Abstract
MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level1. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex2,3,4. These pre-miRNAs are cleaved by the RNase III Dicer5,6,7,8 to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence9. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein10), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer–TRBP with Argonaute 2 (Ago2)11,12, the catalytic engine of RISC. The physical association of Dicer–TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer–TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer–TRBP complex not only in miRNA processing but also as a platform for RISC assembly.
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References
Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004)
Gregory, R. I. et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004)
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)
Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004)
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)
Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34 (2001)
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)
Ketting, R. F. et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15, 2654–2659 (2001)
Sontheimer, E. J. Assembly and function of RNA silencing complexes. Nature Rev. Mol. Cell Biol. 6, 127–138 (2005)
Gatignol, A., Buckler-White, A., Berkhout, B. & Jeang, K. T. Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science 251, 1597–1600 (1991)
Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004)
Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004)
Zhang, H., Kolb, F. A., Jaskiewicz, L., Westhof, E. & Filipowicz, W. Single processing center models for human Dicer and bacterial RNase III. Cell 118, 57–68 (2004)
Liu, Q. et al. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301, 1921–1925 (2003)
Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P. D. A protein sensor for siRNA asymmetry. Science 306, 1377–1380 (2004)
Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004)
Doi, N. et al. Short-interfering-RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors. Curr. Biol. 13, 41–46 (2003)
Zhong, J., Peters, A. H., Lee, K. & Braun, R. E. A double-stranded RNA binding protein required for activation of repressed messages in mammalian germ cells. Nature Genet. 22, 171–174 (1999)
Bernstein, E. et al. Dicer is essential for mouse development. Nature Genet. 35, 215–217 (2003)
Acknowledgements
We thank T. Beer for assistance with the microcapillary HPLC/mass spectrometry. R.S. was supported by a grant from NIH and American Cancer Society. R.I.G. is a fellow of the Jane Coffin Childs Fund for Medical Research.
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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
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Chendrimada, T., Gregory, R., Kumaraswamy, E. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005). https://doi.org/10.1038/nature03868
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DOI: https://doi.org/10.1038/nature03868
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