Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Circular RNA circZbtb20 maintains ILC3 homeostasis and function via Alkbh5-dependent m6A demethylation of Nr4a1 mRNA


Group 3 innate lymphoid cells (ILC3s) play critical roles in innate immunity and gut homeostasis. However, how ILC3 homeostasis is regulated remains elusive. Here, we identified a novel circular RNA, circZbtb20, that is highly expressed in ILC3s and required for their maintenance and function. CircZbtb20 deletion causes reduced ILC3 numbers, increasing susceptibility to C. rodentium infection. Mechanistically, circZbtb20 enhances the interaction of Alkbh5 with Nr4a1 mRNA, leading to ablation of the m6A modification of Nr4a1 mRNA to promote its stability. Nr4a1 initiates Notch2 signaling activation, which contributes to the maintenance of ILC3 homeostasis. Deletion of Alkbh5 or Nr4a1 also impairs ILC3 homeostasis and increases susceptibilities to bacterial infection. Thus, our findings reveal an important role of circular RNA in the regulation of innate lymphoid cell homeostasis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    Eberl, G., Colonna, M., Di Santo, J. P. & McKenzie, A. N. Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology. Science 348, aaa6566 (2015).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Vivier, E. et al. Innate lymphoid cells : 10 years on. Cell 174, 1054–1066 (2018).

    CAS  PubMed  Google Scholar 

  3. 3.

    Serafini, N., Vosshenrich, C. A. & Di Santo, J. P. Transcriptional regulation of innate lymphoid cell fate. Nat. Rev. Immunol. 15, 415–428 (2015).

    CAS  PubMed  Google Scholar 

  4. 4.

    Artis, D. & Spits, H. The biology of innate lymphoid cells. Nature 517, 293–301 (2015).

    CAS  PubMed  Google Scholar 

  5. 5.

    Qiu, J. et al. The aryl hydrocarbon receptor regulates gut immunity through modulation of innate lymphoid cells. Immunity 36, 92–104 (2012).

    CAS  PubMed  Google Scholar 

  6. 6.

    Xia, P. et al. WASH maintains NKp46(+) ILC3 cells by promoting AHR expression. Nat. Commun. 8, 15685 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Liu, B. et al. Long noncoding RNA lncKdm2b is required for ILC3 maintenance by initiation of Zfp292 expression. Nat. Immunol. 18, 499–508 (2017).

    CAS  PubMed  Google Scholar 

  8. 8.

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

    CAS  PubMed  Google Scholar 

  9. 9.

    Zhang, X. O. et al. Complementary sequence-mediated exon circularization. Cell 159, 134–147 (2014).

    CAS  PubMed  Google Scholar 

  10. 10.

    Xia, P. et al. A circular RNA protects dormant hematopoietic stem cells from DNA sensor cGAS-mediated exhaustion. Immunity 48, 688–701.e687 (2018).

    CAS  PubMed  Google Scholar 

  11. 11.

    Guarnerio, J. et al. Oncogenic role of fusion-circRNAs derived from cancer-associated chromosomal translocations. Cell 165, 289–302 (2016).

    CAS  PubMed  Google Scholar 

  12. 12.

    Piwecka, M. et al. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function. Science 357, eaam8526 (2017).

    PubMed  Google Scholar 

  13. 13.

    Liu, C. X. et al. Structure and degradation of circular RNAs regulate PKR activation in innate immunity. Cell 177, 865–880.e821 (2019).

    CAS  PubMed  Google Scholar 

  14. 14.

    Gury-BenAri, M. et al. The spectrum and regulatory landscape of intestinal innate lymphoid cells are shaped by the microbiome. Cell 166, 1231–1246.e1213 (2016).

    CAS  PubMed  Google Scholar 

  15. 15.

    Shaked, I. et al. Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation. Nat. Immunol. 16, 1228–1234 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Mullican, S. E. et al. Abrogation of nuclear receptors Nr4a3 and Nr4a1 leads to development of acute myeloid leukemia. Nat. Med. 13, 730–735 (2007).

    CAS  PubMed  Google Scholar 

  17. 17.

    Liu, X. D. et al. Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction. Nature 567, 525–529 (2019).

  18. 18.

