Article |
Address correspondence to Maria Carmo-Fonseca, Instituto de Medicina Molecular, Faculdade de Medicina, Avenida Prof. Egas Moniz, 1649-028 Lisboa, Portugal. Tel.: 351-21-7934340. Fax: 351-21-7951780. E-mail: carmo.fonseca{at}fm.ul.pt
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Abstract |
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Key Words: mRNA; PABP2; TAP; nucleus; photobleaching
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Introduction |
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In the present work, GFP was fused to two distinct mRNP-binding proteins (poly[A]-binding protein II [PABP2] and TAP) and the mobility of the tagged complexes was analyzed by quantitative photobleaching techniques. PABP2 binds to the growing poly(A) tails formed at the 3' ends of nearly all eukaryotic mRNAs (Wahle, 1991). PABP2 cooperates with the cleavage and polyadenylation specificity factor to stimulate the activity of poly(A) polymerase, the enzyme that catalyses polyadenylation. The cleavage and polyadenylation reactions are currently thought to be coupled to splicing of the last intron and to occur at the same time as, or just before, transcription termination (Bauren et al., 1998; Dye and Proudfoot, 1999, 2001). This implies that GFPPABP2 will bind to nearly terminated and spliced transcripts. Because not all poly(A) RNA is exported from the nucleus, we aimed at achieving a more specific visualization of mRNAs in transit to the cytoplasm by using GFP fused to the export factor TAP. TAP binds directly to the constitutive transport element of viral RNAs and is required for the nucleo-cytoplasmic export of cellular mRNAs in vertebrates, Caenorhabditis elegans, and Saccharomyces cerevisiae (for reviews see Görlich and Kutay, 1999; Nakielny and Dreyfuss, 1999; Conti and Izaurralde, 2001).
Here we have analyzed the effect of energy depletion on the intranuclear mobility of mRNP complexes tagged with GFPPABP2 and GFPTAP. For comparison, we determined the mobility rates of large dextrans microinjected into the nucleus of cells depleted of ATP or incubated at 22°C. The results suggest that energy-dependent processes influence specifically the traffic of mRNPs in the living cell nucleus.
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Results |
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Kinetics of GFPPABP2
Having established that mRNP complexes can be tagged by GFPPABP2, we next performed FRAP experiments to analyze their mobility in the nucleus of living HeLa cells. A defined area in the nucleoplasm was bleached irreversibly by a single, high-powered spot laser pulse. The recovery of fluorescence signal in the bleached area, which is a consequence of the movement of nonbleached molecules into that region, was recorded by time-lapse imaging (Fig. 2).
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For quantitative studies, we selected cells containing the minimal detectable level of GFP fusion proteins in order to avoid potential artifacts caused by overexpression. To monitor the expression level of GFP fusion proteins, the fluorescence intensity of each transfected nucleus was determined using the same detection parameters as those applied during the imaging stage of photobleaching experiments. Because a fraction of poly(A) RNA appears to be retained in the nucleus, where it accumulates in so-called nuclear speckles or clusters of interchromatin granules (Huang et al., 1994), these domains were systematically excluded in all photobleaching measurements.
Quantitative FRAP yields information about the relative mobility of the fluorophore: the effective diffusion coefficient (D) and the fraction of fluorophore that is mobile (White and Stelzer, 1999; Reits and Neefjes, 2001). D indicates the surface area randomly sampled by the fluorophore in a given time, whereas the mobile fraction indicates how much of the fluorophore is available for recovery. To derive both D and the mobile/immobile fractions, the recovery of relative fluorescence intensity within the bleach region is plotted as a function of time (Fig. 3 a, red staining). A function is then fitted to this curve (Fig. 3 a, blue staining). GFP fusion proteins are considered to have a high mobility if they have both a high D and a high mobile fraction.
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For comparison purposes, we analyzed cells expressing GFP, which moves relatively freely throughout the nucleus and cytoplasm. In these cells, the fluorescence signal recovers to the prebleach value in <1 s (Fig. 4 A), and a similar behavior is observed in cells expressing GFPPABP2dm, the mutant form of PABP2 with impaired ability to bind poly(A) RNA (Fig. 4 B). In contrast, GFPPABP2 requires >7 s to recover completely (Fig. 4 C). The calculated D value for GFPPABP2 is 0.6 µm2s-1, whereas for the mutant it is 4.2 µm2s-1. This indicates that the kinetics of wild-type GFP fusion molecules is approximately sevenfold slower than mutant proteins. Taking into account that both wild-type and mutant GFP fusion proteins have the same molecular size but differ in their ability to bind to poly(A) RNA, the retardation of GFPPABP2 molecules is most likely a consequence of binding to mRNP complexes.
