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.
Raffetseder
Ute
U
Division of Nephrology and Clinical Immunology, University Hospital of Aachen, 52057 Aachen, Germany.
Frye
Björn
B
Rauen
Thomas
T
Jürchott
Karsten
K
Royer
Hans-Dieter
HD
Jansen
Petra Lynen
PL
Mertens
Peter R
PR
D017500:Adenovirus E1A Proteins / Q000235:genetics; D017398:Alternative Splicing; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D015723:Gene Library; D005920:Glomerular Mesangium / Q000166:cytology; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques; D051840:Y-Box-Binding Protein 1
D017500:Adenovirus E1A Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D000068103:Serine-Arginine Splicing Factors