Fig. 5

Identification of circSPECC1 coding capacity. A Diagram of circSPECC1 structure and explanation of amino acid alignment between the encoded new protein by circSPECC1 and the parent SPECC1 protein. B Full-length IRES sequences of circSPECC1 or IRES sequences of its different truncated mutants were cloned between Rluc/Luc reporters with independent start (ATG) and stop (TGA) codons, including full-length IRES (165-286nt), IRES-1 (165-225nt) and IRES-2 (225-286nt). C The relative luciferase activity of Luc/Rluc in empty vector, full-length IRES, IRES-1 and IRES-2 vector detected by dual luciferase assay. D Description of vectors used to detect whether circSPECC1 can encode the protein. Control vector: the flanking sequences were deleted and circSPECC1 could not be expressed. SPECC1-415aa-3 × Flag vector: a linearized ORF of circSPECC1 with a 3 × Flag tag was cloned into a linear plasmid and used as a positive control. CircSPECC1-3 × Flag vector: this vector had the flanking sequences, splice acceptor (SA) and splice donor (SD) and could be able to express circSPECC1 and SPECC1-415aa with Flag tag protein. CircSPECC1-3 × Flag-Del vector: the start codon (ATG) was censored, which could express circSPECC1 but not translate SPECC1-415aa. All vectors had CMV promoters. E The expression of SPECC1-415aa in 293 T cells transfected with the above plasmids by western blot assay. F IP and western blot experiments verified that the circSPECC1-3 × Flag plasmid expressed the Flag tag protein in 293 T cells and was specifically enriched by Flag antibody. G Schematic diagram of the new protein SPECC1-415aa translated by circSPECC1 and the secondary spectrum of the specific peptide of SPECC1-415aa-3 × Flag protein after IP-MS. **, P < 0.01; ****, P < 0.0001