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Published online 23 January 2006. doi:10.1083/jcb.200503061
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 172, Number 3, 373-381
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Article

 In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains

Ute Schmidt, Karsten Richter, Axel Bernhard Berger, and Peter Lichter

Division Molecular Genetics, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany

Correspondence to Peter Lichter: m.macleod{at}dkfz.de

The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.

Abbreviations used in this paper: BiFC, bimolecular fluorescence complementation; DRB, 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole; EJC, exon-exon junction complex; FLIP, fluorescence loss in photobleaching; NXF1, nuclear export factor 1; pol II, RNA polymerase II.


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