Co-occupancy of two Pumilio molecules on a single hunchback NRE

  1. Yogesh K. Gupta1,
  2. Tammy H. Lee2,3,
  3. Thomas A. Edwards1,4,
  4. Carlos R. Escalante1,
  5. Lyudmila Y. Kadyrova5,
  6. Robin P. Wharton2,3 and
  7. Aneel K. Aggarwal1
  1. 1Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York 10029, USA
  2. 2Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210, USA
  3. 3Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, Ohio 43210, USA
  4. 4Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
  5. 5Department of Biochemistry and Molecular Biology, Southern Illinois University at Carbondale, Carbondale, Illinois 62901, USA

    Abstract

    Pumilio controls a number of processes in eukaryotes, including the translational repression of hunchback (hb) mRNA in early Drosophila embryos. The Pumilio Puf domain binds to a pair of 32 nucleotide (nt) Nanos response elements (NRE1 and NRE2) within the 3′ untranslated region of hb mRNA. Despite the elucidation of structures of human Pumilio Puf domain in complex with hb RNA elements, the nature of hb mRNA recognition remains unclear. In particular, the site that mediates regulation in vivo is significantly larger than the 8–10-nt RNA elements bound to single Puf molecules in crystal structures. Here we present biophysical and biochemical data that partially resolve the paradox. We show that each NRE is composed of two binding sites (Box A and Box B) and that two Puf domains can co-occupy a single NRE. The Puf domains have a higher affinity for the 3′ Box B site than the 5′ Box A site; binding to the intact NRE appears to be cooperative (at least in some experiments). We suggest that the 2 Pumilio:1 NRE complex is the functional regulatory unit in vivo.

    Keywords:

    Keywords

    Footnotes

    • Reprint requests to: Aneel K. Aggarwal, Department of Structural and Chemical Biology, Mount Sinai School of Medicine Box 1677, 1425 Madison Avenue, New York, NY 10029, USA; e-mail: aneel.aggarwal{at}mssm.edu; fax: (212) 849-2456; or Robin P. Wharton, Department of Molecular Genetics or Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, 982 Biomedical Research Tower, 460 W. 12th Avenue, Columbus, OH 43210, USA; e-mail: robin.wharton{at}osumc.edu.

    • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1327609.

      • Received August 28, 2008.
      • Accepted March 4, 2009.

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    1. Published in Advance April 16, 2009, doi: 10.1261/rna.1327609 RNA Copyright © 2009 RNA Society

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