In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains
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细胞生物学杂志 2006年第1期
Division Molecular Genetics, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany
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--D-ribofuranosylbenzimidazole; EJC, exon-exon junction complex; FLIP, fluorescence loss in photobleaching; NXF1, nuclear export factor 1; pol II, RNA polymerase II.
Introduction
In eukaryotic organisms, transcription is spatially separated from translation by a nuclear envelope. Consequently, gene expression requires nuclear export of mature mRNA. Although the distribution of individual mRNA export factors has been studied, as has that of several nuclear mRNAs, the use of bimolecular fluorescence complementation (BiFC) analysis makes it possible to study the in vivo formation of complexes between different export factors that evidence indicates are functionally associated with RNA. We have used this approach to study the distribution, dynamic behavior, and relationship of Y14–nuclear export factor 1 (NXF1) complexes to RNA synthesis. The assay relies on the reconstitution of fluorescent YFP by the association of two nonfluorescent YFP half-molecules, each linked to one of two proteins, whose interactions are of interest (Hu et al., 2002). Evidence indicates that many or all of the complexes visualized are associated with RNA. Thus, monitoring the interaction of Y14 and NXF1 by BiFC indirectly allows the observation of potentially export-competent mRNA. Y14 is known to bind mRNA as part of the exon–exon junction complex (EJC) at a late stage of splicing (Kataoka and Dreyfuss, 2004) and remains bound to mRNA until translation in the cytoplasm (Dostie and Dreyfuss, 2002). Bound to the EJC, NXF1 (also called TAP) promotes export of the mature mRNA (for reviews see Dreyfuss et al., 2002; Erkmann and Kutay, 2004).
We show that coexpression of the two modified proteins, YC-Y14 and YN-NXF1, carrying the COOH- and NH2-terminal parts of YFP, respectively, allows observation of a characteristic BiFC pattern in cell nuclei. Unexpectedly, BiFC fluorescence accumulated in speckle-associated patches, suggesting an active role for speckles in mRNA processing, although they are otherwise considered mainly as storage sites for splicing and export factors (Reed and Hurt, 2002). Findings also provided insight into the idea that the nuclear retention of RNA is one way in which nature regulates gene expression. Concordantly, it had been found that only a small fraction of all transcribed RNA is exported to the cytoplasm, although most of nuclear polymerase II–derived RNA is maturely spliced and polyadenylated (Gondran et al., 1999; Jackson et al., 2000; Weil et al., 2000). Studies using BiFC to visualize Y14–NXF1 export complexes provide new evidence relating to the nuclear retention of mRNA in vivo.
Results
YC-Y14 and YN-NXF1 reconstitute YFP fluorescence with a characteristic nuclear distributionUpon cotransfection of YC-Y14 and YN-NXF1, MCF7 cells emitted YFP fluorescence depending on BiFC maturation for 2 h at 30°C (Fig. 1 A). Fluorescence was observed in >90% of the cells. The signal was characterized by its nuclear localization and the composition of patchy accumulations embedded in a diffuse background. In nucleoli, the signal level was very low. Immunostaining of the YC epitope (the COOH-terminal area of YFP) essentially colocalized with the BiFC pattern (Fig. 1 A). Y14 tagged by full-length YFP displayed a similar pattern, except that it also stained nucleoli (Fig. 1 B, YFP-Y14). In contrast, patchy accumulations were less obvious with YFP-tagged NXF1, where focal accumulations aligned at the nuclear periphery appeared as a characteristic expression pattern (Fig. 1 B, YFP-NXF1).
BiFC of YFP from YC-Y14 and YN-NXF1 depends on specific interaction of the two BiFC partnersWhen the fusion partners YC-NXF1 and YN-Y14 were cross exchanged, YFP fluorescence was not reconstituted, even though transfection efficiency as revealed by YC immunostaining was still high (unpublished data). Furthermore, BiFC as obtained with YC-Y14 and YN-NXF1 did not occur if NXF1 was mutated by deletion of the NH2-terminal 60 or 371 amino acids (Fig. 1, C and D). Deletion of the first 60 amino acids of NXF1, which are known to interact with the EJC protein E1B-AP5 (Bachi et al., 2000), is supposed to prevent BIFC formation caused by disturbed interaction with Y14, whereas the deletion of the NH2-terminal 371 amino acids totally compromises interaction with RNA (Braun et al., 1999; Bachi et al., 2000; Liker et al., 2000). With both mutations, BiFC occurred in <10% of the cells, even though transfection efficiency was >90%, as determined by YC immunodetection, and the nuclear distribution, which was monitored by full YFP tags (YFP-NXF1 1–371 and YFP-NXF1 1–60), was not seemingly influenced by the mutation (Fig. 1 B). In the few cases of BiFC, fluorescence was very weak and different from the characteristic patchy pattern of BiFC with full-length NXF1. In particular, BiFC with YN-NXF1 1–371 only occurred in cells with shrunken nuclei and abnormally condensed chromatin.
Do YC-Y14 and YN-NXF1 functionally interact with RNAThe aforementioned experiments support the hypothesis that functional interaction of the proteins YC-Y14 and YN-NXF1 is a prerequisite for the reconstitution of fluorescent YFP. As such, BiFC should depend on active mRNA transcription. Accordingly, BiFC was compromised upon RNA polymerase II (pol II) inhibition with either 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) or -amanitin at 37°C, even though expression of the YC epitope still appeared unperturbed (Fig. 2 A; -amanitin not depicted). In contrast, pol II inhibition after BiFC maturation at 30°C did not abolish the signal, which remained stable within 5% for a period of at least 6 h (Fig. 2 B). Immunoprecipitation of YC-Y14 in nuclear extracts of BiFC-matured cells confirmed the expected interactions. Targeted against the YC epitope of YC-Y14, immunoprecipitation coprecipitated YN-NXF1, NXF1, Y14, and radiolabeled RNA (Fig. 3).
BiFC patches colocalize with SC35 domains and the perispeckle regionSignal patches of BiFC from YC-Y14 and YN-NXF1 resembled speckles in respect to size, shape, and number. Speckles are nuclear bodies enriched in splicing and export factors (Spector, 1990; Zhou et al., 2000; Rodrigues et al., 2001). Colocalization of the BiFC patches with speckles was investigated by immunodetection of SC35, a marker protein for speckles (Fig. 4). All BiFC patches colocalized with SC35 domains, and vice versa. However, the BiFC patches were larger than their corresponding SC35 domain, surmounting the speckle perimeter by up to 500 nm. In 10% of the cells, the perispeckle signal was even higher than the SC35-positive center.
Export of BiFC complexes partially depends on ATPExport capability of the BiFC complexes was investigated in digitonin-permeabilized cells. Permeabilization allows observation of unidirectional nuclear export (Kehlenbach, 2003) because soluble factors necessary for reimport become depleted. Upon permeabilization, the whole nuclear level of BiFC fluorescence decreased by two thirds within 5 min (Fig. 5 A) and remained stable for at least 5 min further. However, the remaining YFP fluorescence was immediately extinguished upon administration of 5 mM ATP. To further verify the role of ATP in releasing the Y14–NXF1 complexes, cells were pretreated with sodium azide to deplete cellular levels of ATP. Sodium azide treatment reduced overall BiFC intensity. In particular, the diffuse background appeared extracted and a diffuse rim emerged at the nuclear periphery. Upon digitonin permeabilization, the signal level decreased by 50%. Again, the administration of 5 mM ATP caused the complete loss of fluorescence (Fig. 5 B).
