m6A-dependent biogenesis of circular RNAs in male germ cells

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Leblond, C. P. & Clermont, Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N.Y. Acad. Sci. 55, 548–573 (1952).

  2. 2.

    Oakberg, E. F. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am. J. Anat. 99, 507–516 (1956).

  3. 3.

    Maclean, J. A. 2nd & Wilkinson, M. F. Gene regulation in spermatogenesis. Curr. Top. Dev. Biol. 71, 131–197 (2005).

  4. 4.

    Eddy, E. M. Regulation of gene expression during spermatogenesis. Semin. Cell Dev. Biol. 9, 451–457 (1998).

  5. 5.

    Kleene, K. C. Patterns, mechanisms, and functions of translation regulation in mammalian spermatogenic cells. Cytogenet. Genome Res. 103, 217–224 (2003).

  6. 6.

    Bao, J. et al. UPF2-dependent nonsense-mediated mrna decay pathway is essential for spermatogenesis by selectively eliminating longer 3'UTR transcripts. PLoS Genet. 12, e1005863 (2016).

  7. 7.

    Li, W. et al. Alternative cleavage and polyadenylation in spermatogenesis connects chromatin regulation with post-transcriptional control. BMC Biol. 14, 6 (2016).

  8. 8.

    Kashiwabara, S., Nakanishi, T., Kimura, M. & Baba, T. Non-canonical poly(A) polymerase in mammalian gametogenesis. Biochim. Biophys. Acta 1779, 230–238 (2008).

  9. 9.

    Steger, K. Haploid spermatids exhibit translationally repressed mRNAs. Anat. Embryol. 203, 323–334 (2001).

  10. 10.

    Braun, R. E. Temporal translational regulation of the protamine 1 gene during mouse spermatogenesis. Enzyme 44, 120–128 (1990).

  11. 11.

    Nguyen-Chi, M. & Morello, D. RNA-binding proteins, RNA granules, and gametes: is unity strength? Reproduction 142, 803–817 (2011).

  12. 12.

    Idler, R. K. & Yan, W. Control of messenger RNA fate by RNA-binding proteins: an emphasis on mammalian spermatogenesis. J. Androl. 33, 309–337 (2012).

  13. 13.

    Zhang, Y. et al. MicroRNAs control mRNA fate by compartmentalization based on 3' UTR length in male germ cells. Genome Biol. 18, 105 (2017).

  14. 14.

    Jha, K. N., Tripurani, S. K. & Johnson, G. R. TSSK6 is required for gammaH2AX formation and the histone-to-protamine transition during spermiogenesis. J. Cell Sci. 130, 1835–1844 (2017).

  15. 15.

    Tanaka, H. & Baba, T. Gene expression in spermiogenesis. Cell Mol. Life Sci. 62, 344–354 (2005).

  16. 16.

    Miura, P., Shenker, S., Andreu-Agullo, C., Westholm, J. O. & Lai, E. C. Widespread and extensive lengthening of 3' UTRs in the mammalian brain. Genome Res. 23, 812–825 (2013).

  17. 17.

    MacDonald, C. C. & McMahon, K. W. Tissue-specific mechanisms of alternative polyadenylation: testis, brain, and beyond. Wiley Interdiscip. Rev. RNA 1, 494–501 (2010).

  18. 18.

    Fanourgakis, G., Lesche, M., Akpinar, M., Dahl, A. & Jessberger, R. Chromatoid body protein TDRD6 supports long 3' UTR triggered nonsense mediated mRNA decay. PLoS Genet. 12, e1005857 (2016).

  19. 19.

    Yue, Y., Liu, J. & He, C. RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation. Genes Dev. 29, 1343–1355 (2015).

  20. 20.

    Zhao, X. et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 24, 1403–1419 (2014).

  21. 21.

    Adhikari, S., Xiao, W., Zhao, Y. L. & Yang, Y. G. m(6)A: Signaling for mRNA splicing. RNA Biol. 13, 756–759 (2016).

