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  • The EMBO Journal (1999) 18, 4535 - 4548
  • doi:10.1093/emboj/18.16.4535

Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6dotU5] tri-snRNP

Alexander Gottschalk1, Gitte Neubauer2, Josette Banroques3, Matthias Mann2,4, Reinhard Lührmann1,5 and Patrizia Fabrizio1

  1. Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany
  2. European Molecular Biology Laboratory (EMBL), Protein and Peptide Group, Meyerhofstr. 1, D-69117 Heidelberg, Germany
  3. Centre de Génétique Moléculaire du CNRS, Laboratoire Propre Associé à l'Université Pierre et Marie Curie, 91198 Gif-sur-Yvette, France
  4. Present address: Protein Interaction Laboratory, Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark
  5. Department of Cellular Biochemistry, Max-Planck-Institute of Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany

Correspondence to:

Reinhard Lührmann, E-mail: luehrmann@imt.uni-marburg.de

Patrizia Fabrizio, E-mail: fabrizio@imt.uni-marburg.de

Received 17 May 1999; Accepted 23 June 1999; Revised 23 June 1999

The 25S [U4/U6dotU5] tri-snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre-mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri-snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6dotU5] tri-snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri–snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm-like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA-binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre-mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri-snRNPs and the U2 snRNP are co-precipitated with protein A-tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2dotU4/U6dotU5] tetra-snRNP complex.

  • Keywords:

    • Lsm proteins,
    • mass spectrometry,
    • pre-mRNA splicing,
    • RSE1,
    • snRNP proteins