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Department of Cell Biology, Harvard Medical School, Boston, Massachusetts,1 Department of Biochemistry,2 Department of Physics,6 Center for Structural Biology,3 Howard Hughes Medical Institute,4 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee5
Received 18 July 2004/ Returned for modification 12 August 2004/ Accepted 28 September 2004
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ABSTRACT |
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INTRODUCTION |
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In Saccharomyces cerevisiae, the NTC can be isolated as a distinct unit that is composed of at least 11 proteins, many of which have been identified (6, 27, 33). NTC proteins include Prp19p, Syf1p (Ntc90), Cef1p (Ntc85), Clf1p (Ntc77), Prp46p (Ntc50), Cwc2p (Ntc40), Syf2p (Ntc31), Isy1p (Ntc30), Snt309p (Ntc25), and Ntc20p. NTC components, highly conserved across species, are also present in Schizosaccharomyces pombe and human spliceosomal complexes (reviewed in reference 17).
Accumulating evidence suggests that Prp19p is required for the structural integrity of the NTC and its subsequent association with the spliceosome (3, 27, 33). Prp19p interacts directly with a number of NTC components, including Cef1p, Snt309p, and Cwc2p, and is critical for their assembly into a multiprotein complex (5, 6, 27, 35). Prp19p contains three recognized protein motifs: an N-terminal U-box, a predicted coiled-coil (21), and a WD40 repeat domain at its C terminus (Fig. 1B). The WD40 repeat domain interacts with Cwc2p, whereas the N-terminal part of the protein interacts with Cef1p and Snt309p (27). The U-box, despite lacking zinc-chelating residues, is structurally similar to RING finger domains (28). Like many proteins that contain RING domains, both human and budding yeast Prp19ps exhibit E3 ubiquitin ligase activity in vitro (15, 28). Mutations in the U-box that do not disrupt the structural integrity of Prp19p are still unable to rescue function in vivo, suggesting that this enzymatic activity of Prp19p is essential for some aspect of NTC structure or function (28).
Although the above-mentioned studies have suggested models of NTC organization, a fundamental understanding of the composition and assembly of the complex is lacking. We address details of NTC organization here by examining the stoichiometries of Prp19p and Cef1p within the complex. We demonstrate that while Cef1p is monomeric, Prp19p is tetrameric, and we map the domain required for oligomerization and test the functional consequences of disrupting Prp19p tetramerization in vivo. Our data suggest how Prp19p might provide flexibility for structural rearrangements within the spliceosome while still serving as a binding scaffold for numerous proteins.
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MATERIALS AND METHODS |
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Immunoprecipitations and immunoblotting. Native protein lysates were prepared from approximately 2.4 x108 cells as detailed previously (13). To the resultant 1.0 ml of lysate, 2 µg of antihemagglutinin (anti-HA; 12CA5) or anti-myc (9E10) antibody was added to precipitate tagged proteins. Immunoprecipitates were resolved on Novex NuPAGE 4-to-12% bis-Tris gels using NuPAGE MOPS SDS running buffer (Invitrogen, Carlsbad, Calif.) and transferred by electroblotting to polyvinylidene difluoride membrane (Immobilon P; Millipore Corp., Bedford, Mass.). Anti-HA (12CA5) antibodies and anti-myc (9E10) antibodies were used at 2 µg/ml, and antibodies were detected using horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (0.8 mg/ml; Jackson Immunoresearch Laboratories, West Grove, Pa.) at a dilution of 1:50,000. Immunoblots were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). For immunoblots that were quantitated, detection was performed with an Odyssey instrument (Licor Corp., Lincoln, Nebr.) using the protocol and reagents supplied by the manufacturer. Quantitation was performed with Odyssey version 1.2.
Yeast two-hybrid assays. The yeast two-hybrid system used in this study was described previously (16). Various portions of the PRP19 cDNAs were cloned into the bait plasmid pGBT9 and/or the prey plasmid pGAD424 (Clontech, Palo Alto, Calif.) and sequenced to ensure the absence of PCR-induced mutations and that the correct reading frame had been retained.
Two-hybrid screens were performed as described previously (16). To test for protein interactions, both bait and prey plasmids were cotransformed into S. cerevisiae strain PJ69-4A. ß-Galactosidase reporter enzyme activity in the two-hybrid strains was measured using the Galacto-Star chemiluminescent reporter assay system according to the manufacturer's instructions (Tropix Inc., Bedford, Mass.), with the exception that cells were lysed by glass bead disruption. Each sample was measured in triplicate. Reporter assays were recorded either on the BMG luminometer (Bartlett-Williams Scientific, Chapel Hill, N.C.) or the Mediator Phl luminometer (Aureon Biosystems, Vienna, Austria).