Short-term immobile fractions of BiFC complexes slowly turn over in living cellsTo measure immobile fractions and the recovery speed of BiFC complexes in speckles and the nonspeckle nucleoplasm, FRAP was performed within 2-μm-diam circular regions of interest (Fig. 6). Immobile fractions measured by FRAP were confirmed by fluorescence loss in photobleaching (FLIP; Table I). Because temperature dependence was of special concern in this study, FRAP was also measured for YFP-Y14 to observe a possible shift from 37 to 30°C and RT (Table I). Besides a marginal trend of decreased immobile fraction in the cold, temperature had no significant effect within the error of individual measurements. For the BiFC signal, immobile fractions were much higher in speckles (46%) than in the nonspeckle nucleoplasm (21%). In contrast, recovery time of the mobile fraction was similar in both compartments (1.6 s). Sodium azide treatment significantly increased the immobile fractions (74 and 55%) and also decreased the recovery times. In particular, fluorescence recovery was much slower in speckles compared with the nonspeckle nucleoplasm (Table I). Performed in a timescale of 35 s, FRAP measured the mobility of fluorochromes already established at the time of bleaching. Bleaching all fluorescence of the complete nucleus, in contrast, allowed observation of de novo BiFC formation (Fig. 6). It took roughly 2 h for a steady state of restoration, which resembled the initial state. Separate analysis of the time course of recovery within speckles and the nonspeckle nucleoplasm revealed that speckles reached only 80% of their initial value.
Discussion
Recently, in vivo analysis of nuclear mRNA greatly contributed to our current understanding of RNA dynamics. However, measuring the dynamics of processed endogenous mRNA in vivo has been challenging. Studies that visualize mRNA export factors individually cannot distinguish functionally associated reporters from unbound reporters. We used the BiFC approach to visualize the interaction of Y14 and NXF1. Furthermore, evidence indicates that these complexes are associated with nuclear RNA. Thus, monitoring the interaction of target molecules instead of labeling single molecules by fluorescent protein tags or by FISH probes allows discrimination of false positive backgrounds from noninteracting entities. Compared with fluorescence resonance energy transfer, which is another method used to visualize protein–protein interaction in situ, the signal-to-noise ratio is higher in the BiFC experiments because the signal is measured directly. In contrast, fluorescence resonance energy transfer analysis requires cross talk compensation between donor and acceptor channels, which intrinsically increases statistical noise.
As NXF1 binds to processed mRNA and mediates the interactions of the export complex with the nuclear pore complex, it is considered a mRNA export receptor (Katahira et al., 1999; Bachi et al., 2000; Herold et al., 2001). The combination of the splicing-associated protein Y14 with NXF1 was thought to label spliced mRNA with putative export capacity. BiFC from YC-Y14 and YN-NXF1 reproducibly demonstrated a specific nuclear distribution of the interacting proteins, revealing spatial association with the speckle compartment in particular.
The BiFC approach is particularly well suited for in vivo observations. A peculiar requirement is the maturation at a low temperature (30°C). Because cells still grow and divide at this temperature, mRNA processing and export go on at physiologically acceptable rates. Accordingly, even though we observed a trend in reduction of the immobile fraction of YFP-Y14 in speckle-associated patches, temperature dependence of immobile fractions and recovery times upon photobleaching were not significant. Thus, we consider the shift to 30°C of marginal influence on the analysis of RNA maintenance by the BiFC assay.
Several experiments were performed to demonstrate that the BiFC assay labels functional interaction of the two proteins with RNA. First, the cross exchanged pair YN-Y14–YC-NXF1 did not reconstitute YFP, indicating that BiFC depended on the sterical configuration of the linked proteins, Y14 and NXF1, and that the YC and YN parts did not interact in solution to reconstitute fluorescence. Second, the characteristic BiFC pattern was not observed if the NH2-terminal 60 or 371 amino acids of NXF1 were deleted, even though topological expression of the proteins was not affected. The NH2-terminal part of NXF1 is responsible for RNA binding and interaction with the EJC (Braun et al., 1999; Bachi et al., 2000; Liker et al., 2000). According to Bachi et al. (2000), deletion of the first 371 amino acids blocks nuclear export and impairs viability of the cells. This may explain the bad condition of many cells transfected with YN-NXF1 1–371. BiFC fluorescence, with YN-NXF1 1–371 exclusively seen in shrunken nuclei, may have evolved because of aggregation of the BiFC chimeras. The first 60 amino acids are involved in interaction of NXF1 with the EJC protein E1B-AP5 (Bachi et al., 2000). Because NXF1 1–60 still binds to RNA (Braun et al., 1999, 2001), interaction of YN-NXF1 with YC-Y14 may occur with low efficiency, explaining the few cases where BiFC fluorescence could be monitored, although at very low intensity. Third, immunoprecipitation of YC-Y14 from nuclear extracts not only coprecipitated YN-NXF1 but also Y14, NXF1, and radiolabeled RNA, demonstrating that all expected interactions of YC-Y14 did occur. In particular, this experiment shows directly that the YC tag does not prohibit interaction with NXF1. Fourth, BiFC from YC-Y14 and YN-NXF1 requires pol II activity. Inhibition of pol II by DRB or -amanitin before BiFC maturation prohibited fluorescence reconstitution, whereas already established fluorescence was not affected by pol II inhibition. All together, these experiments suggest that BiFC of YFP from YC-Y14 and YN-NXF1 monitors endogenous mRNA at a terminal state of maturation.
Because BiFC-YFP represents the interaction of YC-Y14 and YN-NXF1, the characteristic BiFC pattern should be part of both YFP-Y14 and YFP-NXF1 distributions. This was the case, as both constructs showed nuclear location with accumulation in specklelike patches. Differences from the characteristic BiFC pattern may be attributable to entities that are not functionally associated with RNA, such as the nucleolar localization of YFP-Y14. The focal accumulations of YFP-NXF1 at the nuclear edge may represent cytoplasmic entities that become docked to nuclear pores before import (Forler et al., 2004).
The BiFC complexes accumulated within speckles and the perispeckle region, suggesting a functional role of the speckle compartment in mRNA processing, including maturation and/or transport. Furthermore, the pronounced enrichment of BiFC complexes in the perispeckle space indicates that speckles associate with a specific nuclear environment.
