In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160.
We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)-labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)-dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility.
Wagner
Stefan
S
Dept. of Cell Biology, S7-214, University of Massachusetts Medical School, 55 Lake Ave., Worcester, MA 01655, USA.
Chiosea
Simion
S
Ivshina
Maria
M
Nickerson
Jeffrey A
JA
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D005091:Exons; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D010539:Permeability; D012313:RNA / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism
D034961:Antigens, Nuclear; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D046911:Macromolecular Substances; D034521:Nuclear Matrix-Associated Proteins; D016601:RNA-Binding Proteins; C084538:RNPS1 protein, human; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; C111710:SRRM1 protein, human; C414618:SRRM2 protein, human; C439768:hnRNP A2; D012313:RNA; D000255:Adenosine Triphosphate