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

Abstract

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.

Bimolecular Fluorescence Complementation: Visualization of Molecular Interactions in Living Cells

“…Likewise, co-occupancy by zinc-finger DNA binding proteins on oligonucleotides has been detected in vitro (Stains, et al, 2005). Complexes between the exon junction complex components Y14 and NXF1 were also detected only in the presence of newly synthesized transcripts (Schmidt, et al, 2006). Therefore, although the association of fluorescent protein fragments in the BiFC assay is bimolecular, this assay is not limited to the visualization of binary interactions.…”

“…Complexes between the exon junction complex components Y14 and NXF1 are formed only during ongoing transcription and are localized to nuclear splicing speckles (Schmidt, et al, 2006). Further studies of the mechanisms regulating the localization of protein complexes will increase our understanding of the roles of compartmentalization in regulating protein function.…”

Efficient Induction of Nuclear Aggresomes by Specific Single Missense Mutations in the DNA-binding Domain of a Viral AP-1 Homolog

“…We examined the ability of Z(R183E)-induced aggresomes to recruit and sequester two cellular proteins, SC35 and nucleolin, whose specific distribution within cell nuclei is well documented (31,(47)(48)(49). SC35 is a spliceosome assembly factor that localizes to multiple discrete, irregularly shaped subnuclear organelles alternatively called "SC35 domains," "splicing speckles," or "interchromatin granules" (47,50).…”

Assembly and mobility of exon–exon junction complexes in living cells

“…This raises the question for which step in mRNA biogenesis speckles play a functional role. In order to address this point we previously used the technique of bimolecular fluorescence complementation (BiFC) (Hu et al 2002) to visualize Y14/NXF1-containing mRNPs (Schmidt et al 2006). The assay is based on the complementation of two fragments of the yellow fluorescent protein, which are fused to Y14 and NXF1, respectively.…”