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Article |
Address correspondence to R.W. Dirks, Dept. of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, Netherlands. Tel.: 31-71-5276026. Fax.: 31-71-5276180. email: r.w.dirks{at}lumc.nl
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Abstract |
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Key Words: RNA transport; mRNA; 2'O-methyl RNA; FRAP; live cell imaging
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Introduction |
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Splicing factors are also considered to be indispensable for retaining pre-mRNAs in the cell nucleus. Pre-mRNAs were shown to accumulate at or near active sites of transcription and to colocalize with splicing factors (Bauren et al., 1996; Dirks et al., 1997; Misteli et al., 1997). Also, transcripts defective in splicing were shown to accumulate at sites of transcription and unable to be exported to the cytoplasm (Custodio et al., 1999). Therefore, it is assumed that most mRNAs are released from their site of synthesis and processing after completion of splicing. These mRNAs move randomly within the cell nucleus with export factors associated with them before they get bound to a nuclear pore complex and enter the cytoplasm. This assumption is supported by fluorescence in situ hybridization and BrUTP labeling studies showing that specific gene transcripts emanate from transcription sites in all directions in cell nuclei (Zachar et al., 1993; Dirks et al., 1995; Macville et al., 1995; Singh et al., 1999). Furthermore, in vivo hybridization studies revealed that poly(A)+ RNA moves randomly in the cell nucleus at a rate compatible with free diffusion (Politz et al., 1998, 1999).
However, various aspects of nuclear RNA export are unclear. These aspects include the role of nuclear compartments in the export process. Speckles, also referred to as SC-35 domains, are nuclear compartments that contain a large number of factors required for mRNA synthesis, processing, and export (Lamond and Spector, 2003). The observation that speckles also contain poly(A)+ RNA led to the suggestion that speckles themselves may play a role in RNA metabolism and export (Carter et al., 1991, 1993). These speculations were supported by the observation that sites of bromouridine incorporation that mark nascent transcripts overlap with speckles (Wei et al., 1999) and that some specific active genes localize at the edges of speckles (Xing et al., 1993, 1995; Smith et al., 1999; Shopland et al., 2003). Furthermore, a number of specific gene transcripts were shown to localize to the inside of speckles (Puvion and Puvion-Dutilleul, 1996; Smith et al., 1999; Johnson et al., 2000; Melcak et al., 2000; Hattinger et al., 2002; Shopland et al., 2002, 2003), indicating that speckles play a direct role in mRNA metabolism and export. However, various lines of evidence argue against a direct role of speckles in gene transcription, RNA processing, and RNA transport. First, in contrast to Wei et al. (1999), several reports indicate that speckles are not labeled after 3H- or bromouridine incorporation (Fakan, 1994; Cmarko et al., 1999). Second, splicing factors were shown to be recruited from speckles to sites of active transcription (Jimenez-Garcia and Spector, 1993; Huang and Spector, 1996; Dirks et al., 1997; Misteli et al., 1997; Zeng et al., 1997; Snaar et al., 1999). Third, poly(A)+ RNA is not exported from speckles when transcription is inhibited and is, therefore, suggested to be a stable population that plays a structural role and acts as a binding site for RNA processing proteins (Huang et al., 1994; Sacco-Bubulya and Spector, 2002). Finally, in vivo hybridization experiments using oligo (dT) probes that hybridize to the poly(A) tails of mRNAs in living cells did not reveal any accumulation of poly(A)+ RNA in speckles at any stage of transport (Politz et al., 1999).
To investigate a possible role for speckles in RNA transport, we analyzed the mobility of poly(A)+ RNA in the nucleoplasm and in nuclear speckles in transcriptionally active and inactive cells. Using 2'O-methyl RNA probes and photobleaching techniques, we demonstrate that poly(A)+ RNA moves throughout the nucleoplasm, though at a much slower rate compared with transport rates determined in previous works using oligodeoxynucleotide probes and compared with proteins that play a role in RNA processing and transport. Furthermore, we present evidence that poly(A)+ RNA transiently interacts with speckle domains independent of transcription but dependent on cellular energy levels.
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Results |
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Next, we photobleached nucleoplasmic areas at some distance from a speckle and analyzed the recovery of fluorescence. The recovery appeared to be 1.5 times faster (Fig. 1, B and D): t1/2
18 s, D
0.04 µm2/s (Table I). These experiments suggest a difference in mobility between the poly(A)+ RNA fractions inside the speckles and poly(A)+ RNA fractions outside the speckles. However, in cells where only a part of a speckle was bleached, the calculated diffusion constants appeared to be in the order of 0.035 µm2/s. The discrepancy in estimated D-values could result from the difference in the number of molecules present in the bleached area relative to those in the vicinity of the bleached area.
