UMMS Affiliation
Department of Cell Biology
Publication Date
3-17-2004
Document Type
Article
Subjects
Active Transport, Cell Nucleus; Adenosine Triphosphate; Antigens, Nuclear; Cell Nucleus; *Exons; Fluorescence Recovery After Photobleaching; Hela Cells; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; Macromolecular Substances; Nuclear Matrix-Associated Proteins; Permeability; RNA; RNA Splicing; RNA-Binding Proteins; Recombinant Fusion Proteins; Ribonucleoproteins
Disciplines
Cell Biology | Life Sciences | Medicine and Health Sciences
Abstract
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.
DOI of Published Version
10.1083/jcb.200307002
Source
J Cell Biol. 2004 Mar 15;164(6):843-50. Epub 2004 Mar 15. Link to article on publisher's site
Journal/Book/Conference Title
The Journal of cell biology
Related Resources
PubMed ID
15024032
Repository Citation
Wagner, Stefan; Chiosea, Simion I.; Ivshina, Mariya P.; and Nickerson, Jeffrey A., "In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160" (2004). Open Access Articles. 918.
https://escholarship.umassmed.edu/oapubs/918