The accumulation of RNA in speckles had already been observed for both microinjected and endogenous RNA that were labeled by FISH with either an oligo d(T) probe or sequence-specific probes (Carter et al., 1991; Zirbel et al., 1993; Melcak et al., 2001; Shopland et al., 2002). Because BiFC has a maturation time that is >2 h (Hu and Kerppola, 2003), the observed accumulation in speckles derives from complexes of rather long nuclear residence time. The poor mobility of BiFC complexes was experimentally revealed by FRAP and FLIP and was further confirmed in cell permeabilization assays. Upon cell permeabilization, roughly two-thirds of the BiFC signal was eluted. The remaining fluorescence of an apparently immobile fraction completely disappeared upon administration of ATP. One might argue that ATP destroys the BiFC complexes. However, YN-NXF1 still coprecipitates with YC-Y14 upon ATP administration, indicating that the effect of ATP is the release of the complexes. ATP-dependent mobilization of splicing- and export-related factors were described for the alternate splicing factor (Misteli et al., 1997) and the EJC protein SRm160 (Wagner et al., 2004). ATP-dependent export was also observed for in vitro–transcribed rab11 mRNA (Ossareh-Nazari et al., 2000).
In cells treated with sodium azide, the diffuse background of BiFC complexes strongly decreased. The speckle-associated accumulations became more prominent, and a rim emerged at the nuclear periphery. This reorganization may be caused by the extraction of a soluble part of BiFC complexes or by the reduced release of bound complexes. Accordingly, a lower fraction of fluorescence was eluted upon digitonin permeabilization. The peripheral rim was broad and not sharply delimited, unlike the focal accumulations of NXF1. It may represent a zone of weak binding for mRNA, which only becomes visible when the diffuse background of presumptively mobile BiFC complexes disappeared.
In vivo analysis of mobility by FRAP was kept simple. Half-life recovery and immobile fractions were read out directly from the recovery plots of bleach-corrected raw data. Neither curve fitting to decipher the complexity of presumptively superposed events nor modeling of diffusion constants wer performed. Thus, the data presented are valuable for direct comparison only. Recovery of YFP-Y14 was faster, compared with BiFC-YFP. Because the YFP-Y14 signal does not represent a pure population of complexed molecules, a fraction of unbound molecules will account for this apparent faster mobility. One might also argue that BiFC formation, by itself, impedes mobility of the complexes.
Measured by FRAP or FLIP, roughly half of the BiFC-forming complexes in the speckle-associated patches were immobile, and the immobile fraction was lower in the nonspeckle nucleoplasm, suggesting a functional association of BiFC complexes with speckles. Further evidence for functional association with speckles compared with haphazard immobilization caused by the artificial BiFC tag is provided by the efficient release of immobile BiFC complexes upon ATP administration in permeabilized cells.
Upon incubation of cells in sodium azide, most of the BiFC signal became immobile. According to the ATP-dependent release in permeabilized cells, ATP depletion may have impaired the release of bound complexes rather than decreased the nuclear export rate. FRAP of diffusible molecules is a fast process. In our study, an approximately steady state was reached within a period of 35 s. In contrast, bleaching the entire pool of fluorescence in a cell allows the observation of fluorescence turnover instead of diffusion. Upon total bleach of a nucleus, the initial BiFC pattern recovered within a period of 2 h, demonstrating its turnover even within BiFC patches.
Previous studies reported that speckles contain poly(A) RNA (Carter et al., 1991; Huang et al., 1994). As shown by Johnson et al. (2000) and Shopland et al. (2002), at least part of this speckle RNA comes from transcription of protein-coding genes. Although possibly all actively transcribed genes associate with the surface of speckles (Huang and Spector, 1991; Johnson et al., 2000), some genes' interaction with speckles is particularly intimate, as their transcription products pervade the associated speckle ("typeI genes;" Smith et al., 1999). Transcripts can even move over and accumulate in the entire speckle compartment of the nucleus (Hattinger et al., 2002). Enrichment of messengers in speckles, however, appears to depend on the particular conditions of either the cell state or the transcript. Thus, observable accumulation throughout the entire speckle compartment of the induced gene p21 transcripts occurred in <20% of the induced cells (Hattinger et al., 2002). Comparing the distribution of collagen 11 and 12 transcripts in cultured fibroblast nuclei, Shopland et al. (2002) found significant different distribution patterns. The collagen 11 product pervades the entire associated speckle. Because transcripts of a splice mutation of collagen 11 that never leave the nucleus also pervade the entire associated speckle (Johnson et al., 2000), one may argue that speckle-associated mRNA is retained before export. This also explains why their prevalence remains practically stable upon transcripition inhibition (Huang et al., 1994; Shopland et al., 2002). Long residence time is also a characteristic feature of the observed BiFC complexes because of the duration of fluorescence maturation. There may be complexes that exit the nucleus quicker. These, however, would not be visible by the assay, as the complex is supposed to decay in the cytoplasm. Although we cannot exclude the possibility that the BiFC complexes are artificially bound within speckles because of their tag, they may equally represent a fraction of normally retained RNA. In any case, the assay reveals a possible involvement of the speckle-associated nuclear space in retention of processed mRNA.
BiFC of YFP from YC-Y14 and YN-NXF1 indicates that mammalian cell nuclei harbor NXF1-associated mRNA with very long residence times, implying that association of RNA with NXF1 is insufficient for immediate export. Because the BiFC signal can be released by ATP, an ATP-dependent mechanism may be involved in the export prohibition of NXF1-associated RNA.
Materials and methods
Plasmid constructionFull-length NXF1 and Y14 sequences were PCR amplified and cloned into peYFP-C1 (CLONTECH Laboratories) using EcoRI–BamHI (New England BioLabs, Inc.) restriction sites. Subsequently, the eYFP was substituted by the 1–154 or the 155–238 fragment of eYFP. These fragments were PCR amplified and inserted using AgeI–XhoI or AgeI–BspEI (New England BioLabs, Inc.) restriction enzymes to generate fusion proteins with the NH2- or COOH-terminal parts of YFP (YN-NXF1, YN-Y14, YC-NXF1, and YC-Y14, respectively). NXF1 deletion mutants (YN-NXF1 1–60 and YN-NXF1 1–371) were linked to full-length YFP or to the NH2-terminal part of YFP using EcoRI–BamHI.
Single transfection of the plasmids used for BiFC (YN-x and YC-x) did not produce fluorescence, indicating that the vectors did not code for full-length YFP.
Cell cultureHuman MCF7 cells were cultured in DME supplemented with 10% FCS. 1 d before transfection, cells were seeded onto coverslips or in Lab-Tek chambers (Nunc) and transfected using Effectene (QIAGEN) according to the manufacturer's instructions. 16–48 h after transfection, cells were washed with PBS and incubated with fresh medium at 30°C for at least 2 h to allow fluorophore maturation. Live cell observations were performed in DME containing 20 mM Hepes, pH 7.0 on a heating stage.
For 32P labeling of RNA (Lerner and Steitz, 1979), cells were washed with prewarmed, phosphate-free DME (Invitrogen) and incubated with 0.4 mCi 32P (GE Healthcare) per 200 ml of phosphate-free DME supplemented with 50 mM Hepes (Invitrogen) for 4 h at 30°C.
ImmunofluorescenceCells were fixed with 4% formaldehyde in PBS with 2% sucrose for 15 min on ice, permeabilized in 0.2% Triton X-100 for 3 min on ice, preincubated with 4% goat serum for 10 min at RT, and incubated with primary antibody (mouse -SC35; Sigma-Aldrich; rabbit -GFP; Abcam pvc) for 30 min at 37°C. The GFP antibody cross reacts with the YC epitope. Samples were washed and incubated with Cy5-labeled secondary antibodies (Dianova) for 30 min at 37°C. Coverslips were mounted with Vectashield containing DAPI (Linaris) for direct observation.