    Hanna, R. N. et al. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C-monocytes. Nat. Immunol. 12, 778–785 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Sekiya, T. et al. Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis. Nat. Immunol. 14, 230–237 (2013).

    CAS  PubMed  Google Scholar 

  20. 20.

    Hanna, R. N. et al. NR4A1 (Nur77) deletion polarizes macrophages toward an inflammatory phenotype and increases atherosclerosis. Circ. Res. 110, 416–427 (2012).

    CAS  PubMed  Google Scholar 

  21. 21.

    Liu, B. et al. An inducible circular RNA circKcnt2 inhibits ILC3 activation to facilitate colitis resolution. Nat. Commun. 11, 4076 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Li, X. et al. Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection. Mol. Cell 67, 214–227.e217 (2017).

    CAS  PubMed  Google Scholar 

  23. 23.

    Conn, S. J. et al. The RNA binding protein quaking regulates formation of circRNAs. Cell 160, 1125–1134 (2015).

    CAS  PubMed  Google Scholar 

  24. 24.

    Errichelli, L. et al. FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons. Nat. Commun. 8, 14741 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Diefenbach, A., Colonna, M. & Koyasu, S. Development, differentiation, and diversity of innate lymphoid cells. Immunity 41, 354–365 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Zheng, Y. et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat. Med. 14, 282–289 (2008).

    CAS  PubMed  Google Scholar 

  27. 27.

    Zhu, P. et al. IL-13 secreted by ILC2s promotes the self-renewal of intestinal stem cells through circular RNA circPan3. Nat. Immunol. 20, 183–194 (2019).

    CAS  PubMed  Google Scholar 

  28. 28.

    Zhang, S. et al. m(6)A demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation program. Cancer Cell 31, 591–606.e596 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Fu, Y., Dominissini, D., Rechavi, G. & He, C. Gene expression regulation mediated through reversible m(6)A RNA methylation. Nat. Rev. Genet. 15, 293–306 (2014).

    CAS  PubMed  Google Scholar 

  30. 30.

    Xiao, Y. et al. An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N(6)-methyladenosine modification. Angew. Chem. 57, 15995–16000 (2018).

    CAS  Google Scholar 

  31. 31.

    Frye, M., Harada, B. T., Behm, M. & He, C. RNA modifications modulate gene expression during development. Science 361, 1346–1349 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Wang, X. et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

    PubMed  Google Scholar 

  33. 33.

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

    CAS  PubMed  Google Scholar 

  34. 34.

    Yang, Q. et al. TCF-1 upregulation identifies early innate lymphoid progenitors in the bone marrow. Nat. Immunol. 16, 1044–1050 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Klose, C. S. N. et al. Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 157, 340–356 (2014).

    CAS  PubMed  Google Scholar 

  36. 36.

    Constantinides, M. G., McDonald, B. D., Verhoef, P. A. & Bendelac, A. A committed precursor to innate lymphoid cells. Nature 508, 397–401 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Guo, X. et al. Innate lymphoid cells control early colonization resistance against intestinal pathogens through ID2-dependent regulation of the microbiota. Immunity 42, 731–743 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Longman, R. S. et al. CX(3)CR1(+) mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22. J. Exp. Med. 211, 1571–1583 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Castellanos, J. G. et al. Microbiota-induced TNF-like ligand 1A drives group 3 innate lymphoid cell-mediated barrier protection and intestinal T cell activation during colitis. Immunity 49, 1077–1089.e1075 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Bauche, D. et al. LAG3(+) regulatory T cells restrain interleukin-23-producing CX3CR1(+) gut-resident macrophages during group 3 innate lymphoid cell-driven colitis. Immunity 49, 342–352.e345 (2018).

    CAS  PubMed  Google Scholar 

  41. 41.

    Lee, J. S. et al. AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch. Nat. Immunol. 13, 144–151 (2011).

    PubMed  PubMed Central  Google Scholar 

  42. 42.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Li, Z. Y. et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nat. Struct. Mol. Biol. 22, 256–264 (2015).

    PubMed  Google Scholar 

  44. 44.

    Li, Q. et al. CircACC1 regulates assembly and activation of AMPK complex under metabolic stress. Cell Metab. 30, 157–173.e157 (2019).

    PubMed  Google Scholar 

  45. 45.

