m6A-dependent biogenesis of circular RNAs in male germ cells


The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at m6A-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with m6A-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of m6A in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and m6A-modified start codons in junction sequences.

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Fig. 1: CircRNA levels increase with progression of spermiogenesis.
Fig. 2: CircRNA accumulation correlates with enhanced splicing at m6A sites.
Fig. 3: A subgroup of circRNAs contain ORFs with m6A-modified start codons.
Fig. 4: Spermiogenesis-enriched circRNAs shift from RNPs to polyribosomes with progression of spermiogenesis.
Fig. 5: ALKBH5 and METTL3 affect circRNA generation through modulating m6A levels.
Fig. 6: Sperm-borne circRNAs are abundant, highly conserved across species and variable with aging and fertility statuses.
Fig. 7: Detection of circRNA junction peptides using LC-MS.


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This study was supported by grants from the NIH (HD071736 and HD085506 to W.Y.) and the Templeton Foundation (PID: 61174 to W.Y.). RNA-seq was conducted in the Single Cell Genomics Core of the University of Nevada, Reno School of Medicine, which was supported, in part, by the NIH COBRE Grant (P30GM110767 to W.Y.). Bioinformatics and RPAD-seq were, in part, carried out in the BGI Co. Ltd, with the support of a grant from the Science, Technology and Innovation Commission of Shenzhen Municipality (JSGG20170824152728492 to C.T.). The human sperm work was supported by grants from the Natural Science Foundation of Guangdong Province (2015A030313884 and 2018A030313528 to Y.T. and W.Q.), the Science and Technology Projects of Guangzhou (201607010137 and 201804010431 to W.Q. and Y.T.) and the Family Planning Research Institute of Guangdong Province (S2014001 to Y.T.).

Author information

W.Y. and C.T. conceived and designed the research. C.T., T.Y., Z.W., Z.X., R.J.W., Y.Z., R.K., H.Z., and D.R.Q. performed experiments on mouse samples. Y.T., X.Z., G.S., W.Z., and W.Q. provided human samples and performed RNA-seq on human samples. C.T., Y.X., N.L., J.W., W.C., and X.W. performed bioinformatics analyses. W.Y. and C.T. wrote the manuscript. All authors reviewed and agreed with the contents of the manuscript.

Correspondence to Chong Tang or Wei Yan.

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The authors declare no competing interests.

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Tang, C., Xie, Y., Yu, T. et al. m6A-dependent biogenesis of circular RNAs in male germ cells. Cell Res 30, 211–228 (2020). https://doi.org/10.1038/s41422-020-0279-8

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