Transcription inhibition and energy starvationPol II transcription was inhibited by incubation in 50 μg/ml DRB or -amanitin in DME for 6 h. For energy starvation, cells were incubated for 20 min at 30°C in PBS containing 10 mM sodium azide, 50 mM of deoxy-glucose, 1 mM MgCl2, and 0.5 mM CaCl2 (Svitkina and Borisy, 1999).
Cell permeabilization assayFor selective permeabilization of the cell membrane, unfixed cells were incubated for 5 min at 30°C with 40 μg/ml digitonin (Calbiochem) in transcription buffer (Stanek et al., 2000), following the protocol used by Adam et al. (1990). In experiments with ATP supply after permeabilization, digitonin was replaced by transcription buffer containing 5 mM ATP.
Cell fractionation, coimmunoprecipitation, and Western blottingTransfected cells were trypsinized, suspended in prewarmed DME, and washed with ice-cold PBS. For the cytoplasmic preparation, the cell membrane was permeabilized for 5 min on ice with digitonin buffer supplemented with complete EDTA-free protease inhibitor cocktail (Roche), 0.1 mM PMSF (Sigma-Aldrich), and 250 mM KCl. Cells were spun down at 800 g for 3 min, the supernatant was taken off, and cells were respun at 15,000 g and saved. The pellet containing the nuclear fraction was resuspended in buffer containing 0.1% NP-40, incubated on ice for 10 min, and spun at 15,000 g for 3 min, and the supernatant was saved.
For immunoprecipitation, 200-μl protein A–Sepharose beads (GE Healthcare) were washed in NP-40 buffer and blocked with 100 μg/ml of Escherichia coli tRNA and 1 mg/ml BSA. Beads were incubated with antibody (-GFP; Abcam pvc) at a ratio of 20:1 for 1 h, in a total volume of 500 μl. Cell extracts were incubated with either antibody-coated beads or beads alone overnight. Beads were washed three times in 1 ml of buffer and resuspended in 300 μl of buffer. All steps were performed at 4°C. Proteins were isolated from the beads and denatured by boiling in Laemmli buffer (2% SDS, 20% glycerol, 20 mM Tris-Cl, pH 8, 2 mM EDTA, 1 mM DTT, and 0.1 mg/ml Bromophenol blue) for 5 min.
For Western blotting, proteins were separated by 10% SDS-PAGE, transferred to a PVDF membrane (Millipore), and detected with rabbit -NXF1 (gift from E. Hurt, University of Heidelberg, Heidelberg, Germany), rabbit -GFP (Abcam pvc), or mouse -Y14 (Abcam pvc). The secondary antibodies used were -rabbit–HRP and -mouse–HRP (DakoCytomation).
RNA purification with TRIzolAfter the immunoprecipitation, 1 ml TRIzol (Invitrogen) was added, mixed, and incubated at RT for 5 min. 200 μl chloroform was added, mixed, and incubated at RT for 15 min. Phases were separated by centrifugation at 15,000 g for 5 min. The upper phase was saved, 10 μg of glycogen was added, and RNA was precipitated with 500 μl isopropanol. The pellet was washed with 70% ethanol, resuspendend in 5 μl of nuclease-free water, and spotted onto a nitrocellulose membrane. Radioactivity was exposed on BioMax MS film (Kodak).
Confocal laser scanning microscopy and image processingFluorescence images were acquired by confocal laser scanning microscopy (model TCS SP2; Leica) with a 63x, NA 1.32, oil objective at pinhole size 1 Airy and a nominal voxel size of 58 x 58 x 284 nm3. DAPI, YFP, and Cy5 were excited by 405-, 514-, and 633-nm laser light and emission was detected at 410–500, 520–590, and 640–700 nm, respectively. Cross talk was minimized by serial acquisition of the fluorescence color channels. The digital images were analyzed with either LSM software (Leica) or analySIS pro 3.2 (Soft Imaging System GmBH). All micrographs in Fig. 5 have been deconvoluted (blind three dimensional) with analySIS pro 3.2. Frequency distributions of intensities were collected either over a regions of interest or through binary masks, which were configured by global thresholding, morphologic filtering, and interactive editing. Two features of interest were compared, which were the BiFC signal in speckles (corresponding to the SC35 domains), including a perispeckle compartment and the nuclear area, except for speckles and nucleoli. Note that the "nonspeckle nucleoplasm" is heterogeneous, as it contains chromatin, nuclear bodies, and parts of the speckle compartment that were not identified as speckles.
FRAPFRAP was performed in two dimensions within the bleach zones of a 2-μm nominal diam positioned over speckles and the nonspeckle nucleoplasm. Image size was 64 x 256 pixels at a lateral pixel size of 116 nm. Lag time between bleaching and the first recovery measurement was 0.625 ms at a line rate of 800 Hz. Beam expander of the illumination path was set to value 1 and the pinhole to 6 Airy.
Four postbleach images were taken every 107 ms, followed by four images every 214 ms and 30 images every 1.07 s. The period of recovery measurement was adjusted to reach saturation (up to 100 s). After subtraction of the mean of image black level, the recovery data were corrected for FLIP effects and postbleach loss of fluorescence during recovery observation and transformed into relative values according to Irel = It/I0 x T0/Tt, with I0 being the initial intensity and It the intensity at the time t in the bleached spot, and T0 and Tt being the total nuclear intensity before bleach and at time t, respectively. Immobile fractions and the period to reach half saturation (half-life) were directly read out from plotted FRAP curves.
FLIPFor FLIP analysis, approximately one half of a cell nucleus was bleached 100 times every 1.6 s at maximum laser intensity and beam expander 1 while the unbleached part of the nucleus was not illuminated. Two dimensional image acquisition before and after bleaching was conducted with pinhole size 6 Airy. For data analysis, the black level was subtracted and the postbleach image corrected for observation-induced bleaching. The factor for bleach correction was calculated from the fluorescence decay, It/I0, of an adjacent unbleached cell observed under the same conditions.
Recovery of BiFC after total bleach of the cell nucleusA complete loss of fluorescence across the entire cell nucleus was achieved with 25 repeated scans at maximum laser intensity. Fluorescence recovery was then observed over 2 h, and images of the same confocal plane were recorded 4 times every 30 s, followed by 8 times every 1 min, 10 times every 5 min, and 6 times every 10 min. The images were black level subtracted and the time-dependent increase in intensity was measured as mean intensity through masks designed for speckles and the nonspeckle nucleoplasm.
Acknowledgments
The support of Tom Kerppola and Malte Wachsmuth is gratefully acknowledged. We would like to thank Harald Herrmann and Daniel Mertens for constructive suggestions. We are grateful to Ed Hurt for providing antibodies against human NXF1/TAP. Katalin Fejes Toth, Michelle Nessling, Don and Ada Olins, and Tamas Fischer are gratefully acknowledged for critical reading of the manuscript and valuable discussions. We also thank Roland Eils for the use of his microscope imaging device.