  22. 22.

    Tang, C. et al. ALKBH5-dependent m6A demethylation controls splicing and stability of long 3'-UTR mRNAs in male germ cells. Proc. Natl. Acad. Sci. USA 115, E325–E333 (2018).

  23. 23.

    Salzman, J. Circular RNA expression: its potential regulation and function. Trends Genet. 32, 309–316 (2016).

  24. 24.

    Chen, L. L. The biogenesis and emerging roles of circular RNAs. Nat. Rev. Mol. Cell Biol. 17, 205–211 (2016).

  25. 25.

    Liang, D. & Wilusz, J. E. Short intronic repeat sequences facilitate circular RNA production. Genes Dev. 28, 2233–2247 (2014).

  26. 26.

    Starke, S. et al. Exon circularization requires canonical splice signals. Cell Rep. 10, 103–111 (2015).

  27. 27.

    Memczak, S. et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333–338 (2013).

  28. 28.

    Song, Y. Z. & Li, J. F. Circular RNA hsa_circ_0001564 regulates osteosarcoma proliferation and apoptosis by acting miRNA sponge. Biochem. Biophys. Res. Commun. 495, 2369–2375 (2018).

  29. 29.

    Weiser-Evans, M. C. M. Smooth muscle differentiation control comes full circle: the circular noncoding RNA, circActa2, functions as a miRNA sponge to fine-tune alpha-SMA expression. Circ. Res. 121, 591–593 (2017).

  30. 30.

    Hansen, T. B., Kjems, J. & Damgaard, C. K. Circular RNA and miR-7 in cancer. Cancer Res. 73, 5609–5612 (2013).

  31. 31.

    Hansen, T. B. et al. Natural RNA circles function as efficient microRNA sponges. Nature 495, 384–388 (2013).

  32. 32.

    Piwecka, M. et al. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function. Science 357, https://doi.org/10.1126/science.aam8526 (2017).

  33. 33.

    Pamudurti, N. R. et al. Translation of CircRNAs. Mol. Cell 66, 9–21 e27 (2017).

  34. 34.

    Legnini, I. et al. Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis. Mol. Cell 66, 22–37 e29 (2017).

  35. 35.

    Yang, Y. et al. Extensive translation of circular RNAs driven by N(6)-methyladenosine. Cell Res. 27, 626–641 (2017).

  36. 36.

    Wang, Y. & Wang, Z. Efficient backsplicing produces translatable circular mRNAs. RNA 21, 172–179 (2015).

  37. 37.

    Yang, Y. & Wang, Z. Constructing GFP-based reporter to study back splicing and translation of circular RNA. Methods Mol. Biol. 1724, 107–118 (2018).

  38. 38.

    Fan, X., Yang, F. & Wang, Z. Pervasive translation of circular RNAs driven by short IRES-like elements. BioRxiv, https://doi.org/10.1101/473207 (2018).

  39. 39.

    Panda, A. C. et al. High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs. Nucleic Acids Res. 45, e116 (2017).

  40. 40.

    Jeck, W. R. et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19, 141–157 (2013).

  41. 41.

    Venables, J. P. Alternative splicing in the testes. Curr. Opin. Genet. Dev. 12, 615–619 (2002).

  42. 42.

    Matzuk, M. M. & Lamb, D. J. The biology of infertility: research advances and clinical challenges. Nat. Med. 14, 1197–1213 (2008).

  43. 43.

    Li, Y. et al. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis. Cell Res. 25, 981–984 (2015).

  44. 44.

    Xiao, W. et al. Nuclear m(6)A reader YTHDC1 regulates mRNA splicing. Mol. Cell 61, 507–519 (2016).

  45. 45.

    Bartosovic, M. et al. N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3'-end processing. Nucleic Acids Res. 45, 11356–11370 (2017).