    Liu, G. et al. Regulation of hepatic lipogenesis by the zinc finger protein Zbtb20. Nat. Commun. 8, 14824 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Fahrner, T. J., Carroll, S. L. & Milbrandt, J. The Ngfi-B protein, an inducible member of the thyroid steroid-receptor family, is rapidly modified posttranslationally. Mol. Cell. Biol. 10, 6454–6459 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Seehus, C. R. et al. The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor. Nat. Immunol. 16, 599–608 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Rankin, L. C. et al. The transcription factor T-bet is essential for the development of NKp46+ innate lymphocytes via the Notch pathway. Nat. Immunol. 14, 389–395 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Mielke, L. A. et al. TCF-1 controls ILC2 and NKp46+RORgammat+ innate lymphocyte differentiation and protection in intestinal inflammation. J. Immunol. 191, 4383–4391 (2013).

    CAS  PubMed  Google Scholar 

  50. 50.

    Wang, L. M. et al. Nr4a1 plays a crucial modulatory role in Th1/Th17 cell responses and CNS autoimmunity. Brain. Behav. Immun. 68, 44–55 (2018).

    CAS  PubMed  Google Scholar 

  51. 51.

    Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

    CAS  PubMed  Google Scholar 

  52. 52.

    Liu, J. et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93–95 (2014).

    CAS  PubMed  Google Scholar 

  53. 53.

    Wang, X. et al. N(6)-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Molinie, B. et al. m(6)A-LAIC-seq reveals the census and complexity of the m(6)A epitranscriptome. Nat. Methods 13, 692–698 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Zheng, Q., Hou, J., Zhou, Y., Li, Z. & Cao, X. The RNA helicase DDX46 inhibits innate immunity by entrapping m(6)A-demethylated antiviral transcripts in the nucleus. Nat. Immunol. 18, 1094–1103 (2017).

    CAS  PubMed  Google Scholar 

  56. 56.

    Winkler, R. et al. m(6)A modification controls the innate immune response to infection by targeting type I interferons. Nat. Immunol. 20, 173–182 (2019).

    CAS  PubMed  Google Scholar 

  57. 57.

    Li, H. B. et al. m(6)A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways. Nature 548, 338–342 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Zhu, X. et al. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci. Rep. 4, 6420 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Liu, B. et al. IL-7Ralpha glutamylation and activation of transcription factor Sall3 promote group 3 ILC development. Nat. Commun. 8, 231 (2017).

    PubMed  PubMed Central  Google Scholar 

  60. 60.

    Liu, B. et al. Yeats4 drives ILC lineage commitment via activation of Lmo4 transcription. J. Exp. Med. 216, 2653–2668 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank Shu Meng, Dongdong Fan, Yan Teng, Junying Jia, and Xiang Shi for technical support. We also thank Jing Li (Cnkingbio Company, Ltd., Beijing, China) for technical support. This work was supported by the Ministry of Science and Technology of China (2020YFA0803501 and 2019YFA0508501), the National Natural Science Foundation of China (31930036, 81921003, 92042302, 31870883, 91940305, 31728006, 81772646, and 31871494), the Strategic Priority Research Programs of the Chinese Academy of Sciences (XDB19030203), the Beijing Natural Science Foundation (5192018), the Biological Resource Program of the Chinese Academy of Science (KFJ-BRP-017-04), and the Young Elite Scientist Sponsorship Program of CAST (2018QNRC001).

Author information




B.L. and N.L. performed experiments; B.L. designed the project, analyzed the data, and wrote the paper; and X.Z. constructed genetic mouse strains. L.Y., B.Y., H.L., P.Z., and T.L. analyzed data; Y.T. initiated the study and analyzed data; and Z.F. initiated the study and organized, designed, and wrote the paper.

Corresponding authors

Correspondence to Benyu Liu or Yong Tian or Zusen Fan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, B., Liu, N., Zhu, X. et al. Circular RNA circZbtb20 maintains ILC3 homeostasis and function via Alkbh5-dependent m6A demethylation of Nr4a1 mRNA. Cell Mol Immunol 18, 1412–1424 (2021).

Download citation


  • circZbtb20
  • ILC3
  • Alkbh5
  • Nr4a1
  • Homeostasis


Quick links