DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures

  1. Feng Guo1,5
  1. 1Department of Biological Chemistry, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA
  2. 2Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles, Los Angeles, California 90095, USA
  3. 3Biomedical Engineering Interdepartmental Program, University of California at Los Angeles, Los Angeles, California 90095, USA
  4. 4California NanoSystems Institute, University of California at Los Angeles, Los Angeles, California 90095, USA
  5. 5Molecular Biology Institute, University of California, Los Angeles, California 90095, USA

Abstract

DiGeorge critical region 8 (DGCR8) is essential for maturation of microRNAs (miRNAs) in animals. In the cleavage of primary transcripts of miRNAs (pri-miRNAs) by the Drosha nuclease, the DGCR8 protein directly binds and recognizes pri-miRNAs through a mechanism currently controversial. Our previous data suggest that DGCR8 trimerizes upon cooperative binding to pri-mir-30a. However, a separate study proposed a model in which a DGCR8 molecule contacts one or two pri-miRNA molecules using its two double-stranded RNA binding domains. Here, we extensively characterized the interaction between DGCR8 and pri-miRNAs using biochemical and structural methods. First, a strong correlation was observed between the association of DGCR8 with pri-mir-30a and the rate of pri-miRNA processing in vitro. Second, we show that the high binding cooperativity allows DGCR8 to distinguish pri-miRNAs from a nonspecific competitor with subtle differences in dissociation constants. The highly cooperative binding of DGCR8 to a pri-miRNA is mediated by the formation of higher-order structures, most likely a trimer of DGCR8 dimers, on the pri-miRNA. These properties are not limited to its interaction with pri-mir-30a. Furthermore, the amphipathic C-terminal helix of DGCR8 is important both for trimerization of DGCR8 on pri-miRNAs and for the cleavage of pri-miRNAs by Drosha. Finally, our three-dimensional model from electron tomography analysis of the negatively stained DGCR8–pri-mir-30a complex directly supports the trimerization model. Our study provides a molecular basis for recognition of pri-miRNAs by DGCR8. We further propose that the higher-order structures of the DGCR8–pri-miRNA complexes trigger the cleavage of pri-miRNAs by Drosha.

Keywords

Footnotes

  • Reprint requests to: Feng Guo, Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA; e-mail: fguo{at}mbi.ucla.edu; fax: (310) 206-7286; or Z. Hong Zhou, Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA; e-mail: hong.zhou{at}ucla.edu; fax: (310) 206-5231.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2111310.

  • Received February 1, 2010.
  • Accepted May 14, 2010.

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  1. Published in Advance June 17, 2010, doi: 10.1261/rna.2111310 RNA 16: 1570-1583 Copyright © 2010 RNA Society

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