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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--D-ribofuranosylbenzimidazole; EJC, exon-exon junction complex; FLIP, fluorescence loss in photobleaching; NXF1, nuclear export factor 1; pol II, RNA polymerase II.
Introduction
In eukaryotic organisms, transcription is spatially separated from translation by a nuclear envelope. Consequently, gene expression requires nuclear export of mature mRNA. Although the distribution of individual mRNA export factors has been studied, as has that of several nuclear mRNAs, the use of bimolecular fluorescence complementation (BiFC) analysis makes it possible to study the in vivo formation of complexes between different export factors that evidence indicates are functionally associated with RNA. We have used this approach to study the distribution, dynamic behavior, and relationship of Y14–nuclear export factor 1 (NXF1) complexes to RNA synthesis. The assay relies on the reconstitution of fluorescent YFP by the association of two nonfluorescent YFP half-molecules, each linked to one of two proteins, whose interactions are of interest (Hu et al., 2002). Evidence indicates that many or all of the complexes visualized are associated with RNA. Thus, monitoring the interaction of Y14 and NXF1 by BiFC indirectly allows the observation of potentially export-competent mRNA. Y14 is known to bind mRNA as part of the exon–exon junction complex (EJC) at a late stage of splicing (Kataoka and Dreyfuss, 2004) and remains bound to mRNA until translation in the cytoplasm (Dostie and Dreyfuss, 2002). Bound to the EJC, NXF1 (also called TAP) promotes export of the mature mRNA (for reviews see Dreyfuss et al., 2002; Erkmann and Kutay, 2004).
We show that coexpression of the two modified proteins, YC-Y14 and YN-NXF1, carrying the COOH- and NH2-terminal parts of YFP, respectively, allows observation of a characteristic BiFC pattern in cell nuclei. Unexpectedly, BiFC fluorescence accumulated in speckle-associated patches, suggesting an active role for speckles in mRNA processing, although they are otherwise considered mainly as storage sites for splicing and export factors (Reed and Hurt, 2002). Findings also provided insight into the idea that the nuclear retention of RNA is one way in which nature regulates gene expression. Concordantly, it had been found that only a small fraction of all transcribed RNA is exported to the cytoplasm, although most of nuclear polymerase II–derived RNA is maturely spliced and polyadenylated (Gondran et al., 1999; Jackson et al., 2000; Weil et al., 2000). Studies using BiFC to visualize Y14–NXF1 export complexes provide new evidence relating to the nuclear retention of mRNA in vivo.
Results
YC-Y14 and YN-NXF1 reconstitute YFP fluorescence with a characteristic nuclear distributionUpon cotransfection of YC-Y14 and YN-NXF1, MCF7 cells emitted YFP fluorescence depending on BiFC maturation for 2 h at 30°C (Fig. 1 A). Fluorescence was observed in >90% of the cells. The signal was characterized by its nuclear localization and the composition of patchy accumulations embedded in a diffuse background. In nucleoli, the signal level was very low. Immunostaining of the YC epitope (the COOH-terminal area of YFP) essentially colocalized with the BiFC pattern (Fig. 1 A). Y14 tagged by full-length YFP displayed a similar pattern, except that it also stained nucleoli (Fig. 1 B, YFP-Y14). In contrast, patchy accumulations were less obvious with YFP-tagged NXF1, where focal accumulations aligned at the nuclear periphery appeared as a characteristic expression pattern (Fig. 1 B, YFP-NXF1).
BiFC of YFP from YC-Y14 and YN-NXF1 depends on specific interaction of the two BiFC partnersWhen the fusion partners YC-NXF1 and YN-Y14 were cross exchanged, YFP fluorescence was not reconstituted, even though transfection efficiency as revealed by YC immunostaining was still high (unpublished data). Furthermore, BiFC as obtained with YC-Y14 and YN-NXF1 did not occur if NXF1 was mutated by deletion of the NH2-terminal 60 or 371 amino acids (Fig. 1, C and D). Deletion of the first 60 amino acids of NXF1, which are known to interact with the EJC protein E1B-AP5 (Bachi et al., 2000), is supposed to prevent BIFC formation caused by disturbed interaction with Y14, whereas the deletion of the NH2-terminal 371 amino acids totally compromises interaction with RNA (Braun et al., 1999; Bachi et al., 2000; Liker et al., 2000). With both mutations, BiFC occurred in <10% of the cells, even though transfection efficiency was >90%, as determined by YC immunodetection, and the nuclear distribution, which was monitored by full YFP tags (YFP-NXF1 1–371 and YFP-NXF1 1–60), was not seemingly influenced by the mutation (Fig. 1 B). In the few cases of BiFC, fluorescence was very weak and different from the characteristic patchy pattern of BiFC with full-length NXF1. In particular, BiFC with YN-NXF1 1–371 only occurred in cells with shrunken nuclei and abnormally condensed chromatin.
Do YC-Y14 and YN-NXF1 functionally interact with RNAThe aforementioned experiments support the hypothesis that functional interaction of the proteins YC-Y14 and YN-NXF1 is a prerequisite for the reconstitution of fluorescent YFP. As such, BiFC should depend on active mRNA transcription. Accordingly, BiFC was compromised upon RNA polymerase II (pol II) inhibition with either 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) or -amanitin at 37°C, even though expression of the YC epitope still appeared unperturbed (Fig. 2 A; -amanitin not depicted). In contrast, pol II inhibition after BiFC maturation at 30°C did not abolish the signal, which remained stable within 5% for a period of at least 6 h (Fig. 2 B). Immunoprecipitation of YC-Y14 in nuclear extracts of BiFC-matured cells confirmed the expected interactions. Targeted against the YC epitope of YC-Y14, immunoprecipitation coprecipitated YN-NXF1, NXF1, Y14, and radiolabeled RNA (Fig. 3).
BiFC patches colocalize with SC35 domains and the perispeckle regionSignal patches of BiFC from YC-Y14 and YN-NXF1 resembled speckles in respect to size, shape, and number. Speckles are nuclear bodies enriched in splicing and export factors (Spector, 1990; Zhou et al., 2000; Rodrigues et al., 2001). Colocalization of the BiFC patches with speckles was investigated by immunodetection of SC35, a marker protein for speckles (Fig. 4). All BiFC patches colocalized with SC35 domains, and vice versa. However, the BiFC patches were larger than their corresponding SC35 domain, surmounting the speckle perimeter by up to 500 nm. In 10% of the cells, the perispeckle signal was even higher than the SC35-positive center.
Export of BiFC complexes partially depends on ATPExport capability of the BiFC complexes was investigated in digitonin-permeabilized cells. Permeabilization allows observation of unidirectional nuclear export (Kehlenbach, 2003) because soluble factors necessary for reimport become depleted. Upon permeabilization, the whole nuclear level of BiFC fluorescence decreased by two thirds within 5 min (Fig. 5 A) and remained stable for at least 5 min further. However, the remaining YFP fluorescence was immediately extinguished upon administration of 5 mM ATP. To further verify the role of ATP in releasing the Y14–NXF1 complexes, cells were pretreated with sodium azide to deplete cellular levels of ATP. Sodium azide treatment reduced overall BiFC intensity. In particular, the diffuse background appeared extracted and a diffuse rim emerged at the nuclear periphery. Upon digitonin permeabilization, the signal level decreased by 50%. Again, the administration of 5 mM ATP caused the complete loss of fluorescence (Fig. 5 B).
