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In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160
Address correspondence to Jeffrey A. Nickerson, Dept. of Cell Biology, S7-214, University of Massachusetts Medical School, 55 Lake Ave., Worcester, MA 01655. Tel.: (508) 856-2312. Fax: (508) 856-1033. email: jeffrey.nickerson{at}umassmed.edu
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.
Key Words: nuclear matrix; RNPS1; RNA export; RNA splicing; nuclear protein mobility
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