  46. 46.

    Wang, C. X. et al. METTL3-mediated m6A modification is required for cerebellar development. PLoS Biol. 16, e2004880 (2018).

  47. 47.

    Zheng, G. et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49, 18–29 (2013).

  48. 48.

    Tatomer, D. C. & Wilusz, J. E. An unchartered journey for ribosomes: circumnavigating circular RNAs to produce proteins. Mol. Cell 66, 1–2 (2017).

  49. 49.

    Maezawa, S., Yukawa, M., Alavattam, K. G., Barski, A. & Namekawa, S. H. Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis. Nucleic Acids Res. 46, 593–608 (2018).

  50. 50.

    O'Donnell, L. Mechanisms of spermiogenesis and spermiation and how they are disturbed. Spermatogenesis 4, e979623 (2014).

  51. 51.

    Yan, W. Male infertility caused by spermiogenic defects: lessons from gene knockouts. Mol. Cell Endocrinol. 306, 24–32 (2009).

  52. 52.

    Xu, H., Yuan, S. Q., Zheng, Z. H. & Yan, W. The cytoplasmic droplet may be indicative of sperm motility and normal spermiogenesis. Asian J. Androl. 15, 799–805 (2013).

  53. 53.

    Xu, K. et al. Mettl3-mediated m(6)A regulates spermatogonial differentiation and meiosis initiation. Cell Res. 27, 1100–1114 (2017).

  54. 54.

    Lin, Z. et al. Mettl3-/Mettl14-mediated mRNA N(6)-methyladenosine modulates murine spermatogenesis. Cell Res. 27, 1216–1230 (2017).

  55. 55.

    Yuan, S., Zheng, H., Zheng, Z. & Yan, W. Proteomic analyses reveal a role of cytoplasmic droplets as an energy source during epididymal sperm maturation. PLoS One 8, e77466 (2013).

  56. 56.

    Gruner, H., Cortes-Lopez, M., Cooper, D. A., Bauer, M. & Miura, P. CircRNA accumulation in the aging mouse brain. Sci. Rep. 6, 38907 (2016).

  57. 57.

    Schuster, A. et al. SpermBase: a database for sperm-borne RNA contents. Biol. Reprod. 95, 99 (2016).

  58. 58.

    Yamauchi, Y. & Ward, M. A. Preservation of ejaculated mouse spermatozoa from fertile C57BL/6 and infertile Hook1/Hook1 mice collected from the uteri of mated females. Biol. Reprod. 76, 1002–1008 (2007).

  59. 59.

    Bao, J. et al. RAN-binding protein 9 is involved in alternative splicing and is critical for male germ cell development and male fertility. PLoS Genet. 10, e1004825 (2014).

  60. 60.

    Capel, B. et al. Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell 73, 1019–1030 (1993).

  61. 61.

    Jeck, W. R. & Sharpless, N. E. Detecting and characterizing circular RNAs. Nat. Biotechnol. 32, 453–461 (2014).

  62. 62.

    Panda, A. C. et al. High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs. Nucleic Acids Res. https://doi.org/10.1093/nar/gkx297 (2017).

  63. 63.

    Yang, J., Morales, C. R., Medvedev, S., Schultz, R. M. & Hecht, N. B. In the absence of the mouse DNA/RNA-binding protein MSY2, messenger RNA instability leads to spermatogenic arrest. Biol. Reprod. 76, 48–54 (2007).

  64. 64.

    Gou, L. T. et al. Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis. Cell Res. 24, 680–700 (2014).

  65. 65.

    Watanabe, T., Cheng, E. C., Zhong, M. & Lin, H. Retrotransposons and pseudogenes regulate mRNAs and lncRNAs via the piRNA pathway in the germline. Genome Res. 25, 368–380 (2015).

  66. 66.

    Liang, D. et al. The output of protein-coding genes shifts to circular RNAs when the pre-mRNA processing machinery is limiting. Mol. Cell 68, 940–954 e943 (2017).

