated into CUC (Start Mut #1 and #2), and the non-canonical start codons CUG at downstream of the AAGAAG hexamers were further mutated into CUC (Start Mut #3). The effects of these mutations on protein production were determined with western blotting using similar procedure as panel c, with relative protein changes represented by bar graphs (N.S. not significant). f The back-splicing reporter of rcPFAS was co-transfected into 293T cells with the expression vectors of various trans-acting factors that bind to the newly identified IRES-like elements. The cells were collected and analyzed using same procedures as panel (c). g The circRNA with mutated IRES-like hexamer AAGAAG (Mut2) was co-expressed with the same set of trans-acting factors, and the production of rolling circle translation were measured using same experimental conditions described in panel (f).