DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs

A Retraction to this article was published on 31 October 2014

Abstract

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences1,2,3,4,5,6. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs7. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits8,9,10 in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68−/− and p72−/− embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72−/− MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.

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Figure 1: Embryonic and neonatal lethality of p68 and p72 knockout mice.
Figure 2: The role of RNA helicases p72 and p68 in miRNA or rRNA processing.
Figure 3: In vivo association of the Drosha complex with pri-miRNAs through p68–p72.
Figure 4: Biochemical isolation of mouse Drosha-containing complex.
Figure 5: Purified mDrosha complex mediates processing of miRNA and rRNA in vitro.

References

  1. 1

    Carthew, R. W. Gene regulation by microRNAs. Curr. Opin. Genet. Dev. 16, 203–208 (2006).

    CAS  Article  Google Scholar 

  2. 2

    Cullen, B. R. Transcription and processing of human microRNA precursors. Mol. Cell 16, 861–865 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Filipowicz, W., Jaskiewicz, L., Kolb, F. A. & Pillai, R. S. Post-transcriptional gene silencing by siRNAs and miRNAs. Curr. Opin. Struct. Biol. 15, 331–341 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Kim, V. N. MicroRNA biogenesis: coordinated cropping and dicing. Nature Rev. Mol. Cell Biol. 6, 376–385 (2005).

    CAS  Article  Google Scholar 

  5. 5

    Valencia-Sanchez, M. A., Liu, J., Hannon, G. J. & Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Zamore, P. D. & Haley, B. Ribo-gnome: the big world of small RNAs. Science 309, 1519–1524 (2005).

    CAS  Article  Google Scholar 

  7. 7

    Gregory, R. I. et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Hirling, H., Scheffner, M., Restle, T. & Stahl, H. RNA helicase activity associated with the human p68 protein. Nature 339, 562–564 (1989).

    CAS  Article  Google Scholar 

  9. 9

    Huang, Y. & Liu, Z. R. The ATPase, RNA unwinding, and RNA binding activities of recombinant p68 RNA helicase. J. Biol. Chem. 277, 12810–12815 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Lamm, G. M., Nicol, S. M., Fuller-Pace, F. V. & Lamond, A. I. p72: a human nuclear DEAD box protein highly related to p68. Nucleic Acids Res. 24, 3739–3747 (1996).

    CAS  Article  Google Scholar 

  11. 11

    Auboeuf, D., Honig, A., Berget, S. M. & O'Malley, B. W. Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science 298, 416–419 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Bates, G. J. et al. The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor. EMBO J. 24, 543–553 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Causevic, M. et al. Overexpression and poly-ubiquitylation of the DEAD-box RNA helicase p68 in colorectal tumours. Oncogene 20, 7734–7743 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Dubey, P. et al. The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68. J. Exp. Med. 185, 695–705 (1997).

    CAS  Article  Google Scholar 

  15. 15

    Endoh, H. et al. Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor alpha. Mol. Cell Biol. 19, 5363–5372 (1999).

    CAS  Article  Google Scholar 

  16. 16

    Kircher, S. G., Kim, S. H., Fountoulakis, M. & Lubec, G. Reduced levels of DEAD-box proteins DBP-RB and p72 in fetal Down syndrome brains. Neurochem. Res. 27, 1141–1146 (2002).

    CAS  Article  Google Scholar 

  17. 17

    Watanabe, M. et al. A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor alpha coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA. EMBO J. 20, 1341–1352 (2001).

    CAS  Article  Google Scholar 

  18. 18

    Boehm, M. & Slack, F. A developmental timing microRNA and its target regulate life span in C. elegans. Science 310, 1954–1957 (2005).

    CAS  Article  Google Scholar 

  19. 19

    Yang, W. J. et al. Dicer is required for embryonic angiogenesis during mouse development. J. Biol. Chem. 280, 9330–9335 (2005).

    CAS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

    Wu, H., Xu, H., Miraglia, L. J. & Crooke, S. T. Human RNase III is a 160-kDa protein involved in preribosomal RNA processing. J. Biol. Chem. 275, 36957–36965 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Han, J. et al. The Drosha–DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Ni, J. Q., Liu, L. P., Hess, D., Rietdorf, J. & Sun, F. L. Drosophila ribosomal proteins are associated with linker histone H1 and suppress gene transcription. Genes Dev. 20, 1959–1973 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Kitagawa, H. et al. The chromatin-remodeling complex WINAC targets a nuclear receptor to promoters and is impaired in Williams syndrome. Cell 113, 905–917 (2003).

    CAS  Article  Google Scholar 

  25. 25

    Yanagisawa, J. et al. Nuclear receptor function requires a TFTC-type histone acetyl transferase complex. Mol. Cell 9, 553–562 (2002).

    CAS  Article  Google Scholar 

  26. 26

    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).

    CAS  Article  Google Scholar 

  27. 27

    Ogilvie, V. C. et al. The highly related DEAD box RNA helicases p68 and p72 exist as heterodimers in cells. Nucleic Acids Res. 31, 1470–1480 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Bernstein, E. et al. Dicer is essential for mouse development. Nature Genet. 35, 215–217 (2003).

    CAS  Article  Google Scholar 

  29. 29

    Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Otte, A. P. & Kwaks, T. H. Gene repression by Polycomb group protein complexes: a distinct complex for every occasion? Curr. Opin. Genet. Dev. 13, 448–454 (2003).

    CAS  Article  Google Scholar 

  31. 31

    Perissi, V. & Rosenfeld, M. G. Controlling nuclear receptors: the circular logic of cofactor cycles. Nature Rev. Mol. Cell Biol. 6, 542–554 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Iggo, R. D. & Lane, D. P. Nuclear protein p68 is an RNA-dependent ATPase. EMBO J. 8, 1827–1831 (1989).

    CAS  Article  Google Scholar 

  33. 33

    Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha–DGCR8 complex. Cell 125, 887–901 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Fujiki, R. et al. Ligand-induced transrepression by VDR through association of WSTF with acetylated histones. EMBO J 24, 3881–3894 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Ohtake, F. et al. Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature 423, 545–550 (2003).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank T. Watanabe, H. Shiina, J. Miyamoto, K. Sekine, R. Fujiki, Y. Imai and S. Tanaka for generation of knockout mice, and H. Higuchi for manuscript preparation. This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and priority areas from the Ministry of Education, Culture, Sports, Science and Technology (to S.K.).

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The experimental concept was developed by S.K., B.W.O.M, S.T., K.T., H.E. and H.K. T.F., K.Y. and S.F. conducted most of experiments, and I.K., K.Y., M.M., M.N., T.N., C.A., Y.Y., T.K., C.F. and S.T. contributed materials and supported several experiments. S.K., T.F., K.Y. and S.F. wrote the manuscript.

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Correspondence to Shigeaki Kato.

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The authors declare no competing financial interests.

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Supplementary Figure S1, S2, S3, S4 and S5 (PDF 765 kb)

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Fukuda, T., Yamagata, K., Fujiyama, S. et al. DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs. Nat Cell Biol 9, 604–611 (2007). https://doi.org/10.1038/ncb1577

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