It is likely that the majority of the poly(A)+ RNA that moves into the bleached area initially comes from the immediate vicinity of the bleached spot. To determine the freedom of movement of poly(A)+ RNA throughout the nucleoplasm, including speckles, we applied FLIP. A spot in the nucleoplasm was repeatedly bleached for 3 s with 10-s time intervals in which images of the cell were recorded. Fig. 2 shows the result of a typical FLIP experiment. Loss of total nuclear fluorescence was imaged (Fig. 2 A) and measured over time (Fig. 2 B). After 400 s from the first bleach, 85% of the nuclear poly(A)+ RNA fluorescence was lost. Similar results were obtained when a region inside a speckle was repeatedly photobleached, suggesting that the mobility of poly(A)+ RNA in the nucleoplasm and speckles is very similar. The fraction of
15% of the total amount of fluorescence that is still present in the nucleus after 400 s bleaching suggests the presence of a relatively immobile population of poly(A)+ RNA, which is in agreement with the result of the FRAP experiments.
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No significant loss of signal was observed when the bleached spot was set in nucleoli or cytoplasm (unpublished data). These results imply that most poly(A)+ RNA is moving throughout the nucleoplasm, except for nucleoli. However, we cannot exclude that low amounts of poly(A)+ RNA move through nucleoli. Prolonged periods of photobleaching a spot in a nucleolus resulted in some loss of fluorescent signal in the nucleoplasm, which could be due to the photobleaching of fluorescent probe either present in nucleoli or in the nucleoplasm above or below nucleoli.
2'OMe (U)22 localization and kinetics are dependent on the presence of poly(A) tails
To confirm that 2'OMe (U)22 probe molecules hybridize specifically to the poly(A) tail of RNAs, cells were treated with cordycepin, which prevents poly(A) addition but does not block RNA synthesis (Darnell et al., 1971; Mendecki et al., 1972; Calado and Carmo-Fonseca, 2000). It was predicted that after cordycepin treatment the 2'OMe (U)22 probe would not localize to poly(A)+ rich speckle domains and would reveal a fast movement consistent with free diffusion. U2OS cells were incubated with cordycepin for 16 h, injected with 2'OMe (U)22 probe, and imaged by confocal microscopy. Consistent with our prediction, the 2'OMe (U)22 probe revealed a diffuse staining in the nucleus excluding nucleoli (Fig. 3, A and B). Also, as determined by FRAP (Fig. 3, A and C) and FLIP (Fig. 3, B and D), the mobility of the 2'OMe (U)22 probe was significantly increased in cordycepin-treated cells compared with nontreated cells. These findings demonstrate that the 2'OMe (U)22 probe binds with high specificity to the poly(A) tail of RNAs. Furthermore, the results show that the preferential association of the 2'OMe (U)22 probe with speckles results from their interaction with poly(A)+ RNA in living cells. This result is consistent with our finding that the 2'OMe (U)22 probe is highly specific for poly(A)+ RNA in fixed cells as determined by RNase controls (Molenaar et al., 2001).
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To measure the mobility of SF2/ASF-GFP relative to the mobility of poly(A)+ RNA, we microinjected U2OS cells stably expressing SF2/ASF-GFP with the 2'OMe (U)22-TAMRA probe and analyzed them by FRAP. SF2/ASF-GFP and the probe 2'OMe (U)22-TAMRA were photobleached simultaneously in a speckle, and the fluorescence recovery of both fluorophores was imaged separately at each time point in a time series to prevent cross talk. Fig. 8 A illustrates that the fluorescence recovery of SF2/ASF-GFP in a photobleached speckle precedes that of poly(A)+ RNA. Hence, SF2/ASF-GFP seems significantly more mobile than poly-(A)+RNA. This difference in fluorescence recovery was confirmed by comparing the calculated FRAP curves obtained from eight cells (Fig. 8 D).
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Because Aly and Tap have been implicated in playing roles in mRNA export and Aly has been shown to accumulate in nuclear speckles, we expected that the kinetics of Aly and Tap movement would correlate with the kinetic behavior of poly(A)+ movement. Fig. 8 C shows the localization pattern of Aly-GFP as observed in U2OS cells and a speckle that has been photobleached and subsequently imaged at regular time intervals afterwards. As shown, a full recovery of fluorescence is obtained within 1 min after photobleaching (Fig. 8 C). Also, the FRAP curve that has been generated after measuring recovery values in speckles from 10 cells shows that a near full recovery is obtained within 1 min (Fig. 8 E).
