Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

A dicer-independent miRNA biogenesis pathway that requires Ago catalysis

Abstract

The nucleolytic activity of animal Argonaute proteins is deeply conserved, despite its having no obvious role in microRNA-directed gene regulation. In mice, Ago2 (also known as Eif2c2) is uniquely required for viability, and only this family member retains catalytic competence. To investigate the evolutionary pressure to conserve Argonaute enzymatic activity, we engineered a mouse with catalytically inactive Ago2 alleles. Homozygous mutants died shortly after birth with an obvious anaemia. Examination of microRNAs and their potential targets revealed a loss of miR-451, a small RNA important for erythropoiesis. Though this microRNA is processed by Drosha (also known as Rnasen), its maturation does not require Dicer. Instead, the pre-miRNA becomes loaded into Ago and is cleaved by the Ago catalytic centre to generate an intermediate 3′ end, which is then further trimmed. Our findings link the conservation of Argonaute catalysis to a conserved mechanism of microRNA biogenesis that is important for vertebrate development.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Ago2 is essential for extra-embryonic development
Figure 2: Ago2 catalysis is essential for development
Figure 3: Mature miR-451 expression depends on Ago2 catalysis
Figure 4: Non-canonical biogenesis of miR-451.
Figure 5: Ago2 catalysis is required for miR-451 biogenesis

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Sequencing data has been deposited in GEO and assigned accession number GSE21370.

References

  1. Hutvagner, G. & Simard, M. J. Argonaute proteins: key players in RNA silencing. Nature Rev. Mol. Cell Biol. 9, 22–32 (2008)

    Article  CAS  Google Scholar 

  2. Joshua-Tor, L. The Argonautes. Cold Spring Harb. Symp. Quant. Biol. 71, 67–72 (2006)

    Article  CAS  Google Scholar 

  3. Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001)

    Article  CAS  Google Scholar 

  4. Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W. & Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888 (2001)

    Article  CAS  Google Scholar 

  5. Yuan, Y. R. et al. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419 (2005)

    Article  CAS  Google Scholar 

  6. Martinez, J. & Tuschl, T. RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev. 18, 975–980 (2004)

    Article  CAS  Google Scholar 

  7. Schwarz, D. S., Tomari, Y. & Zamore, P. D. The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Curr. Biol. 14, 787–791 (2004)

    Article  CAS  Google Scholar 

  8. Malone, C. D. & Hannon, G. J. Small RNAs as guardians of the genome. Cell 136, 656–668 (2009)

    Article  CAS  Google Scholar 

  9. Yigit, E. et al. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell 127, 747–757 (2006)

    Article  CAS  Google Scholar 

  10. Bohmert, K. et al. AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17, 170–180 (1998)

    Article  CAS  Google Scholar 

  11. Baumberger, N. & Baulcombe, D. C. Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl Acad. Sci. USA 102, 11928–11933 (2005)

    Article  ADS  CAS  Google Scholar 

  12. Qi, Y., Denli, A. M. & Hannon, G. J. Biochemical specialization within Arabidopsis RNA silencing pathways. Mol. Cell 19, 421–428 (2005)

    Article  CAS  Google Scholar 

  13. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009)

    Article  CAS  Google Scholar 

  14. Yekta, S., Shih, I. H. & Bartel, D. P. MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596 (2004)

    Article  ADS  CAS  Google Scholar 

  15. Davis, E. et al. RNAi-mediated allelic trans-interaction at the imprinted Rtl1/Peg11 locus. Curr. Biol. 15, 743–749 (2005)

    Article  CAS  Google Scholar 

  16. Harfe, B. D., McManus, M. T., Mansfield, J. H., Hornstein, E. & Tabin, C. J. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb. Proc. Natl Acad. Sci. USA 102, 10898–10903 (2005)

    Article  ADS  CAS  Google Scholar 

  17. Sekita, Y. et al. Role of retrotransposon-derived imprinted gene, Rtl1, in the feto-maternal interface of mouse placenta. Nature Genet. 40, 243–248 (2008)

    Article  CAS  Google Scholar 

  18. Hornstein, E. et al. The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature 438, 671–674 (2005)

    Article  ADS  CAS  Google Scholar 

  19. Tolia, N. H. & Joshua-Tor, L. Slicer and the argonautes. Nature Chem. Biol. 3, 36–43 (2007)

    Article  ADS  CAS  Google Scholar 

  20. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004)

    Article  ADS  CAS  Google Scholar 

  21. Rivas, F. V. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nature Struct. Mol. Biol. 12, 340–349 (2005)

    Article  CAS  Google Scholar 

  22. Song, J. J., Smith, S. K., Hannon, G. J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004)

    Article  ADS  CAS  Google Scholar 

  23. Azuma-Mukai, A. et al. Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing. Proc. Natl Acad. Sci. USA 105, 7964–7969 (2008)

    Article  ADS  CAS  Google Scholar 

  24. Ender, C. et al. A human snoRNA with microRNA-like functions. Mol. Cell 32, 519–528 (2008)

    Article  CAS  Google Scholar 

  25. Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P. & Blelloch, R. Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev. 22, 2773–2785 (2008)

    Article  CAS  Google Scholar 

  26. Tam, O. H. et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534–538 (2008)

    Article  ADS  CAS  Google Scholar 

  27. Watanabe, T. et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543 (2008)

    Article  ADS  CAS  Google Scholar 

  28. Kaneda, M., Tang, F., O’Carroll, D., Lao, K. & Surani, M. A. Essential role for Argonaute2 protein in mouse oogenesis. Epigenetics Chromatin 2, 9 (2009)

