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.

Rights and Permissions

Citation: J Cell Biol. 2004 Mar 15;164(6):843-50. Epub 2004 Mar 15. Link to article on publisher's site

DOI of Published Version

10.1083/jcb.200307002

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

The Journal of cell biology

PubMed ID

15024032

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