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. 2020 Apr 2;19:71. doi: 10.1186/s12943-020-01179-5

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Evaluation of the coding ability of circFNDC3B. a Upper panel, the putative open reading frame (ORF) in circFNDC3B. The ORF implied that it took more than one whole circle of circFNDC3B. Lower panel, the sequences of putative ORF are shown. b The putative IRES activity of circFNDC3B was tested. Left panel, IRES sequences in circFNDC3B or its different truncation/mutation were cloned between the Rluc and Luc reporter genes with independent start (AUG) and stop (UGA) codons. Right panel, the relative luciferase activity of Luc/Rluc in the above vectors was tested. c Four vectors were constructed. Lv-vector; Lv-circFNDC3B-flag: flag-labeled circFNDC3B sequence was cloned into a CMV-induced expression vector; Lv-circFNDC3B-flag-mut: flag-labeled circFNDC3B sequence with start codon mutant (ATG → ACG) was cloned into a CMV-induced expression vector; Lv-circFNDC3B-218aa: flag-labeled circFNDC3B-218aa sequence was cloned into a CMV-induced expression vector. FCF and BCF are sequences which could circularize the sequence of circRNA. d Relative RNA expression of circFNDC3B and linearFNDC3B were detected by qRT-PCR. e The expression level of flag-label circFNDC3B-218aa and FNDC3B were detected by Western blotting analysis. f The lysates from indicated cells were separated by SDS-PAGE. Protein bands near 25 kDa were excised manually and summited for identification by LC-MS/MS. g circFNDC3B-218aa junction-specific peptide (MIQTCKVIEDSTGVR) was identified. ***P < 0.001