Fig. 1

High-throughput sequencing and mass spectrometry of translatable circRNAs in mouse testes. a Flow chart of bioinformatic analysis and mass spectrometry detection of translatable circRNAs in mouse testes. b Flow chart of the detection of translatable circRNAs in mouse testes. The ORFs of mouse circRNAs were predicted by two or three different algorithms and cross verified by peptide identification using mass spectrometry. The existing Uniprot peptides were removed and manually corrected before human-mouse homology analysis. c Illustration of the annotated genomic region of Rsrc1, the putative linear and head-to-tail splicing form, and the validation strategy for the circRsrc1 are shown. Divergent primers (solid arrows) detected the circular form in cDNA but not in gDNA of mouse testes. Convergent primers (hollow arrows) spanning between exons 2 and 3 of Rsrc1 specifically detected the linear splicing form. Sanger sequencing following PCR was performed using the indicated divergent primers. d LC–MS/MS analysis was performed to identify specific peptide sequences translated by circRsrc1 in mouse testes. The predicted 161aa peptide sequence is listed; the one in bold blue is the specific peptide identified by mass spectrometry. e Diagram of circRsrc1-HA vector; circRsrc1 with an HA tag before stop codon was cloned into a plasmid carrying reverse complementary sequences. f Western blotting of cells transfected with circRsrc1-HA or circRsrc1-ATGmut plasmids mutated at the predicted start codon. β-tubulin was used as the internal control