Next, we determined the dissociation kinetics of Aly-GFP from speckles by FLIP. Repeated bleaching of Aly-GFP in a defined area using high laser power revealed that the majority of Aly-GFP fluorescence was lost from the nucleus within 80 s (Fig. 8 F). When we performed similar experiments with cells expressing the RNA export factor Tap-GFP and its cofactor p15, which distributes more or less homogeneously throughout the nucleoplasm, we observed a loss of nuclear fluorescence within 60 s (Fig. 8 F). These results show that the transport factors Tap and Aly move more rapidly through the cell nucleus compared with the 2'OMe (U)22 probe hybridized to poly(A)+ RNA (Fig. 2) and suggest that there is a significant fraction of unbound Tap and Aly present in cell nuclei.
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Discussion |
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Previously, oligo (dT) probes have been used to study the movement of poly(A)+ RNA in living cells and, on the basis of these studies, it was concluded that the majority of poly(A)+ RNA is diffusing freely throughout the interchromatin space in cell nuclei (Politz et al., 1998, 1999). However, dependent on the detection method used, different diffusion coefficients for poly(A)+ RNA movement were estimated. Most recently, by using a caged fluorescein-labeled oligo (dT) probe, a diffusion coefficient of 0.6 µm2/s was estimated for poly(A)+ RNA movement (Politz et al., 1999). However, earlier, a diffusion coefficient of 9 µm2/s was measured by fluorescence correlation spectroscopy (Politz et al., 1998). By measuring fluorescence recovery rates in photobleached areas, we report that poly(A)+ RNA is moving through the nucleoplasm at a significantly slower rate of 0.03 µm2/s. Therefore, we compared the mobility of 2'OMe RNA probes with that of oligodeoxynucleotide probes under identical conditions. We conclude that the discrepancies in rates of poly(A)+ RNA movement that have been measured in this and previous works can be explained by the differences in hybridization properties in living cells between oligodeoxynucleotide and 2'OMe RNA probes (Molenaar et al., 2001). We suggest that our estimate more accurately reflects the in vivo situation. Our data demonstrate that under conditions of in vivo poly(A)+ RNA imaging, a relatively large fraction of oligo (dT) is highly mobile in the cell nucleus and therefore unbound or transiently bound to poly(A)+ RNA. Importantly, we have shown that the localization and kinetics of the 2'OMe (U)22 probe is fully dependent on the presence of a poly(A) tail and that there is at best a very small fraction of unbound probe present that may have led to an overestimation of the poly(A)+ RNA diffusion coefficient. Because hybridization in a living cell is in principle a reversible kinetic process, we cannot exclude that there is some rate of exchange between probe and target molecules during our measurements that can lead to a slight overestimation of the diffusion rate of poly(A)+ RNA.
Recently, it was suggested that the RNA binding proteins PABP2 and TAP move at rates similar to rapidly diffusing poly(A)+ RNA (Calapez et al., 2002). We show that poly(A)+ RNA molecules move at significantly slower rates than previously anticipated, but that PABP2, TAP, and Aly move much more rapidly through the nucleus than poly(A)+ RNA. This finding suggests that a substantial proportion of these proteins is not bound to RNA but is diffusing rapidly throughout the cell nucleus to be available for newly synthesized transcripts.
It should be noted that FRAP analysis provides only an average value for poly(A)+ RNA mobility. Some poly-(A)+ RNAs may move, within a certain range, faster or slower. Previously, we estimated that abundantly synthesized HCMV IE transcripts move through the nucleus at a diffusion rate of 0.13 µm2/s (Snaar et al., 2002), which is fourfold faster than what we measured for poly(A)+ RNA. In any case, our observations are consistent with studies suggesting that poly(A)+ RNA is not transported by free diffusion but, at least at some stages, by an energy-dependent mechanism (Dargemont and Kühn, 1992; Jarmolowski et al., 1994; Calado et al., 2000; Miralles et al., 2000; Snaar et al., 2002).
