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.
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
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
J. Biol. Chem.
Date: May. 16, 2003
PubMed ID: 12604611
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