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To compare the nuclear mobility of GFPPABP2 bound to RNP complexes with that of exogenous particles with similar size, FRAP experiments were performed on cells microinjected in the nucleus with fluorescein-labeled dextrans of 0.58 and 2 x 106 D. These probes, which are not degraded or metabolized by cells, have been previously used for photobleaching recovery measurements within the nucleus (Seksek et al., 1997). The estimated D value for the 580-kD dextran is 0.95 µm2s-1, whereas for the 2,000-kD dextran, D is 0.53 µm2s-1 (Fig. 6). In both cases, the recovery was incomplete, with immobile pools of 5 and 14%, respectively. This implies that Brownian motion is insufficient to sustain constant movement of large particles within the size range of mRNPs. Thus, the finding that poly(A) RNP complexes tagged with GFPPABP2 are completely mobile in the nucleus (immobile fraction is 0%) argues that mRNP movement involves more than simple diffusional processes.
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Energy-dependent mobility of GFPTAP
Next, photobleaching experiments were performed using a GFP-tagged version of the mRNA export factor TAP. TAP associates with cellular mRNPs and is thought to promote their export by interacting with nuclear pore proteins during translocation (Bachi et al., 2000). The NH2-terminal region of the TAP protein includes a noncanonical RNP-type RNA binding domain that exhibits general RNA binding activity (Liker et al., 2000) and four leucine-rich repeats, which play an essential role in TAP-mediated export of mRNA (Braun et al., 2001).
A TAP fragment comprising residues 371619 (which excludes both the RNA binding domain and the leucine-rich repeats; Bachi et al., 2000) was fused to GFP and used in photobleaching experiments (Fig. 11). The recovery curve shows that this protein moves with kinetics similar to that of GFPPABP2dm (D values of 4.3 and 4.2 µm2s-1, respectively), suggesting that it does not assemble into a larger macromolecular complex. In contrast, the fluorescence recovery of GFP fused to full-length TAP was significantly slower (D = 1.2 µm2s-1), as expected from its binding to mRNP complexes. Compared with the diffusion coefficient of GFPPABP2 (0.6 µm2s-1), GFPTAP has a faster diffusion rate. FLIP analysis shows that this apparent higher mobility is caused by the presence of two distinct GFPTAP populations in the nucleus, a slow-moving fraction of molecules bound to mRNP complexes and a fast-moving population of unbound molecules (to be described in detail elsewhere). In contrast, GFPPABP2 forms a single population of molecules, the vast majority of which are bound to poly(A) RNPs (Fig. 5).
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Discussion |
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Here we estimated a diffusion coefficient of 0.6 ± 0.03 µm2s-1 for RNP complexes containing GFPPABP2 in the nucleus. A very similar value (0.6 ± 0.1 µm2s-1) was reported by Politz et al. (1999) using fluorescein-labeled oligo(dT) hybridized to nuclear poly(A) RNA. The diffusion coefficient for a particle in a solution is given by the Stokes-Einstein formula, D = kT/6Rh, which correlates the hydrodynamic behavior of a sphere with the absolute temperature T, the viscosity of the solution
, the Boltzmann constant k, and the hydrodynamic radius of the particle Rh (for a recent review see Reits and Neefjes, 2001). Because viscosity is highly dependent on temperature, this equation implies that changes in diffusion coefficients are expected in response to temperature variation. Because the viscosity of water increases
30% when temperature is lowered from 37°C to 2223°C, the diffusion coefficient for a particle in the nucleus should decrease by a similar order of magnitude. Consistent with this prediction, we observe a reduction of
25% in the diffusion coefficients of the 2,000-kD dextran and the mutant form of PABP2 when the temperature is lowered from 37°C to 22°C. Likewise, an
30% reduction in recovery rate was reported for soluble GFP targeted to the endoplasmic reticulum when FRAP experiments were performed at 37°C and 23°C (Reits and Neefjes, 2001). In contrast to these results, other studies did not detect a significant effect of reducing the temperature to 23°C on the mobility rate of either GFP-tagged nuclear proteins (Phair and Misteli, 2000) or nuclear poly(A) RNA hybridized with fluorescein-labeled oligo(dT) (Politz et al., 1999).