Short-term immobile fractions of BiFC complexes slowly turn over in living cellsTo measure immobile fractions and the recovery speed of BiFC complexes in speckles and the nonspeckle nucleoplasm, FRAP was performed within 2-μm-diam circular regions of interest (Fig. 6). Immobile fractions measured by FRAP were confirmed by fluorescence loss in photobleaching (FLIP; Table I). Because temperature dependence was of special concern in this study, FRAP was also measured for YFP-Y14 to observe a possible shift from 37 to 30°C and RT (Table I). Besides a marginal trend of decreased immobile fraction in the cold, temperature had no significant effect within the error of individual measurements. For the BiFC signal, immobile fractions were much higher in speckles (46%) than in the nonspeckle nucleoplasm (21%). In contrast, recovery time of the mobile fraction was similar in both compartments (1.6 s). Sodium azide treatment significantly increased the immobile fractions (74 and 55%) and also decreased the recovery times. In particular, fluorescence recovery was much slower in speckles compared with the nonspeckle nucleoplasm (Table I). Performed in a timescale of 35 s, FRAP measured the mobility of fluorochromes already established at the time of bleaching. Bleaching all fluorescence of the complete nucleus, in contrast, allowed observation of de novo BiFC formation (Fig. 6). It took roughly 2 h for a steady state of restoration, which resembled the initial state. Separate analysis of the time course of recovery within speckles and the nonspeckle nucleoplasm revealed that speckles reached only 80% of their initial value.
Discussion
Recently, in vivo analysis of nuclear mRNA greatly contributed to our current understanding of RNA dynamics. However, measuring the dynamics of processed endogenous mRNA in vivo has been challenging. Studies that visualize mRNA export factors individually cannot distinguish functionally associated reporters from unbound reporters. We used the BiFC approach to visualize the interaction of Y14 and NXF1. Furthermore, evidence indicates that these complexes are associated with nuclear RNA. Thus, monitoring the interaction of target molecules instead of labeling single molecules by fluorescent protein tags or by FISH probes allows discrimination of false positive backgrounds from noninteracting entities. Compared with fluorescence resonance energy transfer, which is another method used to visualize protein–protein interaction in situ, the signal-to-noise ratio is higher in the BiFC experiments because the signal is measured directly. In contrast, fluorescence resonance energy transfer analysis requires cross talk compensation between donor and acceptor channels, which intrinsically increases statistical noise.
As NXF1 binds to processed mRNA and mediates the interactions of the export complex with the nuclear pore complex, it is considered a mRNA export receptor (Katahira et al., 1999; Bachi et al., 2000; Herold et al., 2001). The combination of the splicing-associated protein Y14 with NXF1 was thought to label spliced mRNA with putative export capacity. BiFC from YC-Y14 and YN-NXF1 reproducibly demonstrated a specific nuclear distribution of the interacting proteins, revealing spatial association with the speckle compartment in particular.
The BiFC approach is particularly well suited for in vivo observations. A peculiar requirement is the maturation at a low temperature (30°C). Because cells still grow and divide at this temperature, mRNA processing and export go on at physiologically acceptable rates. Accordingly, even though we observed a trend in reduction of the immobile fraction of YFP-Y14 in speckle-associated patches, temperature dependence of immobile fractions and recovery times upon photobleaching were not significant. Thus, we consider the shift to 30°C of marginal influence on the analysis of RNA maintenance by the BiFC assay.
Several experiments were performed to demonstrate that the BiFC assay labels functional interaction of the two proteins with RNA. First, the cross exchanged pair YN-Y14–YC-NXF1 did not reconstitute YFP, indicating that BiFC depended on the sterical configuration of the linked proteins, Y14 and NXF1, and that the YC and YN parts did not interact in solution to reconstitute fluorescence. Second, the characteristic BiFC pattern was not observed if the NH2-terminal 60 or 371 amino acids of NXF1 were deleted, even though topological expression of the proteins was not affected. The NH2-terminal part of NXF1 is responsible for RNA binding and interaction with the EJC (Braun et al., 1999; Bachi et al., 2000; Liker et al., 2000). According to Bachi et al. (2000), deletion of the first 371 amino acids blocks nuclear export and impairs viability of the cells. This may explain the bad condition of many cells transfected with YN-NXF1 1–371. BiFC fluorescence, with YN-NXF1 1–371 exclusively seen in shrunken nuclei, may have evolved because of aggregation of the BiFC chimeras. The first 60 amino acids are involved in interaction of NXF1 with the EJC protein E1B-AP5 (Bachi et al., 2000). Because NXF1 1–60 still binds to RNA (Braun et al., 1999, 2001), interaction of YN-NXF1 with YC-Y14 may occur with low efficiency, explaining the few cases where BiFC fluorescence could be monitored, although at very low intensity. Third, immunoprecipitation of YC-Y14 from nuclear extracts not only coprecipitated YN-NXF1 but also Y14, NXF1, and radiolabeled RNA, demonstrating that all expected interactions of YC-Y14 did occur. In particular, this experiment shows directly that the YC tag does not prohibit interaction with NXF1. Fourth, BiFC from YC-Y14 and YN-NXF1 requires pol II activity. Inhibition of pol II by DRB or -amanitin before BiFC maturation prohibited fluorescence reconstitution, whereas already established fluorescence was not affected by pol II inhibition. All together, these experiments suggest that BiFC of YFP from YC-Y14 and YN-NXF1 monitors endogenous mRNA at a terminal state of maturation.
Because BiFC-YFP represents the interaction of YC-Y14 and YN-NXF1, the characteristic BiFC pattern should be part of both YFP-Y14 and YFP-NXF1 distributions. This was the case, as both constructs showed nuclear location with accumulation in specklelike patches. Differences from the characteristic BiFC pattern may be attributable to entities that are not functionally associated with RNA, such as the nucleolar localization of YFP-Y14. The focal accumulations of YFP-NXF1 at the nuclear edge may represent cytoplasmic entities that become docked to nuclear pores before import (Forler et al., 2004).
The BiFC complexes accumulated within speckles and the perispeckle region, suggesting a functional role of the speckle compartment in mRNA processing, including maturation and/or transport. Furthermore, the pronounced enrichment of BiFC complexes in the perispeckle space indicates that speckles associate with a specific nuclear environment.
