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DGCR8 HITS-CLIP reveals novel functions for the Microprocessor


The Drosha–DGCR8 complex (Microprocessor) is required for microRNA (miRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as the endonuclease. Using high-throughput sequencing and cross-linking immunoprecipitation (HITS-CLIP) we identified RNA targets of DGCR8 in human cells. Unexpectedly, miRNAs were not the most abundant targets. DGCR8-bound RNAs also comprised several hundred mRNAs as well as small nucleolar RNAs (snoRNAs) and long noncoding RNAs. We found that the Microprocessor controlled the abundance of several mRNAs as well as of MALAT1. By contrast, DGCR8-mediated cleavage of snoRNAs was independent of Drosha, suggesting the involvement of DGCR8 in cellular complexes with other endonucleases. Binding of DGCR8 to cassette exons is a new mechanism for regulation of the relative abundance of alternatively spliced isoforms. These data provide insights in the complex role of DGCR8 in controlling the fate of several classes of RNAs.

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Figure 1: CLIP for endogenous and overexpressed DGCR8 in HEK 293T cells.
Figure 2: DGCR8 binds and controls the stability of the long intergenic noncoding RNA MALAT1.
Figure 3: Binding of DGCR8 to snoRNAs affects their abundance independently of Drosha.
Figure 4: Microprocessor binding to mRNAs.
Figure 5: Binding of DGCR8 to cassette exons can modulate the relative abundance of alternatively spliced isoforms.

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  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

    Zeng, Y., Yi, R. & Cullen, B.R. Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. EMBO J. 24, 138–148 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Morlando, M. et al. Primary microRNA transcripts are processed co-transcriptionally. Nat. Struct. Mol. Biol. 15, 902–909 (2008).

    CAS  Article  Google Scholar 

  5. 5

    Pawlicki, J.M. & Steitz, J.A. Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production. J. Cell Biol. 182, 61–76 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Yi, R., Qin, Y., Macara, I.G. & Cullen, B.R. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011–3016 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Lund, E., Guttinger, S., Calado, A., Dahlberg, J.E. & Kutay, U. Nuclear export of microRNA precursors. Science 303, 95–98 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Bohnsack, M.T., Czaplinski, K. & Gorlich, D. Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA 10, 185–191 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Bernstein, E., Caudy, A.A., Hammond, S.M. & Hannon, G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).

    CAS  Article  Google Scholar 

  10. 10

    Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Shiohama, A., Sasaki, T., Noda, S., Minoshima, S. & Shimizu, N. Molecular cloning and expression analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region. Biochem. Biophys. Res. Commun. 304, 184–190 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Landthaler, M., Yalcin, A. & Tuschl, T. The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14, 2162–2167 (2004).

    CAS  Article  Google Scholar 

  13. 13

    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 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

    Davis, B.N. & Hata, A. Regulation of microRNA biogenesis: a miRiad of mechanisms. Cell Commun. Signal. 7, 18 (2009).

    Article  Google Scholar 

  16. 16

    Winter, J., Jung, S., Keller, S., Gregory, R.I. & Diederichs, S. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat. Cell Biol. 11, 228–234 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Krol, J., Loedige, I. & Filipowicz, W. The widespread regulation of microRNA biogenesis, function and decay. Nat. Rev. Genet. 11, 597–610 (2010).

    CAS  Article  Google Scholar 

  18. 18

    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 

  19. 19

    Faller, M., Matsunaga, M., Yin, S., Loo, J.A. & Guo, F. Heme is involved in microRNA processing. Nat. Struct. Mol. Biol. 14, 23–29 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Faller, M. et al. DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures. RNA 16, 1570–1583 (2010).

    CAS  Article  Google Scholar 

  21. 21

    Licatalosi, D.D. et al. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464–469 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Sanford, J.R. et al. Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts. Genome Res. 19, 381–394 (2009).

    CAS  Article  Google Scholar 

  23. 23

    Chi, S.W., Zang, J.B., Mele, A. & Darnell, R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479–486 (2009).

    CAS  Article  Google Scholar 

  24. 24

    Zisoulis, D.G. et al. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 17, 173–179 (2010).

    CAS  Article  Google Scholar 

  25. 25

    Konig, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).

    Article  Google Scholar 

  26. 26

    Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

    CAS  Article  Google Scholar 

  27. 27

    Han, J. et al. Posttranscriptional crossregulation between Drosha and DGCR8. Cell 136, 75–84 (2009).

    CAS  Article  Google Scholar 

  28. 28

    Kadener, S. et al. Genome-wide identification of targets of the drosha-pasha/DGCR8 complex. RNA 15, 537–545 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Triboulet, R., Chang, H.M., LaPierre, R.J. & Gregory, R.I. Post-transcriptional control of DGCR8 expression by the Microprocessor. RNA 15, 1005–1011 (2009).

    CAS  Article  Google Scholar 

  30. 30

    Ule, J., Jensen, K., Mele, A. & Darnell, R.B. CLIP: A method for identifying protein-RNA interaction sites in living cells. Methods 37, 376–386 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Licatalosi, D.D. & Darnell, R.B. RNA processing and its regulation: global insights into biological networks. Nat. Rev. Genet. 11, 75–87 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Karginov, F.V. et al. Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases. Mol. Cell 38, 781–788 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Ji, P. et al. MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer. Oncogene 22, 8031–8041 (2003).

    Article  Google Scholar 

  34. 34

    Tollervey, D. & Kiss, T. Function and synthesis of small nucleolar RNAs. Curr. Opin. Cell Biol. 9, 337–342 (1997).

