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The Microprocessor complex mediates the genesis of microRNAs

Abstract

MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory genes that regulate the expression of homologous target-gene transcripts. They have been implicated in the control of cell death and proliferation in flies1,2, haematopoietic lineage differentiation in mammals3, neuronal patterning in nematodes4 and leaf and flower development in plants5,6,7,8. miRNAs are processed by the RNA-mediated interference machinery. Drosha is an RNase III enzyme that was recently implicated in miRNA processing. Here we show that human Drosha is a component of two multi-protein complexes. The larger complex contains multiple classes of RNA-associated proteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha and the double-stranded-RNA-binding protein, DGCR8, the product of a gene deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution studies revealed that both components of this smaller complex, termed Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs from the primary miRNA transcript.

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Figure 1: Isolation of Drosha-containing complexes.
Figure 2: Composition and miRNA processing activity of Superose 6 gel-filtration fractions.
Figure 3: Microprocessor is the Drosha–DGCR8 complex mediating miRNA processing.
Figure 4: Reconstitution of Microprocessor by using recombinant Drosha and DGCR8.
Figure 5: A role for Microprocessor in vivo in miRNA processing.

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Acknowledgements

We thank V. N. Kim for the Drosha cDNA; O. Delattre and A. I. Lamond for the gift of EWS and DDX17 antibodies, respectively; K. Nishikura for providing recombinant Drosha; and T. Beer (Wistar Proteomocs Facility) for expertise in the microcapillary HPLC/mass spectrometry. R.S. was supported by grants from the NIH and the American Cancer Institute. R.G. is a fellow of the Jane Coffin Child Memorial Fund for Medical Research.

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Correspondence to Ramin Shiekhattar.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

Diagrammatic depiction of Drosha-associated proteins and their domain structures. (PDF 47 kb)

Supplementary Figure 2

The bulk of miRNA processing activity resides in Drosha/DGCR8 complex. (PDF 281 kb)

Supplementary Figure 3

Analysis of miRNA precursors. (PDF 425 kb)

Supplementary Figure Legends (DOC 20 kb)

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Gregory, R., Yan, Kp., Amuthan, G. et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004). https://doi.org/10.1038/nature03120

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