Consistent with in situ hybridization studies on fixed cells, we observed that poly(A)+ RNA concentrates in speckles in living cells. Notably, this pattern was not observed when we or others (Politz et al., 1998) used an oligodeoxynucleotide probe instead of a 2'OMe RNA probe for detecting poly(A)+ RNA. Microinjected TAMRA-labeled oligo (dT)40 probe revealed a dispersed localization throughout the cell nucleus, but not a staining of speckles. Interestingly, our FRAP and FLIP analyses revealed that the poly(A)+ RNA population inside speckles is mobile and in continuous flux with the nucleoplasm. Only a small amount of the poly(A)+ population that reside in speckles appears to be immobile. Importantly, our observations show that the poly(A)+ population found in speckles is not a stable population of RNAs as suggested previously (Huang et al., 1994). Even when gene transcription is inhibited, poly(A)+ RNA molecules that remain in the nucleus continue to associate and dissociate from speckles and to move throughout the entire nucleus. This observation is consistent with the finding that RNA transport from nucleus to cytoplasm is not dependent on ongoing transcription (Huang and Spector, 1996). Hence, due to their dynamic behavior, it is less likely that the population of poly(A)+ RNA that is localized to speckles plays an essential role in the core organization of speckles by creating binding sites for RNA processing proteins (Sacco-Bubulya and Spector, 2002). In this context, it is worth mentioning that poly(A)+ RNA is not required for the assembly of nuclear speckles in the nuclei of early G1 cells (Ferreira et al., 1994; Gama-Carvalho et al., 1997). Nevertheless, it cannot be excluded that speckle maintenance is a dynamic process and that poly(A)+ RNA plays a role in stabilizing these compartments like mobile heterochromatin protein 1 is responsible for establishing stable heterochromatin domains in cell nuclei (Cheutin et al., 2003; Festenstein et al., 2003).
Another, not necessarily exclusive, possibility is that nuclear poly(A)+ RNA is present in speckles for nonstructural reasons. Because various specific gene transcripts have been found to associate with or to localize to speckles, it has been suggested that speckles play a role in the posttranscriptional processing of RNAs (Xing et al., 1995; Bridge et al., 1996; Smith et al., 1999; Johnson et al., 2000; Hattinger et al., 2002; Shopland et al., 2003). A role for speckles in posttranscriptional splicing is consistent with the observation that splicing factors present in the diffuse nuclear pool of cells lacking speckles are not competent to perform pre-mRNA splicing (Sacco-Bubulya and Spector, 2002). However, there is compelling evidence that the majority of pre-mRNAs are processed cotranscriptionally (Bauren and Wieslander, 1994; Wuarin and Schibler, 1994; Tennyson et al., 1995), and therefore, it is not expected that a high concentration of non- or partially spliced transcripts is present in speckles. Nevertheless, our FRAP and FLIP results show that nearly all nuclear poly(A)+ RNAs move through speckle domains and are therefore consistent with the suggestion that a quality control for correctly spliced or export-competent RNAs takes place in speckles (Johnson et al., 2000). Also, it cannot be excluded that many of the most active, rapidly transcribed genes may have substantial posttransciptional processing. For these purposes, transcripts may transiently interact with some speckle components, and then be released to the nucleoplasm. Interestingly, the import of poly(A)+ RNA into speckles appears to be temperature, and thus energy dependent, which is consistent with the observation that the uptake of microinjected adenovirus pre-mRNAs by speckles was precluded when cells were incubated at 4°C or ATP depleted (Melcak et al., 2001; Kopsky et al., 2002).
In conclusion, speckles may fulfill different functions in the cell nucleus. In addition to playing a role in the assembly, supply, storage, and, possibly, recycling of RNA processing complexes, speckles may represent a checkpoint for whether or not RNAs are appropriately processed and assembled in transport-competent complexes.
The apparently immobile pool of poly(A)+ RNA residing in speckles and nucleoplasm may represent very slow moving RNAs, structural RNAs, as well as incorrectly or slowly processed RNAs. However, the immobile pool may also reflect storage of specific mRNAs. Many mature mRNAs were observed to accumulate in cell nuclei to higher levels than the corresponding precursors (Gondran et al., 1999), and it has been suggested that the nucleus may function as a reservoir for these mRNAs until they are required in the cytoplasm and released by some stimulus. The mechanism by which these mRNAs are retained in the nucleus has not yet been determined though it was shown that some mRNAs are tightly associated with a nuclear matrix structure that remained after nuclear extraction (Gondran et al., 1999). Future work may shed some light on the role that immobile poly(A)+ RNA plays in the cell nucleus.
