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Splicing factor SRp30c interaction with Y-box protein-1 confers nuclear YB-1 shuttling and alternative splice site selection.

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.

Pubmed ID: 12604611

Authors

  • Raffetseder U
  • Frye B
  • Rauen T
  • J├╝rchott K
  • Royer HD
  • Jansen PL
  • Mertens PR

Journal

The Journal of biological chemistry

Publication Data

May 16, 2003

Associated Grants

None

Mesh Terms

  • Adenovirus E1A Proteins
  • Alternative Splicing
  • Binding Sites
  • CCAAT-Enhancer-Binding Proteins
  • Cell Division
  • Cell Line
  • Cell Nucleus
  • Cytoplasm
  • DNA, Complementary
  • DNA-Binding Proteins
  • Gene Library
  • Glomerular Mesangium
  • HeLa Cells
  • Humans
  • Microscopy, Fluorescence
  • NFI Transcription Factors
  • Nuclear Proteins
  • Phosphoproteins
  • Plasmids
  • Precipitin Tests
  • Protein Binding
  • Protein Isoforms
  • Protein Structure, Tertiary
  • RNA, Messenger
  • RNA-Binding Proteins
  • Transcription Factors
  • Two-Hybrid System Techniques
  • Y-Box-Binding Protein 1