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 RIS Download
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