The accumulation of RNA in speckles had already been observed for both microinjected and endogenous RNA that were labeled by FISH with either an oligo d(T) probe or sequence-specific probes (Carter et al., 1991; Zirbel et al., 1993; Melcak et al., 2001; Shopland et al., 2002). Because BiFC has a maturation time that is >2 h (Hu and Kerppola, 2003), the observed accumulation in speckles derives from complexes of rather long nuclear residence time. The poor mobility of BiFC complexes was experimentally revealed by FRAP and FLIP and was further confirmed in cell permeabilization assays. Upon cell permeabilization, roughly two-thirds of the BiFC signal was eluted. The remaining fluorescence of an apparently immobile fraction completely disappeared upon administration of ATP. One might argue that ATP destroys the BiFC complexes. However, YN-NXF1 still coprecipitates with YC-Y14 upon ATP administration, indicating that the effect of ATP is the release of the complexes. ATP-dependent mobilization of splicing- and export-related factors were described for the alternate splicing factor (Misteli et al., 1997) and the EJC protein SRm160 (Wagner et al., 2004). ATP-dependent export was also observed for in vitro–transcribed rab11 mRNA (Ossareh-Nazari et al., 2000).
In cells treated with sodium azide, the diffuse background of BiFC complexes strongly decreased. The speckle-associated accumulations became more prominent, and a rim emerged at the nuclear periphery. This reorganization may be caused by the extraction of a soluble part of BiFC complexes or by the reduced release of bound complexes. Accordingly, a lower fraction of fluorescence was eluted upon digitonin permeabilization. The peripheral rim was broad and not sharply delimited, unlike the focal accumulations of NXF1. It may represent a zone of weak binding for mRNA, which only becomes visible when the diffuse background of presumptively mobile BiFC complexes disappeared.
In vivo analysis of mobility by FRAP was kept simple. Half-life recovery and immobile fractions were read out directly from the recovery plots of bleach-corrected raw data. Neither curve fitting to decipher the complexity of presumptively superposed events nor modeling of diffusion constants wer performed. Thus, the data presented are valuable for direct comparison only. Recovery of YFP-Y14 was faster, compared with BiFC-YFP. Because the YFP-Y14 signal does not represent a pure population of complexed molecules, a fraction of unbound molecules will account for this apparent faster mobility. One might also argue that BiFC formation, by itself, impedes mobility of the complexes.
Measured by FRAP or FLIP, roughly half of the BiFC-forming complexes in the speckle-associated patches were immobile, and the immobile fraction was lower in the nonspeckle nucleoplasm, suggesting a functional association of BiFC complexes with speckles. Further evidence for functional association with speckles compared with haphazard immobilization caused by the artificial BiFC tag is provided by the efficient release of immobile BiFC complexes upon ATP administration in permeabilized cells.
Upon incubation of cells in sodium azide, most of the BiFC signal became immobile. According to the ATP-dependent release in permeabilized cells, ATP depletion may have impaired the release of bound complexes rather than decreased the nuclear export rate. FRAP of diffusible molecules is a fast process. In our study, an approximately steady state was reached within a period of 35 s. In contrast, bleaching the entire pool of fluorescence in a cell allows the observation of fluorescence turnover instead of diffusion. Upon total bleach of a nucleus, the initial BiFC pattern recovered within a period of 2 h, demonstrating its turnover even within BiFC patches.
Previous studies reported that speckles contain poly(A) RNA (Carter et al., 1991; Huang et al., 1994). As shown by Johnson et al. (2000) and Shopland et al. (2002), at least part of this speckle RNA comes from transcription of protein-coding genes. Although possibly all actively transcribed genes associate with the surface of speckles (Huang and Spector, 1991; Johnson et al., 2000), some genes' interaction with speckles is particularly intimate, as their transcription products pervade the associated speckle ("typeI genes;" Smith et al., 1999). Transcripts can even move over and accumulate in the entire speckle compartment of the nucleus (Hattinger et al., 2002). Enrichment of messengers in speckles, however, appears to depend on the particular conditions of either the cell state or the transcript. Thus, observable accumulation throughout the entire speckle compartment of the induced gene p21 transcripts occurred in <20% of the induced cells (Hattinger et al., 2002). Comparing the distribution of collagen 11 and 12 transcripts in cultured fibroblast nuclei, Shopland et al. (2002) found significant different distribution patterns. The collagen 11 product pervades the entire associated speckle. Because transcripts of a splice mutation of collagen 11 that never leave the nucleus also pervade the entire associated speckle (Johnson et al., 2000), one may argue that speckle-associated mRNA is retained before export. This also explains why their prevalence remains practically stable upon transcripition inhibition (Huang et al., 1994; Shopland et al., 2002). Long residence time is also a characteristic feature of the observed BiFC complexes because of the duration of fluorescence maturation. There may be complexes that exit the nucleus quicker. These, however, would not be visible by the assay, as the complex is supposed to decay in the cytoplasm. Although we cannot exclude the possibility that the BiFC complexes are artificially bound within speckles because of their tag, they may equally represent a fraction of normally retained RNA. In any case, the assay reveals a possible involvement of the speckle-associated nuclear space in retention of processed mRNA.
BiFC of YFP from YC-Y14 and YN-NXF1 indicates that mammalian cell nuclei harbor NXF1-associated mRNA with very long residence times, implying that association of RNA with NXF1 is insufficient for immediate export. Because the BiFC signal can be released by ATP, an ATP-dependent mechanism may be involved in the export prohibition of NXF1-associated RNA.
Materials and methods
Plasmid constructionFull-length NXF1 and Y14 sequences were PCR amplified and cloned into peYFP-C1 (CLONTECH Laboratories) using EcoRI–BamHI (New England BioLabs, Inc.) restriction sites. Subsequently, the eYFP was substituted by the 1–154 or the 155–238 fragment of eYFP. These fragments were PCR amplified and inserted using AgeI–XhoI or AgeI–BspEI (New England BioLabs, Inc.) restriction enzymes to generate fusion proteins with the NH2- or COOH-terminal parts of YFP (YN-NXF1, YN-Y14, YC-NXF1, and YC-Y14, respectively). NXF1 deletion mutants (YN-NXF1 1–60 and YN-NXF1 1–371) were linked to full-length YFP or to the NH2-terminal part of YFP using EcoRI–BamHI.
Single transfection of the plasmids used for BiFC (YN-x and YC-x) did not produce fluorescence, indicating that the vectors did not code for full-length YFP.
Cell cultureHuman MCF7 cells were cultured in DME supplemented with 10% FCS. 1 d before transfection, cells were seeded onto coverslips or in Lab-Tek chambers (Nunc) and transfected using Effectene (QIAGEN) according to the manufacturer's instructions. 16–48 h after transfection, cells were washed with PBS and incubated with fresh medium at 30°C for at least 2 h to allow fluorophore maturation. Live cell observations were performed in DME containing 20 mM Hepes, pH 7.0 on a heating stage.
For 32P labeling of RNA (Lerner and Steitz, 1979), cells were washed with prewarmed, phosphate-free DME (Invitrogen) and incubated with 0.4 mCi 32P (GE Healthcare) per 200 ml of phosphate-free DME supplemented with 50 mM Hepes (Invitrogen) for 4 h at 30°C.
ImmunofluorescenceCells were fixed with 4% formaldehyde in PBS with 2% sucrose for 15 min on ice, permeabilized in 0.2% Triton X-100 for 3 min on ice, preincubated with 4% goat serum for 10 min at RT, and incubated with primary antibody (mouse -SC35; Sigma-Aldrich; rabbit -GFP; Abcam pvc) for 30 min at 37°C. The GFP antibody cross reacts with the YC epitope. Samples were washed and incubated with Cy5-labeled secondary antibodies (Dianova) for 30 min at 37°C. Coverslips were mounted with Vectashield containing DAPI (Linaris) for direct observation.