    Article  Google Scholar 

  29. Ma, J. et al. MicroRNA activity is suppressed in mouse oocytes. Curr. Biol. 20, 265–270 (2010)

    Article  CAS  Google Scholar 

  30. Murchison, E. P. et al. Critical roles for Dicer in the female germline. Genes Dev. 21, 682–693 (2007)

    Article  CAS  Google Scholar 

  31. Suh, N. et al. MicroRNA function is globally suppressed in mouse oocytes and early embryos. Curr. Biol. 20, 271–277 (2010)

    Article  CAS  Google Scholar 

  32. Alisch, R. S., Jin, P., Epstein, M., Caspary, T. & Warren, S. T. Argonaute2 is essential for mammalian gastrulation and proper mesoderm formation. PLoS Genet. 3, e227 (2007)

    Article  Google Scholar 

  33. Morita, S. et al. One Argonaute family member, Eif2c2 (Ago2), is essential for development and appears not to be involved in DNA methylation. Genomics 89, 687–696 (2007)

    Article  CAS  Google Scholar 

  34. Sasaki, T., Shiohama, A., Minoshima, S. & Shimizu, N. Identification of eight members of the Argonaute family in the human genome. Genomics 82, 323–330 (2003)

    Article  CAS  Google Scholar 

  35. Rossant, J. & Cross, J. C. in Mouse development: Pattering, Morphogenesis and Organogenesis (eds Rossant, J. & Tam, P. P. L.) 155–180 (Academic Press, 2002)

    Book  Google Scholar 

  36. Rossant, J. & Cross, J. C. Placental development: lessons from mouse mutants. Nature Rev. Genet. 2, 538–548 (2001)

    Article  CAS  Google Scholar 

  37. Papapetrou, E. P., Korkola, J. E. & Sadelain, M. A genetic strategy for single and combinatorial analysis of miRNA function in mammalian hematopoietic stem cells. Stem Cells 28, 287–296 (2009)

    Google Scholar 

  38. Dore, L. C. et al. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc. Natl Acad. Sci. USA 105, 3333–3338 (2008)

    Article  ADS  CAS  Google Scholar 

  39. Kim, V. N., Han, J. & Siomi, M. C. Biogenesis of small RNAs in animals. Nature Rev. Mol. Cell Biol. 10, 126–139 (2009)

    Article  CAS  Google Scholar 

  40. Siolas, D. et al. Synthetic shRNAs as potent RNAi triggers. Nature Biotechnol. 23, 227–231 (2004)

    Article  Google Scholar 

  41. Chendrimada, T. P. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005)

    Article  ADS  CAS  Google Scholar 

  42. Wang, H. W. et al. Structural insights into RNA processing by the human RISC-loading complex. Nature Struct. Mol. Biol. 16, 1148–1153 (2009)

    Article  CAS  Google Scholar 

  43. Song, J. J. et al. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nature Struct. Biol. 10, 1026–1032 (2003)

    Article  CAS  Google Scholar 

  44. Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D. J. Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209–213 (2008)

    Article  ADS  CAS  Google Scholar 

  45. Diederichs, S. & Haber, D. A. Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression. Cell 131, 1097–1108 (2007)

    Article  CAS  Google Scholar 

  46. O’Carroll, D. et al. A Slicer-independent role for Argonaute 2 in hematopoiesis and the microRNA pathway. Genes Dev. 21, 1999–2004 (2007)

    Article  Google Scholar 

  47. Bandres, E. et al. microRNA-451 regulates macrophage migration inhibitory factor production and proliferation of gastrointestinal cancer cells. Clin. Cancer Res. 15, 2281–2290 (2009)

    Article  CAS  Google Scholar 

  48. Pfeffer, S. et al. Identification of microRNAs of the herpesvirus family. Nature Methods 2, 269–276 (2005)

    Article  CAS  Google Scholar 

  49. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003)

    Article  ADS  CAS  Google Scholar 

  50. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo: A Laboratory Manual 3rd edn (Cold Spring Harbor Press, 2003)

    Google Scholar 

  51. Nagy, A. & Rossant, J. in Gene Targeting: A Practical Approach 2nd edn (ed. Joyner, A. L.) 189–192 (Oxford Univ. Press, 2000)

    Google Scholar 

  52. Socolovsky, M. et al. Ineffective erythropoiesis in Stat5a-/-5b-/- mice due to decreased survival of early erythroblasts. Blood 98, 3261–3273 (2001)

    Article  CAS  Google Scholar 

  53. Liu, Y. et al. Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo . Blood 108, 123–133 (2006)

    Article  CAS  Google Scholar 

  54. Aravin, A. & Tuschl, T. Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett. 579, 5830–5840 (2005)

    Article  CAS  Google Scholar 

  55. Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F. & Hannon, G. J. Processing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235 (2004)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Mosquera, S. Y. Kim, D. Frendewey and A. Economides for help with generating mutants and caring for animals. A. Nagy, M. Shen, J. Murn, V. Vagin and A. Aravin provided helpful discussion. N. Kim, D. Littman, I. Ibarra and E. Wagenblast provided critical reagents, and O. Tam, R. Sachidanandam, Z. Xuan, D. McCombie and M. Rooks provided support for generation and analysis of deep sequencing data. This work was supported by grants from the NIH and by a gift from K. W. Davis.

Author Contributions S.C. and G.J.H. planned experiments, interpreted data and wrote the paper. S.C. and C.O.D.S. performed studies, and M.M.W.C. provided critical unpublished reagents and discussion.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Gregory J. Hannon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1 and Supplementary Figures S1-S8 with legends. (PDF 6132 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cheloufi, S., Dos Santos, C., Chong, M. et al. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584–589 (2010). https://doi.org/10.1038/nature09092

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09092

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing