Wagner, StefanChiosea, Simion I.Ivshina, Mariya P.Nickerson, Jeffrey A.2022-08-232022-08-232004-03-172008-08-15J Cell Biol. 2004 Mar 15;164(6):843-50. Epub 2004 Mar 15. <a href="http://dx.doi.org/10.1083/jcb.200307002">Link to article on publisher's site</a>0021-9525 (Print)10.1083/jcb.20030700215024032https://hdl.handle.net/20.500.14038/42583We 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.en-USActive Transport, Cell NucleusAdenosine TriphosphateAntigens, NuclearCell Nucleus*ExonsFluorescence Recovery After PhotobleachingHela CellsHeterogeneous-Nuclear Ribonucleoprotein Group A-BHumansMacromolecular SubstancesNuclear Matrix-Associated ProteinsPermeabilityRNARNA SplicingRNA-Binding ProteinsRecombinant Fusion ProteinsRibonucleoproteinsCell BiologyLife SciencesMedicine and Health SciencesIn vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160Journal Articlehttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1917&context=oapubs&unstamped=1https://escholarship.umassmed.edu/oapubs/918579807oapubs/918