In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains.
The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.
Schmidt
Ute
U
Division Molecular Genetics, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany.
Richter
Karsten
K
Berger
Axel Bernhard
AB
Lichter
Peter
P
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D042581:Intranuclear Space / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D019810:Sodium Azide / Q000494:pharmacology; D014162:Transfection
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C122111:NXF1 protein, human; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; C406243:RBM8A protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; C054562:yellow fluorescent protein, Bacteria; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D012319:RNA Polymerase II; D004072:Digitonin