Contrasting to the observed 25% reduction in the recovery rates of both the large dextran and the RNA bindingdeficient GFPPABP2 mutant, lowering the temperature from 37°C to 22°C caused an
60% reduction in the diffusion coefficient of wild-type GFPPABP2, and a similar effect was observed after depleting the cells of ATP. Thus, energy-depleting treatments act specifically on the mobility of the fusion protein that binds to poly(A) RNA, arguing against a general effect caused by an alteration of the intranuclear environment induced by these treatments. Using a very different technical approach, Politz et al. (1999) measured the movement of nuclear poly(A) RNA and estimated the same apparent diffusion coefficient at both 37°C and 23°C. The method devised by these authors consisted of hybridizing chemically masked (caged) fluorescein to poly(A) RNA, using laser spot photolysis to uncage the fluorochrome, and measuring the radial distance that the signal traveled from the uncaging site. The radial movement of signal was calculated and plotted over time, and the diffusion coefficient was estimated from the slope of the unweighted least-squares fit line (Politz et al., 1999). The different methods used for both poly(A) RNA visualization and quantitative analysis are probably on the basis of the discrepancy between the conclusions of this earlier study and our present data.
Although the mechanism underlying the movement of poly(A) RNPs within the nucleus remains unknown, a parallel can de drawn with the process of translocation through the nuclear pore complex. Early studies showed that energy depletion blocked nuclear import and arrested the molecules in transit at the nuclear pore complexes (Newmeyer and Forbes, 1988; Richardson et al., 1988). Later, GTP hydrolysis by Ran was shown to drive transport, presumably by powering the actual translocation through the pore. However, more recent evidence revealed that the translocation step, per se, is not directly coupled to GTP hydrolysis. Rather, it appears that the translocation process occurs by facilitated diffusion, at least for relatively small cargoes (for review see Görlich and Kutay, 1999). According to this view, it has been proposed that energy depletion inhibits nuclear import by blocking the terminal release of molecules in transit from the nuclear pore into the nucleoplasm (Ribbeck and Görlich, 2001).
Although at present the mechanistic details involved in mRNP traffic inside the nucleus are completely unknown, one possibility is that in order to escape immobilization imposed by the densely packed nuclear interior, mRNPs rely on ATP-dependent enzymatic activities. Putative candidates to maintain mRNP particles in a mobile state are ATP-dependent RNA helicases, such as Dbp5p, which participates in the export of mRNAs out of the nucleus (Schmitt et al., 1999; Strahm et al., 1999). Although Dbp5p was first predominantly detected in the cytoplasmic side of nuclear pore complexes, a recent study shows that in C. tentans, this helicase binds mRNPs cotranscriptionally and accompanies the particle to the pores (Zhao et al., 2002). Additional candidates include actin-related ATPases and actomyosin-based molecular motors. A nuclear isoform of myosin I has been recently identified in a complex with RNA polymerase II (Pestic-Dragovich et al., 2000), and it is well established that mRNAs in the cytoplasm can be transported by an actomyosin-driven mechanism (Long et al., 1997; Takizawa et al., 1997). Furthermore, an increasing number of reports describe the association of actin and/or actin-related proteins (Arps) with chromatin complexes (for review see Boyer and Peterson, 2000). Actin has also been found associated with Balbiani ring mRNA (Percipalle et al., 2001) and with the nucleoplasmic filaments of nuclear pore complexes (Hofmann et al., 2001), thus suggesting an involvement in the RNA export pathway. The experimental approach described in this study should be applicable for future screening and identification of proteins that play a role in the intranuclear mobility of mRNP complexes.
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Materials and methods |
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All GFP fusion constructs were obtained by subcloning into the appropriate pEGFP-C vector (CLONTECH Laboratories, Inc.). We have previously described the construction and properties of GFPPABP2 and GFPPABP2dm (Calado and Carmo-Fonseca, 2000; Calado et al., 2000). GFPTAP and GFPTAP 371619 were also previously described (Bachi et al., 2000). The pEGFP-coilinPABP2 plasmid was constructed by subcloning the cDNA of coilin into the pEGFP-PABP2 vector. The cDNA of coilin was obtained from pBluescript SK(-) coilin (Bohmann et al., 1995) through restriction with BamHI and PvuII and purification with the Qiaex II kit (QIAGEN). This double restriction deletes the DNA sequence that encodes for the 108 COOH-terminal amino acids of coilin. As a result, PABP2 is fused to a COOH-terminally truncated form of coilin. The pEGFP-PABP2 vector was first restricted with Sal I and the protruding ends generated were filled in. This vector was then restricted with Bgl II and ligated to the coilin-coding fragment. DNA for transfection was purified using the plasmid DNA midiprep kit (QIAGEN). HeLa cells were transfected with FuGene6 reagent (Roche Biochemicals) using 1 µg of DNA and analyzed 1624 h after transfection. FITC-labeled dextrans with average molecular sizes of 580 and 2,000 kD (Sigma-Aldrich) were diluted to 200 µg/ml in water and microinjected into the nucleus of HeLa cells as previously described (Almeida et al., 1998). Actinomycin D (Sigma-Aldrich) was used at 5 µg/ml from a stock solution of 5 mg/ml in DMSO. A stock solution of 0.6 M 2-deoxy-D-glucose was prepared in water and sodium azide was used from a 1 M stock solution freshly prepared in DME/F-12. Energy depletion experiments were performed by incubating cells in 12 mM 2-deoxy-D-glucose and 20 mM sodium azide for 3060 min at 37°C. To stain selectively active mitochondria, the cells were incubated with rhodamine 123 (Molecular Probes) for 510 min.
Cellular fractionation and immunoprecipitation
Immunopurification of hnRNP complexes from HeLa cells transfected with pEGFP-PABP2 or pEGFP-PABP2dm was performed according to Piñol-Roma et al. (1990). In brief, cells were rinsed twice with cold PBS, scraped with a rubber policeman into 1 ml of cold immunopurification buffer (IPB), and lysed by passing through a 25-gauge needle. The nuclei were pelleted, resuspended in 500 µl IPB, disrupted by sonicating, and layered in an equal volume of 30% sucrose cushion. After centrifugation at 4,000 g for 15 min at 4°C to remove nucleoli, chromatin, and other insoluble nuclear structures, the nucleoplasm-enriched fraction that overlays the cushion was collected. When required, RNase A was directly added onto this fraction at the final concentration of 100 µg/ml and digestion was allowed to proceed at 30°C for 10 min (Piñol-Roma, 1999).
Prior to immunoprecipitation, antihnRNP C (4F4; Choi and Dreyfuss, 1984) or anti-GFP (Roche Biochemicals) monoclonal antibodies were covalently conjugated to protein ASepharose beads (CL-4B; Amersham Biosciences) or protein GSepharose beads (Sigma-Aldrich), respectively. For the immunoprecipitation, 25 µl (packed volume) of beads were used for each 10-cm plate. The beads were first washed with IPB and then incubated with the nucleoplasmic fraction for 10 min at 4°C with rocking. After washing, the immunoprecipitation complexes were eluted by boiling the beads in SDS-PAGE sample buffer. Immunoprecipitates were analyzed by Western blotting using antihnRNP C and anti-GFP antibodies. SDS-PAGE and Western blotting were performed as previously described (Almeida et al., 1998).
Electrophoretic mobility shift assay
A radiolabeled probe was synthesized by 5' end labeling of an A250 oligonucleotide with [-32P]ATP using T4 polynucleotide kinase (Fermentas). PABP2 was purified from calf thymus as previously described (Wahle et al., 1993). 10 nM 32P-labeled A250 probe was mixed with an equimolar amount of PABP2 diluted in 50 mM Tris (pH 8.0), 100 mM KCl, 10% glycerin, 0.5 mM DTT, 0.01% NP-40, 0.2 mg/ml BSA, 2.6% PVA, and 2 mM MgCl2 and incubated for 30 min at either 37°C or room temperature (22°C). A 1,000-fold excess (10 µM) of unlabeled A250 oligonucleotide was then added to the mix. Aliquots were removed at the indicated times and directly loaded onto the running gel. Complexes were resolved by electrophoresis through nondenaturing 6% polyacrylamide gels (80:1 acrylamide/bisacrylamide ratio). Gels were run at 4°C for 2 h at 20 V/cm and analyzed using the Molecular Dynamics PhosphorImager.
Confocal microscopy and image analysis
Live cells were imaged at 37°C or 22°C, maintained by a heating/cooling frame (LaCon GbR) in conjunction with an objective heater (PeCon GmbH). Images were acquired on a ZEISS LSM 510 with the planapochromat 63x/1.4 objective. EGFP fluorescence was detected using the 488-nm laser line of an Ar laser (25 mW nominal output) in conjunction with an LP 505 filter. Each FRAP analysis started with three image scans, followed by a single bleach pulse of 37 ms on a spot with a diameter of 25 pixels (0.71-µm radius). A series of 97 single-section images were then collected at 78-ms intervals, with the first image acquired 2 ms after the end of bleaching. Image size was 512 x 50 pixels and the pixel width was 57 nm. For imaging, the laser power was attenuated to 0.10.2% of the bleach intensity.
For each time series, the background and nuclear regions were identified using an implementation of the ICM segmentation algorithm (Besag, 1986; Fwu and Djuriç, 1996) in Mathworks Matlab software. The average fluorescence in the nucleus T(t) and the average fluorescence in the bleached region I(t) were calculated for each background-subtracted image at time t after bleaching. FRAP recovery curves were normalized according to Phair and Misteli (2000),
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Quantitative FRAP analysis
Nucleoplasmic diffusion coefficients were determined essentially as described previously (Axelrod et al., 1976; Phair and Misteli, 2000). We assume that fluorescent molecules can be either mobile (with a diffusion coefficient D) or immobile. The initial concentration of fluorophore inside the bleached zone is C0, being the percentage of immobile molecules (ranging from 0 to 1). Total fluorescence observed inside the bleached spot is then the sum of the fluorescence of the mobile molecules and the fluorescence of the immobile ones. After bleach, the immobile fraction fluorescence is constant inside the bleached spot (Axelrod et al., 1976):
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(Axelrod et al., 1976), where D is the characteristic time of diffusion and is related with the diffusion coefficient by D = w2/4
D. The total normalized fluorescence is then
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The bleaching characteristics of the laser were estimated using fixed cells expressing GFP. The post-bleaching concentration of fluorophore at distance r from the center of the bleached region was fitted to a Gaussian laser profile (Axelrod et al., 1976),
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The series solution for the fluorescence recovery was truncated after 40 terms (Phair and Misteli, 2000), assuring that neglected terms made an insignificant contribution. We estimated the bleach constant, the immobile fraction, and the characteristic diffusion time for each set of FRAP replicates using a weighted least squares algorithm (Bell et al., 1996) implemented in Mathematica (WOLFRAM Research).
FLIP analysis
For FLIP experiments, cells were repeatedly bleached at intervals of 3.5 s and imaged between bleach pulses. Bleaching was performed by 278-ms bleach pulses on a spot with a diameter of 35 pixels (2.1-µm radius). Repetitive bleach pulses were achieved by taking advantage of the trigger interface for LSM 510. An electronic oscillator circuit was built to create pulses with a user-defined frequency. When connected to the LSM 510, it would then trigger the bleaching events. A series of 350 images was collected for each cell with laser power attenuated to 1% of the bleach intensity. Nuclear fluorescence of selected areas in FLIP experiments was measured using the ROI mean function of the LSM 510 physiology package. The data were then background subtracted and normalized to correct the loss of fluorescence caused by imaging, in a way similar to FRAP, but using an adjacent cell to estimate T(t) and T0. Loss of fluorescence due to imaging could reach 2025% over the time course of the experiment.
Nuclear fluorescence data from FLIP experiments was fitted to a single exponential with rate constant R (Fig. 5 A) and to a nonlinear curve corresponding to the sum of two exponentials,
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Footnotes |
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A. Calado's present address is Institut für Biochemie, ETH Zentrum, 8092 Zürich, Switzerland.
* Abbreviations used in this paper: FLIP, fluorescence loss induced by photobleaching; IPB, immunopurification buffer; mRNP, messenger ribonucleoprotein particle; PABP2, poly(A)-binding protein II.
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Acknowledgments |
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This study was supported by Fundação para a Ciência e Tecnologia, Portugal, and by the European Commission (contract BMH4-98-3147). A. Calado was a fellow from the Gulbenkian PhD program.
Submitted: 11 March 2002
Revised: 29 October 2002
Accepted: 31 October 2002
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