Multimerization of Staufen1 in live cells

  1. Luc Desgroseillers
  1. Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada
  • 2 Present addresses: Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA;

  • 3 Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA;

  • 4 Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada;

  • 5 McGill Cancer Center, McGill University, Montréal, Québec H3G 1Y6, Canada.

  1. 1 These authors contributed equally to this work.

Abstract

Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 self-association. Protein–protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast two-hybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein–protein interaction.

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Footnotes

  • Reprint requests to: Luc DesGroseillers, Département de Biochimie, Université de Montréal, PO Box 6128, Centre Ville, Montréal, Québec H3C 3J7, Canada; e-mail: luc.desgroseillers{at}umontreal.ca; fax: (514) 343-2210.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1664210.

    • Received March 30, 2009.
    • Accepted November 19, 2009.
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