Assembly and mobility of exon–exon junction complexes in living cells

  1. Ute Schmidt1,2,5,
  2. Kang-Bin Im2,
  3. Carola Benzing1,
  4. Snjezana Janjetovic1,
  5. Karsten Rippe3,
  6. Peter Lichter1 and
  7. Malte Wachsmuth2,4
  1. 1Division of Molecular Genetics, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
  2. 2Cell Biophysics Group, Institut Pasteur Korea, Seoul 136-791, Republic of Korea
  3. 3Research Group Genome Organization and Function, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
  4. 4Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany

    Abstract

    The exon–exon junction complex (EJC) forms via association of proteins during splicing of mRNA in a defined manner. Its organization provides a link between biogenesis, nuclear export, and translation of the transcripts. The EJC proteins accumulate in nuclear speckles alongside most other splicing-related factors. We followed the establishment of the EJC on mRNA by investigating the mobility and interactions of a representative set of EJC factors in vivo using a complementary analysis with different fluorescence fluctuation microscopy techniques. Our observations are compatible with cotranscriptional binding of the EJC protein UAP56 confirming that it is involved in the initial phase of EJC formation. RNPS1, REF/Aly, Y14/Magoh, and NXF1 showed a reduction in their nuclear mobility when complexed with RNA. They interacted with nuclear speckles, in which both transiently and long-term immobilized factors were identified. The location- and RNA-dependent differences in the mobility between factors of the so-called outer shell and inner core of the EJC suggest a hypothetical model, in which mRNA is retained in speckles when EJC outer-shell factors are missing.

    Keywords:

    Keywords

    Footnotes

    • 5 Present address: RNA Trafficking Group, Institut de Génétique Moléculaire de Montpellier CNRS-UMR 5535, 1919 Route de Mende, 34293 Montpellier Cedex 5, France.

    • Reprint requests to: Malte Wachsmuth, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany; e-mail: malte.wachsmuth{at}embl.de; fax: 49-6221-387-8512.

    • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1387009.

      • Received September 23, 2008.
      • Accepted January 30, 2009.
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