    CAS  Article  Google Scholar 

  35. 35

    Kiss, T. SnoRNP biogenesis meets pre-mRNA splicing. Mol. Cell 23, 775–776 (2006).

    CAS  Article  Google Scholar 

  36. 36

    Taft, R.J. et al. Small RNAs derived from snoRNAs. RNA 15, 1233–1240 (2009).

    CAS  Article  Google Scholar 

  37. 37

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

    CAS  Article  Google Scholar 

  38. 38

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

    CAS  Article  Google Scholar 

  39. 39

    Wang, Y., Medvid, R., Melton, C., Jaenisch, R. & Blelloch, R. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat. Genet. 39, 380–385 (2007).

    CAS  Article  Google Scholar 

  40. 40

    Shenoy, A. & Blelloch, R. Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8. PLoS ONE 4, e6971 (2009).

    Article  Google Scholar 

  41. 41

    Chong, M.M. et al. Canonical and alternate functions of the microRNA biogenesis machinery. Genes Dev. 24, 1951–1960 (2010).

    CAS  Article  Google Scholar 

  42. 42

    Lin, Y.T. & Sullivan, C.S. Expanding the role of Drosha to the regulation of viral gene expression. Proc. Natl. Acad. Sci. USA 108, 11229–11234 (2011).

    CAS  Article  Google Scholar 

  43. 43

    Wu, H., Xu, H., Miraaglia, 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 

  44. 44

    Liang, T.J. & Qin, C.Y. The emerging role of microRNAs in immune cell development and differentiation. APMIS 117, 635–643 (2009).

    CAS  Article  Google Scholar 

  45. 45

    Scott, M.S., Avolio, F., Ono, M., Lamond, A.I. & Barton, G.J. Human miRNA precursors with box H/ACA snoRNA features. PLoS Comput. Biol. 5, e1000507 (2009).

    Article  Google Scholar 

  46. 46

    Shiohama, A., Sasaki, T., Noda, S., Minoshima, S. & Shimizu, N. Nucleolar localization of DGCR8 and identification of eleven DGCR8-associated proteins. Exp. Cell Res. 313, 4196–4207 (2007).

    CAS  Article  Google Scholar 

  47. 47

    Stark, K.L. et al. Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model. Nat. Genet. 40, 751–760 (2008).

    CAS  Article  Google Scholar 

  48. 48

    Fenelon, K. et al. Deficiency of Dgcr8, a gene disrupted by the 22q11.2 microdeletion, results in altered short-term plasticity in the prefrontal cortex. Proc. Natl. Acad. Sci. USA 108, 4447–4452 (2011).

    CAS  Article  Google Scholar 

  49. 49

    Michlewski, G. & Caceres, J.F. Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis. Nat. Struct. Mol. Biol. 17, 1011–1018 (2010).

    CAS  Article  Google Scholar 

  50. 50

    Caceres, J.F., Misteli, T., Screaton, G.R., Spector, D.L. & Krainer, A.R. Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity. J. Cell Biol. 138, 225–238 (1997).

    CAS  Article  Google Scholar 

  51. 51

    Guil, S. & Caceres, J.F. The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a. Nat. Struct. Mol. Biol. 14, 591–596 (2007).

    CAS  Article  Google Scholar 

  52. 52

    Flicek, P. et al. Ensembl's 10th year. Nucleic Acids Res. 38, D557–D562 (2010).

    CAS  Article  Google Scholar 

  53. 53

    Slater, G.S. & Birney, E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 6, 31 (2005).

    Article  Google Scholar 

  54. 54

    Fujita, P.A. et al. The UCSC Genome Browser database: update 2011. Nucleic Acids Res. 39, D876–D882 (2011).

    CAS  Article  Google Scholar 

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We are grateful to S. Heras for discussions and critical reading of the manuscript; N. Kim (Seoul National University) for Flag-Drosha, dominant negative DGCR8 and Drosha expression vectors; B. Seraphin (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg) for the Dcp1 antibody and R. Blelloch (University of California, San Francisco) for Dicer knockout and Dicer flox/flox cell lines. This work was supported by the Medical Research Council and by the Wellcome Trust (084057/Z/07/Z to J.F.C.; A.S., G.M. and S.M. were partially funded by this). E.E. and M.P. were supported by grants from the Spanish Ministry of Science and by the Sandra Ibarra Foundation (BIO2008-01091, BIO2011-23920 and CSD2009-00080). S.M. was the recipient of a European Molecular Biology Organization long-term postdoctoral fellowship. M.P. is supported by the Novo Nordisk Foundation. J.F.C is a recipient of a Wellcome Trust Senior Investigator Award (grant 095518/Z/11/Z).

Author information




S.M. and J.F.C. conceived, designed and interpreted the experiments. S.M., G.M. and A.S. performed experiments and analyzed data. M.P. and E.E. performed all bioinformatics analysis, including mapping of the CLIP tags to the genome and statistical analysis. J.F.C. supervised the project. All authors wrote the manuscript.

Corresponding author

Correspondence to Javier F Cáceres.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1,3 and Supplementary Note (PDF 3023 kb)

Supplementary Table 2

List of alternative splicing changes in Dgcr8 knockout mouse embryonic stem cells. (XLS 1187 kb)

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Macias, S., Plass, M., Stajuda, A. et al. DGCR8 HITS-CLIP reveals novel functions for the Microprocessor. Nat Struct Mol Biol 19, 760–766 (2012).

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