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Materials and methods |
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Construction and expression of GFP fusion proteins
The cDNA encoding SF2/ASF was generated by RT-PCR and cloned into the pEGFP-C1 vector (CLONTECH Laboratories, Inc.) using the EcoRI and BamHI restriction sites as described previously (Molenaar et al., 2001). The cDNA encoding PABP2 was subcloned from a construct provided by E. Wahle (Martin-Luther-Universitat Halle, Halle, Germany) in the pEGFP-C1 vector. The constructs coding for TAP-GFP and p15 were provided by E. Izaurralde (European Molecular Biology Laboratory, Heidelberg, Germany; Braun et al., 2001), and the construct ALY-GFP was a gift from J. Katahira (Osaka University, Osaka, Japan; Zhou et al., 2000). SF2/ASF-GFP was transfected stably into U2OS cells. All other constructs were transiently expressed in U2OS cells using DOTAP (Roche Diagnostics GmbH). Cells were analyzed 2448 h after transfection and were selected for moderate expression and protein-specific localization.
Cell culture and microinjection
U2OS (human osteosarcoma) cells were cultured on coverslips in 3.5-cm petri dishes (Mattek) in RPMI 1640, without phenol red supplemented with 5% FCS, 0.03% glutamine, and 1000 U/ml penicillin/streptomycin and buffered with 25 mM Hepes buffer to pH 7.2 (all from Life Technologies). Cordycepin (Sigma-Aldrich) was used at 50 µg/ml. Microinjection of probes was performed as described previously (Molenaar et al., 2001). Cells showing moderate levels of fluorescence were selected and analyzed by digital fluorescence microscopy on the day of microinjection.
Live cell imaging
Cells were monitored using a confocal microscope system (model TCS/SP2; Leica). Cells were scanned in 2D in time, with a pinhole setting of 2.5 Airy. During the experiment, the temperature of the cells was maintained at 37°C using a heated ring surrounding the culture chamber (Harvard App. Inc.) and a microscope objective heater (Bioptechs) unless indicated otherwise. The 543-nm He Ne laser was used for TAMRA excitation with the emission window set between 560630 nm. GFP was scanned with the 488-nm line of an Argon laser with the emission window set between 500540 nm. In the double-labeling experiment, GFP and TAMRA were sequentially scanned to avoid cross talk. Images were acquired using a 100x NA 1.4PL APO lens and analyzed with Leica software. Images were further analyzed using Leica software and Adobe Photoshop. To show colocalization, masked images were obtained using the Leica multi-color software package.
Photobleaching experiments and quantitative analysis
For spot bleaching in FRAP and FLIP analysis, the laser beam parking option on the confocal microscope was used. The 543- and the 488-nm lasers were set at 100%, and the duration of the spot bleaching was set such that bleaching resulted in a nearly complete loss of fluorescence in the defined area. In practice, TAMRA-labeled probes were bleached for 5 s and GFP fusion proteins for 3 s. Subsequent images where recorded before, just after, and at different time intervals after bleaching. The length of the time intervals was established depending on the speed of recovery of the fluorescence. For example, for imaging poly(A)+ RNA, 24 images were acquired in three series with increasing time intervals (10 images every 2 s, 10 images every 10 s, and then 4 images every 30 s). For imaging GFPs and control probes, the time intervals were shorter (indicated in the FRAP curves in Results). Quantitative analysis of fluorescence intensities was performed using Leica software and Excel. FRAP recovery intensities were corrected for background intensities. The total cellular fluorescence was measured before and immediately after the bleach pulse to correct for loss of fluorescence during the bleach pulse and during imaging. For determination of the t1/2 values and for the immobile fractions, the intensity values before bleaching were set at 100% and the intensity values directly after bleaching were set at 0%. All intensities measured during recovery were transformed to relative intensities. The t1/2 values, indicating the time points at which 50% of the end-fluorescence intensity (Fend) was reached, were determined from the FRAP curves. Estimation of the effective diffusion coefficients (Deff) from FRAP experiments was performed as described by Yguerabide et al. (1982) using the formula D = ßw2/4t1/2, where w is the radius of the bleached area at e2 intensity and ß is a parameter that depends on the percent bleach. The values for ß were determined for each experiment from a theoretical plot generated from data presented by Yguerabide et al. (1982), and w was estimated using fixed cells expressing GFP. For FLIP experiments, cells were repeatedly imaged and bleached at intervals of 2 s for measuring loss of GFP signal and of 3 s for measuring loss of TAMRA fluorescence. For curve fitting, the program CurveExpert 1.3 has been used.
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Acknowledgments |
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This work was supported by the Netherlands Organization of Scientific Research program 4D Imaging of Living Cells and Tissues (grant 901-34-144).
Submitted: 29 October 2003
Accepted: 22 March 2004
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References |
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