  67. 67.

    Wojtas, M. N. et al. Regulation of m(6)A transcripts by the 3'–>5' RNA helicase YTHDC2 is essential for a successful meiotic program in the mammalian germline. Mol. Cell 68, 374–387 e312 (2017).

  68. 68.

    Hsu, P. J. et al. Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis. Cell Res. 27, 1115–1127 (2017).

  69. 69.

    Hsu, P. J. & He, C. High-resolution mapping of N (6)-methyladenosine using m(6)A crosslinking immunoprecipitation sequencing (m(6)A-CLIP-Seq. Methods Mol. Biol. 1870, 69–79, https://doi.org/10.1007/978-1-4939-8808-2_5 (2019).

  70. 70.

    Grozhik, A. V., Linder, B., Olarerin-George, A. O. & Jaffrey, S. R. Mapping m(6)A at individual-nucleotide resolution using crosslinking and immunoprecipitation (miCLIP). Methods Mol. Biol. 1562, 55–78 (2017).

  71. 71.

    Linder, B. et al. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12, 767–772 (2015).

  72. 72.

    Gur, Y. & Breitbart, H. Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes. Genes Dev. 20, 411–416 (2006).

  73. 73.

    Gur, Y. & Breitbart, H. Protein translation in mammalian sperm. Soc. Reprod. Fertil. Suppl. 65, 391–397 (2007).

  74. 74.

    Zhao, C. et al. Role of translation by mitochondrial-type ribosomes during sperm capacitation: an analysis based on a proteomic approach. Proteomics 9, 1385–1399 (2009).

  75. 75.

    Yan, W. et al. Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei. Biol. Reprod. 78, 896–902 (2008).

  76. 76.

    Ro, S., Park, C., Sanders, K. M., McCarrey, J. R. & Yan, W. Cloning and expression profiling of testis-expressed microRNAs. Dev. Biol. 311, 592–602 (2007).

  77. 77.

    Gao, Y., Wang, J. & Zhao, F. CIRI: an efficient and unbiased algorithm for de novo circular RNA identification. Genome Biol. 16, 4 (2015).

  78. 78.

    Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

  79. 79.

    Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

  80. 80.

    Vitting-Seerup, K., Porse, B. T., Sandelin, A. & Waage, J. spliceR: an R package for classification of alternative splicing and prediction of coding potential from RNA-seq data. BMC Bioinform. 15, 81 (2014).

  81. 81.

    Kong, L. et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res. 35, W345–W349 (2007).

  82. 82.

    Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

  83. 83.

    Zhao, B. S. et al. m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition. Nature 542, 475–478 (2017).

  84. 84.

    Ye, C. Y. et al. Full-length sequence assembly reveals circular RNAs with diverse non-GT/AG splicing signals in rice. RNA Biol. 14, 1055–1063 (2017).

  85. 85.

    Nicol, J. W., Helt, G. A., Blanchard, S. G. Jr., Raja, A. & Loraine, A. E. The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics 25, 2730–2731 (2009).

Download references

Acknowledgements

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Supplementary information, Figure S1

Supplementary information, Figure S2

Supplementary information, Figure S3

Supplementary information, Figure S4

Supplementary information, Figure S5

Supplementary information, Figure S6

Supplementary information, Figure S7

Supplementary information, Figure S8

Supplementary information, Figure S9

Supplementary information, Figure S10

Supplementary information, Figure S11

Supplementary information, Figure S12

Supplementary information, Figure S13

Supplementary information, Figure S14

Supplementary information, Table S1

Supplementary information, Table S2

Supplementary information, Table S3

Supplementary information, Table S4

Supplementary information, Table S5

Supplementary information, Table S6

Supplementary information, Table S7

Supplementary information, Table S8

Supplementary information, Table S9

Supplementary information, Table S10

Supplementary information, Table S11

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation