Article |
Address correspondence to K.M. Neugebauer, Pfotenhauerstrasse 108, 01307 Dresden, Germany. Tel.: (49) 351-210 2589. Fax: (49) 351-210 1209. email: neugebau{at}mpi-cbg.de
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Abstract |
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Key Words: Cajal body; snRNP; pre-mRNA splicing; coilin; FRET
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Introduction |
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The assembly of U4/U6 spliceosomal snRNPs involves the annealing of the U4 and U6 snRNAs and stepwise assembly of U4/U6 snRNP specific proteins; subsequently, the U4/U6 snRNP associates with the U5 snRNP to produce the U4/U6U5 tri-snRNP that is active in splicing (Will and Lührmann, 2001). During splicing, snRNPs undergo extensive structural rearrangement; in particular, the tri-snRNP disassembles and U4/U6 snRNAs unwind (Staley and Guthrie, 1998). Thus, if disassembled snRNPs participate in subsequent rounds of splicing, the U4/U6 snRNP and the U4/U6U5 tri-snRNP have to be reassembled (Fig. 1). In yeast, U4/U6 snRNA annealing is promoted by Prp24p and the U6-associated LSm proteins, and the interaction between the Prp24p COOH-terminal domain CT-10 and LSm proteins is important for the U4/U6 snRNP assembly (Mayes et al., 1999; Raghunathan and Guthrie, 1998; Rader and Guthrie, 2002). Similarly, the human LSm proteins were shown to promote annealing of in vitrosynthesized U4 and U6 snRNAs (Achsel et al., 1999). Recently, SART3 (also named p110) was shown to be the human homologue of yeast Prp24p (Bell et al., 2002; Rader and Guthrie, 2002). SART3 was shown to bind directly to the U6 snRNA and to promote U4/U6 snRNP assembly in nuclear extracts (Bell et al., 2002). Given their demonstrated functions in vitro, and because Prp24p binds to LSm proteins, it is likely that SART3 and LSm proteins act synergistically to promote U4/U6 snRNP assembly. In contrast to the LSm proteins, however, SART3 associates exclusively with U6 and U4/U6 snRNPs and is not present in the U4/U6U5 tri-snRNP (Bell et al., 2002). Therefore, SART3 can be used as a marker for specific steps in U4/U6 snRNP assembly.
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Results |
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SART3 preferentially interacts with the U4/U6 snRNP proteins in CBs
Each snRNP contains one or more snRNAs associated with seven conserved Sm proteins (LSm in the case of the U6 snRNP) plus proteins specific for each snRNP (for review see Will and Lührmann, 2001). Biochemical studies have identified five U4/U6 snRNP proteins: 15.5K/NHPX, 61K, hPrp3 (90K), hPrp4 (60K), and USA-Cyp (20K) (Horowitz et al., 1997; Lauber et al., 1997; Nottrott et al., 1999; Makarova et al., 2002). The hPrp3 and hPrp4 proteins form, together with USACyp, a stable complex that interacts with U4/U6 snRNA duplex but not with free U4 or U6 snRNAs (Nottrott et al., 2002). The 61K protein binds directly to the U4 snRNA independent of this complex (Nottrott et al., 2002). To localize the transient complex between SART3 and the U4/U6 snRNP, we tagged three U4/U6 snRNP-specific proteins, hPrp3, hPrp4, and 61K, with CFP or YFP. Western blot analysis revealed that ratios of fluorescently tagged proteins to their endogenous counterparts were 0.25 for 61K, 0.79 for hPrp4 and 2.9 for SART3 (see Fig. S1).
To determine whether the tagged U4/U6 snRNP proteins and SART3 are able to assemble into U4/U6 snRNPs, fluorescently tagged proteins were transiently expressed in HeLa cells and cellular RNA was metabolically labeled with [32P]orthophosphate. Assembled snRNPs were detected by immunoprecipitation with anti-GFP antibodies and coprecipitated snRNAs were analyzed by gel electrophoresis and autoradiography (Fig. 3 B). As a positive control, the anti-Sm antibody precipitated all major snRNAs; the relative abundance of U1, U2, U4, U5, and U6 snRNAs immunoprecipitated by anti-Sm antibodies was unaffected by transfection with any of the expression constructs (unpublished data). The anti-GFP antibodies precipitated preferentially the U4 and U6 snRNAs, indicating that the fluorescent protein tags did not interfere with U4/U6 snRNP assembly.
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In MEF 42coilin/ cells, CBs were successfully restored by expression of mouse coilin tagged with photoactivatable GFP (PAGFP). If not activated by short wavelength laser, PAGFP does not emit fluorescence in visible spectra (Patterson and Lippincott-Schwartz, 2002) and does not affect FRET measurements (unpublished data). When coexpressed with PAGFPmcoilin, YFPSART3, and CFP61K were distributed throughout the nucleoplasm and concentrated in the reconstituted CBs (Fig. 5 A). FRET efficiencies in the nucleoplasm decreased to 25.2 ± 2.7% whereas FRET in CBs increased to 39.4 ± 0.4%. These data indicate that CBs are not strictly required for snRNP assembly but, when CBs are present, they concentrate snRNP assembly intermediates.
SART3 interacts with U6 snRNP LSm proteins in the nucleoplasm
We next mapped the interaction of SART3 with U6 snRNP proteins. As a first step toward localizing SART3U6 snRNP complexes by FRET, SART3CFP was coexpressed with YFP-tagged components of the U6 snRNP. Six of the LSm proteins (LSm2, -3, -4, -6, -7, and -8), which assemble as a hetero-heptamer on the 3' end of the U6 snRNA (Achsel et al., 1999; Mayes et al., 1999), were tested individually for FRET with SART3. It was shown previously that LSm proteins expressed with fluorescence protein tags retain their ability to form the heteromeric complex (Ingelfinger et al., 2002). Moreover, fluorescently tagged SART3 and LSm7 were incorporated into U4/U6 snRNPs (Fig. 3 B). Within the cell nucleus, each YFP-tagged LSm protein was detected both in the nucleoplasm and CBs (Stanek et al., 2003; and data not shown). FRET efficiencies were measured between SART3 and LSm proteins in the nucleoplasm and in CBs within the same cell (Fig. 6 A). Interestingly, FRET efficiencies varied among the LSm proteins, with the highest FRET signals reproducibly obtained from the SART3LSm7 pair (Fig. 6 B). However, for all tagged LSm proteins, a positive FRET signal with SART3 in the nucleoplasm was observed. Surprisingly, none of the SART3LSm pairs produced FRET in CBs (Fig. 6 B).
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Discussion |
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Because the detection of FRET between two fluorescent molecules depends strongly on their proximity (Siegel et al., 2000), this technique can provide strong evidence that two proteins interact, either directly or indirectly, within the cell. The conditions for detecting specific FRET in CBs were identified by examining the protein coilin (Fig. 2) that has been shown to bind to itself in vitro and in a yeast two-hybrid assay (Hebert and Matera, 2000). Recently, it was shown by FRET that coilin interacts with itself in CBs in living cells (Dundr et al., 2004). Using fixed samples, we show here that coilin interacts with itself both in CBs and the nucleoplasm. Moreover, coilincoilin FRET detection was dependent on the position of the fluorescent protein tag, such that only when both partners were tagged on their NH2 termini or COOH termini was FRET observed. This provides an important negative control for the possibility that FRET signals could be generated nonspecifically by the high concentration of chromophores in CBs. Interestingly, coilincoilin interactions were detectable by FRET in both the nucleoplasm and the CB (Fig. 2). Thus, although coilin is a prominent and relatively stable component of CBs (Handwerger et al., 2003; Sleeman et al., 2003; Stanek et al., 2003; Dundr et al., 2004), its role in CB assembly must not be limited to coilin self-interaction, which can also occur in the nucleoplasm.
A similar FRET approach was used to map the subnuclear location of SART3 interactions with the U6 and U4/U6 snRNPs. In CBs, robust FRET signals were measured between SART3 and specific components of the U4/U6 snRNP, namely hPrp3, hPrp4, and 61K; to a lesser extent, these FRET signals were detected in the nucleoplasm (Figs. 4 and 5). These data likely reflect the interaction of SART3 with U4/U6 snRNP complexes rather than free proteins, because similar levels of FRET were detected between SART3 and all three proteins that assemble in step-wise fashion on the U4/U6 snRNP (Nottrott et al., 2002). This interpretation is also supported by the recent finding that the NH2-terminal HAT domain of SART3 interacts directly with hPrp3 and not hPrp4 or 61K (Medenbach et al., 2004). Indeed, FRET between SART3 and the three U4/U6 snRNP proteins was observed when the fluorescent protein was placed on the NH2 terminus of SART3; placement of the tag at the SART3 COOH terminus led to a significant reduction of FRET between full-length SART3 and U4/U6 snRNP-specific proteins. We cannot exclude the formal possibility that complexes in CBs undergo conformational changes more favorable for FRET; however, high FRET signals between SART3 and 61K were detected in the nucleoplasm of cells lacking coilin and CBs. Therefore, any conformational changes influencing FRET detection of the SART3U4/U6 snRNP complex must not be coilin or CB specific. Thus, CBs are enriched in complexes of SART3 with the U4/U6 snRNP.
SART3 is also a component of the U6 snRNP, containing seven LSm proteins (LSm 28), which are not present in any other spliceosomal snRNP (Stevens et al., 2001; Will and Lührmann, 2001; Bell et al., 2002). Positive FRET interactions between SART3 and LSm proteins were detected exclusively in nucleoplasm (Fig. 6). Because SART3 and LSm proteins bind directly to the U6 snRNA (Achsel et al., 1999; Medenbach et al., 2004), we anticipate that the FRET observed between SART3 and LSm proteins in the nucleoplasm is representative of the pool of SART3 associated with the U6 snRNP. Interestingly, robust FRET between SART3 and LSm proteins was only detected with LSm7 (Fig. 6) even though two-hybrid studies indicate that yeast Prp24p can bind LSm2, -3, -4, -5, -7, and -8 through the CT10 domain (Rader and Guthrie, 2002). This may be due to structural constraints within the U6 snRNP and/or fluorescent tags themselves that might favor high FRET between SART3 and LSm7 only. A second possibility is that SART3, unlike yeast Prp24p, preferentially binds LSm7. Consistent with the expectation that the CT10 domain mediates interactions with LSm7, reduced FRET signals were observed when full-length SART3 was tagged on the NH2 terminus and when the COOH-terminal domains of SART3 were deleted (Fig. 6). Note that SART3 and LSm proteins also bind the minor U6atac snRNA and promote assembly of the U4atac/U6atac snRNP (Damianov et al., 2004); therefore a small component of FRET measured between SART3 and LSm7 may represent this less abundant pool of intermediates.
Because SART3 is required for accumulation of U6 snRNPs in CBs (Stanek et al., 2003), detection of SART3LSm protein interactions in the nucleoplasm was expected. Surprisingly, FRET between SART3 and LSm proteins was not observed in CBs (Fig. 6), even though both SART3 and LSm proteins were present in CBs. Because SART3 has not been detected in any U4 snRNA-containing complex lacking U6 (Bell et al., 2002), we anticipate that U6 must be present in the observed CB-specific complex (see Discussion). This finding suggests that U4/U6 snRNP assembly is accompanied by a conformational change, which is unfavorable for FRET between SART3 and LSm proteins.
The observation that the two distinct pools of SART3 (SART3U6 snRNP and SART3U4/U6 snRNP) are unequally distributed between nucleoplasm and CBs leads us to propose that the nucleoplasmic SART3U6 snRNP complex translocates to CBs where U4/U6 snRNP assembly occurs. This is consistent with the previous finding that U6 snRNP accumulation in CBs is SART3-dependent (Stanek et al., 2003). An alternative is that SART3U4/U6 snRNPs detected in CBs were assembled in the nucleoplasm and subsequently translocated to the CB. However, the reduction of U6 snRNP levels in CBs upon overexpression of the dominant negative mutant of SART3 is not accompanied by a decrease of U4 snRNA levels (Table I), suggesting that the U4 snRNA is recruited to CBs independently of the U6 snRNP and/or U4/U6 snRNP assembly, as was shown in Xenopus oocytes (Gerbi et al., 2003). Thus, it is currently unknown how the U4 snRNA is targeted to CBs. These, and previous, results imply that the SART3U6 snRNP complex meets the U4 snRNA in the CB. However, the coilin deficient situation that lacks CBs indicates that CBs are not absolutely essential for U4/U6 snRNP formation, which can also occur in the nucleoplasm. Because the U5 snRNA has been localized to CBs as well (Carmo-Fonseca et al., 1992; Matera and Ward, 1993), tri-snRNP formation may also occur in CBs and lead to the release of SART3 from the U4/U6 snRNP. This model is supported by the observation that SART3 has shorter residency time in the CB than the snRNP proteins SmB and SmD1 (Dundr et al., 2004). Taken together, these data indicate that distinct steps in snRNP assembly are compartmentalized within the cell nucleus and strongly support the role of CBs in U4/U6 snRNP assembly.
Some cells lack morphologically defined CBs, suggesting that CBs per se are not required for cell survival. Indeed, while coilin expression is required for the concentration of many CB-specific components in nuclear bodies, coilin is not strictly required for viability or for snRNA base modification (Tucker et al., 2001; Jady et al., 2003). We show here that the SART3U4/U6 complex forms in the nucleoplasm of coilin/ cells (Fig. 5), indicating that coilin is not required for complex formation and that U4/U6 snRNP assembly likely occurs in the nucleoplasm of cells lacking CBs. Moreover, complementation of the coilin deficiency in these cells through transient transfection of a coilin expression construct restored the localization of SART3U4/U6 snRNP intermediates to CBs.
The observation that specific events in snRNP assembly are compartmentalized in cell nuclei suggests that the concentration of snRNPs in CBs may confer certain cellular advantages. More CBs are present in transformed cells and in actively dividing cells compared with quiescent cells within tissues (Boudonck et al., 1998). In neurons, CB number correlates with cell size (Pena et al., 2001). We speculate that recruitment of snRNP assembly intermediates to CBs may support high metabolic activity of cells. On the one hand, concentration of nonfunctional snRNPs in CBs may increase splicing efficiency, by sequestering them away from nucleoplasm where they might compete with active snRNPs in the splicing process. On the other hand, the concentration of inactive snRNPs in CBs might either promote efficient snRNP assembly or alternatively contribute to the accuracy of snRNP assembly in a manner analogous to SMN protein function, in which proper assembly of Sm and LSmprotein heteroheptamers onto snRNAs is regulated (Yong et al., 2004). Indeed, CBs may self-assemble in highly active cells; in this case, CB formation would be coilin dependent and driven by high levels of splicing and/or de novo snRNP synthesis.
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Materials and methods |
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FRET measurement
HeLa cells, mouse embryonic fibroblasts 42coilin/ and 26coilin+/+ (gift of A. Greg Matera) were cultured in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum (GIBCO BRL), penicillin, and streptomycin (GIBCO BRL). Cells were transfected with fluorescent protein-tagged constructs using Fugene 6 (Roche), grown for 2428 h and fixed in 4% PFA/Pipes for 10 min at room temperature. After rinsing with MgPBS (PBS supplemented with 10 mM Mg2+) and water, cells were embedded in glycerol containing 2.5% 1,4-Diazabicyclo [2.2.2]octane (DABCO; Sigma Aldrich) as an antifade reagent. FRET was measured by acceptor photobleaching method (Bastiaens et al., 1996; Karpova et al., 2003). FRET measurement was performed on the Leica TCS SP2 confocal microscope using the FRET acceptor photobleaching protocol (Leica). The HCX PL APO 100x/1.400.7 oil CS and HCX PL APO 63x/1.320.6 oil CS objectives and Ar 20m Watt laser were used. The 454-nm laser line was used for CFP detection, the laser line 514 nm was used for detection and photobleaching of YFP. For YFP detection, laser intensity was set to 2%. YFP was bleached by three consecutive pulses using 20% laser intensity. Minimal CFP bleaching (02%) was observed and was not taken into account for the calculation of FRET efficiency. The gain of the photomultiplier detectors was adjusted to obtain the optimal dynamic range. The CFP fluorescence was measured before (CFPbefore) and after (CFPafter) the YFP bleaching and apparent FRET efficiency calculated according to the equation FRETefficiency[%] = (CFP after CFP before) x 100/CFPafter. First, a region in the nucleoplasm was bleached and FRET efficiency measured. In the same cell, the region (of the same area as in the nucleoplasm) was then bleached around a selected CB. The efficiency of FRET was measured in CB area only. Unbleached regions of the nucleoplasm and CB of the given cell were always used as a negative control. Mean of unbleached regions were 100% for each pair tested. 10 cells were measured in each experiment. Standard deviations of individual experiments reflecting differences between individual cells were 2030% of the mean. Experiments were done in duplicates or triplicates and average of means ± standard error of the mean are presented.
Immunoprecipitation and Western blotting
HeLa cells were grown on a 15-cm Petri dish, transfected with either GFP, GFPSART3, SART3GFP, GFPhPrp3, GFPhPrp4, CFP61K, and CFPLSm7, and grown for 28 h. Cells were labeled before harvesting for 12 h with [32P]orthophosphate (100 µCi/plate). Cells were placed on ice, washed three times with ice cold MgPBS, scraped in NET-2 buffer (50 mM TRIS-Cl, pH 7.5, 150 mM NaCl, 0.05% Nonidet P-40) supplemented with complete mix of protease inhibitors (Roche) and sonicated three times for 30 s on ice. The cell extract was centrifuged at 13,000 rpm and supernatant was incubated either with a monoclonal anti-Sm antibody (Y12) in the case of nontransfected cells or with goat antiGFP antibodies (raised against bacterially expressed full-length EGFP and obtained from David Drechsel, MPI-CBG, Dresden, Germany) for 4 h at 4°C. RNA was extracted using phenol/chloroform, resolved on denaturing polyacrylamide gel, and snRNAs detected by Phosphoimager FLA-3000 film (Fuji).
Alternatively, cells expressing YFPSART3, CFP61K, or YFPhPrp4 were extracted in NET-2 buffer and 10 µg of total protein were resolved on 7.5% polyacrylamide gel and proteins detected by Western blotting. Polyclonal antisera specific for the COOH terminus of SART3 (Stanek et al., 2003), 61K (Makarova et al., 2002), and anti-hPrp4 (Nottrott et al., 2002) were used in this analysis.