Transcription inhibition and energy starvationPol II transcription was inhibited by incubation in 50 μg/ml DRB or -amanitin in DME for 6 h. For energy starvation, cells were incubated for 20 min at 30°C in PBS containing 10 mM sodium azide, 50 mM of deoxy-glucose, 1 mM MgCl2, and 0.5 mM CaCl2 (Svitkina and Borisy, 1999).
Cell permeabilization assayFor selective permeabilization of the cell membrane, unfixed cells were incubated for 5 min at 30°C with 40 μg/ml digitonin (Calbiochem) in transcription buffer (Stanek et al., 2000), following the protocol used by Adam et al. (1990). In experiments with ATP supply after permeabilization, digitonin was replaced by transcription buffer containing 5 mM ATP.
Cell fractionation, coimmunoprecipitation, and Western blottingTransfected cells were trypsinized, suspended in prewarmed DME, and washed with ice-cold PBS. For the cytoplasmic preparation, the cell membrane was permeabilized for 5 min on ice with digitonin buffer supplemented with complete EDTA-free protease inhibitor cocktail (Roche), 0.1 mM PMSF (Sigma-Aldrich), and 250 mM KCl. Cells were spun down at 800 g for 3 min, the supernatant was taken off, and cells were respun at 15,000 g and saved. The pellet containing the nuclear fraction was resuspended in buffer containing 0.1% NP-40, incubated on ice for 10 min, and spun at 15,000 g for 3 min, and the supernatant was saved.
For immunoprecipitation, 200-μl protein A–Sepharose beads (GE Healthcare) were washed in NP-40 buffer and blocked with 100 μg/ml of Escherichia coli tRNA and 1 mg/ml BSA. Beads were incubated with antibody (-GFP; Abcam pvc) at a ratio of 20:1 for 1 h, in a total volume of 500 μl. Cell extracts were incubated with either antibody-coated beads or beads alone overnight. Beads were washed three times in 1 ml of buffer and resuspended in 300 μl of buffer. All steps were performed at 4°C. Proteins were isolated from the beads and denatured by boiling in Laemmli buffer (2% SDS, 20% glycerol, 20 mM Tris-Cl, pH 8, 2 mM EDTA, 1 mM DTT, and 0.1 mg/ml Bromophenol blue) for 5 min.
For Western blotting, proteins were separated by 10% SDS-PAGE, transferred to a PVDF membrane (Millipore), and detected with rabbit -NXF1 (gift from E. Hurt, University of Heidelberg, Heidelberg, Germany), rabbit -GFP (Abcam pvc), or mouse -Y14 (Abcam pvc). The secondary antibodies used were -rabbit–HRP and -mouse–HRP (DakoCytomation).
RNA purification with TRIzolAfter the immunoprecipitation, 1 ml TRIzol (Invitrogen) was added, mixed, and incubated at RT for 5 min. 200 μl chloroform was added, mixed, and incubated at RT for 15 min. Phases were separated by centrifugation at 15,000 g for 5 min. The upper phase was saved, 10 μg of glycogen was added, and RNA was precipitated with 500 μl isopropanol. The pellet was washed with 70% ethanol, resuspendend in 5 μl of nuclease-free water, and spotted onto a nitrocellulose membrane. Radioactivity was exposed on BioMax MS film (Kodak).
Confocal laser scanning microscopy and image processingFluorescence images were acquired by confocal laser scanning microscopy (model TCS SP2; Leica) with a 63x, NA 1.32, oil objective at pinhole size 1 Airy and a nominal voxel size of 58 x 58 x 284 nm3. DAPI, YFP, and Cy5 were excited by 405-, 514-, and 633-nm laser light and emission was detected at 410–500, 520–590, and 640–700 nm, respectively. Cross talk was minimized by serial acquisition of the fluorescence color channels. The digital images were analyzed with either LSM software (Leica) or analySIS pro 3.2 (Soft Imaging System GmBH). All micrographs in Fig. 5 have been deconvoluted (blind three dimensional) with analySIS pro 3.2. Frequency distributions of intensities were collected either over a regions of interest or through binary masks, which were configured by global thresholding, morphologic filtering, and interactive editing. Two features of interest were compared, which were the BiFC signal in speckles (corresponding to the SC35 domains), including a perispeckle compartment and the nuclear area, except for speckles and nucleoli. Note that the "nonspeckle nucleoplasm" is heterogeneous, as it contains chromatin, nuclear bodies, and parts of the speckle compartment that were not identified as speckles.
FRAPFRAP was performed in two dimensions within the bleach zones of a 2-μm nominal diam positioned over speckles and the nonspeckle nucleoplasm. Image size was 64 x 256 pixels at a lateral pixel size of 116 nm. Lag time between bleaching and the first recovery measurement was 0.625 ms at a line rate of 800 Hz. Beam expander of the illumination path was set to value 1 and the pinhole to 6 Airy.
Four postbleach images were taken every 107 ms, followed by four images every 214 ms and 30 images every 1.07 s. The period of recovery measurement was adjusted to reach saturation (up to 100 s). After subtraction of the mean of image black level, the recovery data were corrected for FLIP effects and postbleach loss of fluorescence during recovery observation and transformed into relative values according to Irel = It/I0 x T0/Tt, with I0 being the initial intensity and It the intensity at the time t in the bleached spot, and T0 and Tt being the total nuclear intensity before bleach and at time t, respectively. Immobile fractions and the period to reach half saturation (half-life) were directly read out from plotted FRAP curves.
FLIPFor FLIP analysis, approximately one half of a cell nucleus was bleached 100 times every 1.6 s at maximum laser intensity and beam expander 1 while the unbleached part of the nucleus was not illuminated. Two dimensional image acquisition before and after bleaching was conducted with pinhole size 6 Airy. For data analysis, the black level was subtracted and the postbleach image corrected for observation-induced bleaching. The factor for bleach correction was calculated from the fluorescence decay, It/I0, of an adjacent unbleached cell observed under the same conditions.
Recovery of BiFC after total bleach of the cell nucleusA complete loss of fluorescence across the entire cell nucleus was achieved with 25 repeated scans at maximum laser intensity. Fluorescence recovery was then observed over 2 h, and images of the same confocal plane were recorded 4 times every 30 s, followed by 8 times every 1 min, 10 times every 5 min, and 6 times every 10 min. The images were black level subtracted and the time-dependent increase in intensity was measured as mean intensity through masks designed for speckles and the nonspeckle nucleoplasm.
Acknowledgments
The support of Tom Kerppola and Malte Wachsmuth is gratefully acknowledged. We would like to thank Harald Herrmann and Daniel Mertens for constructive suggestions. We are grateful to Ed Hurt for providing antibodies against human NXF1/TAP. Katalin Fejes Toth, Michelle Nessling, Don and Ada Olins, and Tamas Fischer are gratefully acknowledged for critical reading of the manuscript and valuable discussions. We also thank Roland Eils for the use of his microscope imaging device.
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