In situ hybridization
Digoxigenin-labeled probes directed against human U4 snRNA were obtained by PCR as described previously (Bell et al., 2002) using pSPU4b (Black and Pinto, 1989) as a template. HeLa cells were transfected with SART3GFP or CT10
RRMGFP using Fugene 6, and after 24 h fixed in 4% PFA for 10 min. Cells were permeabilized with 0.5% Triton X-100 for 5 min and in the case of untransfected control incubated with anti-coilin antibody (5P10; Almeida et al., 1998) provided by M. Carmo-Fonseca (University of Lisbon, Lisbon, Portugal) followed by treatment with antimouse antibody conjugated with FITC (Jackson ImmunoResearch Laboratories). Cells were then fixed in 4% PFA for 5 min, quenched for 5 min in 0.1 M glycine/0.2 M Tris, pH 7.4, and incubated with dig-labeled anti-U4 probe in 2x SSC/50% formamide/10% dextran sulfate/1% BSA for 60 min at 37°C. After washing in 2x SSC/50% formamide, 2x SSC, and 1x SSC, the probe was detected by anti-digoxygenin antibody conjugated with TRITC (Jackson ImmunoResearch Laboratories). Images were collected using the DeltaVision microscope system (Applied Precision) coupled with Olympus IX70 microscope. Stacks of 25 optical sections with z-step set to 200 nm were collected per sample and subjected to mathematical deconvolution (SoftWorx; Applied Precision).
Indirect immunofluorescence
HeLa cells were transfected with SART3GFP or CT10
RRMGFP using Fugene 6. After 24 h the cells were fixed in 4% PFA (Sigma-Aldrich) for 10 min, permeabilized for 5 min with 0.2% Triton X-100 (Sigma-Aldrich), and incubated with anti-61K antibody (Makarova et al., 2002; gift of R. Lührmann, MPI, Göttingen, Germany) and in the case of untransfected control also with monoclonal antibody anti-coilin (5P10). Secondary antirabbit antibodies conjugated with TRITC and antimouse antibodies conjugated with FITC (Jackson ImmunoResearch Laboratories) were used. Images were collected using DeltaVision microscope system as described above. MEF 42coilin/ cotransfected with PAGFPmcoilin, CFP61K, and YFPSART3 were fixed and permeabilized as above and mcoilin was detected by rabbit anti-coilin antibodies (R288; gift of E.K.L. Chan, University of Florida, Gainesville, FL); Andrade et al., 1991) and antirabbit antibodies conjugated with Cy5 (Jackson ImmunoResearch Laboratories). Cy5 was detected on Leica TCS SP2 confocal microscope after FRET measurement.
Measurement of fluorescence intensities
Fluorescence intensities were quantified with SoftWorx software using deconvolved images (see In situ hybridization) as described previously (Stanek et al., 2003). The optical sections were merged and the intensities in random regions of the nucleoplasm divided by the region area was taken as the value to which the intensities within the CBs were compared. CB area was defined by SART3EGFP constructs or by coilin labeling; intensities of U4 snRNA or 61K protein were measured within the CB and divided by the CB area. Data were collected from 20 cells.
Online supplemental material
Figure S1 shows Western blot analysis of fluorescently tagged proteins. HeLa cells were transfected with 61K, hPrp4, or SART3 proteins tagged fluorescently and cell extracts were made after 24 h. Proteins were resolved by SDS-PAGE, transfered to a nitrocellulose membrane, and tagged proteins as well as their endogenous counterparts were detected by appropriate antibodies: (lanes 1 and 2) anti-61K; (lanes 3 and 4) anti-hPrp4; and (lanes 5 and 6) anti-SART3. Extract from nontransfected cells (NT; lines 1, 3, and 5) was used as a negative control. Intensities of protein bands were determined by TotalLab. Online supplemental materials are available at http://www.jcb.org/cgi/content/full/jcb.200405160/DC1.
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Acknowledgments |
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This work was supported by grants from the American Cancer Society (RPG-00-110-01-MGO) and the Deutsche Forschungsgemeinschaft (NE 909/1-1) and by the Max Planck Gesellschaft.
Note added in proof. Evidence that the U4/U6 snRNP accumulates in CBs when tri-snRNP formation is inhibited was recently published (Schaffert, W., M. Hossbach, R. Heintzmann, T. Aschel, and R. Lührmann. 2004. EMBO J. 23:30003009).
Submitted: 27 May 2004
Accepted: 5 August 2004
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