Expression of recombinant fusion proteins.
Three constructs spanning the different domains of Prp19p were produced: PRP19 1-58, PRP19 66-141, and PRP19 165-503, along with the full-length protein and the W88A and W88P mutants in the context of the full-length protein. Proteins were expressed in Rosetta(DE3) cells (Novagen). Cells were grown to an optical density at 600 nm of 0.7 and induced with 1 mM isopropyl-ß-D-thiogalactopyranoside. Cells were lysed in 20 mM Tris HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, and 5 mM BME, and proteins were purified using Ni2+-nitrilotriacetic acid columns (QIAGEN) following the manufacturer's instructions. Proteins were eluted from the column by using an imidazole gradient from 20 to 900 mM. In the case of Prp19p 66-141, the His6 tag was cleaved by incubation with thrombin (25 U/
10 mg of protein) for 14 h at 4°C. Proteins were further purified using a Mono-Q 10/10 column (20 mM Tris [pH 8.0], elution 10 to 600 mM NaCl, and 1 mM dithiothreitol [DTT]) (Amersham Pharmacia Biotech).
Gel filtration.
Gel filtration was performed using a 10/30 S200 column (Amersham Pharmacia Biotech). Running buffer was 20 mM Tris (pH 8.0), 100 mM NaCl, and 1 mM DTT. The protein running concentration was 5 mg/ml.
CD and secondary structure estimation. Circular dichroism (CD) measurements were recorded using a Jasco J-810 (Jasco Inc.) spectropolarimeter. Protein was dialyzed into 5 mM sodium phosphate (pH 8.0), 10 mM NaCl, and 1 mM DTT. Spectra were obtained using the average of three scans over the range of 190 to 260 nm with a step size of 0.2 nm and a bandwidth of 1 nm. Secondary structure estimates were obtained using the program K2d (1).
Analytical ultracentrifugation. Sedimentation velocity experiments were conducted with an Optima XLA apparatus (Beckman-Coulter, Fullerton, Calif.), with a four-hole An60Ti rotor and double sector cells with charcoal-filled Epon centerpieces (path length, 1.2 cm) and quartz windows. The value of the radial distance of the bottom of the cell was between 7.13 and 7.15 cm. For sedimentation velocity experiments, samples (397 µl) and reference solutions (400 µl) were loaded into cells. The experiments were conducted at 25°C at a rotor speed of 129,024 x g. Concentration profiles were measured at 280 nm. Partial specific volumes and molecular masses for Prp19p and Prp19p fragments were calculated based on their amino acid compositions using the program Sednterp (20). The velocity scans were analyzed with the program Sedfit (version 8.7) (31). For each analysis, 300 scans collected approximately 2 min apart were analyzed. All size distributions were solved on a radial grid of 1,000 radius values between the meniscus and bottom, a confidence level of P = 0.95, a resolution of n = 300, and sedimentation coefficients between 0.1 and 20 s.
Electron microscopy and image processing. Uranyl formate-stained samples were prepared for electron microscopy as described previously (26). Images were taken with a Philips Tecnai T12 electron microscope at an acceleration voltage of 120 kV with a magnification of x52,000 and a defocus of –1.5 µm using low-dose procedures. After inspection with a JEOL JFO-3000 laser diffractometer, drift-free images were digitized with a Zeiss SCAI scanner using a step size of 7 µm. Three- by three-pixel images were averaged to yield a pixel size of 0.4 nm at the specimen level. A total of 9,725 His6-Prp19p particles were selected from 28 micrographs, and 4,507 His6-Prp19p 165-503 particles were selected from 9 micrographs using WEB, the display program associated with the SPIDER program suite (9), which was used for the subsequent image-processing steps. The His6-Prp19p particles were windowed into 128- by 128-pixel images and subjected to 10 rounds of multireference alignment and K-means classification specifying 200 output classes. Due to the extensive flexibility of the Prp19p tetramers, many class averages only showed three WD40 repeats, whereas the fourth WD40 repeat was averaged out. Therefore, only a subset of class averages is shown below in Fig. 5, in which all four WD40 repeats are clearly resolved. The His6-Prp19p 165-503 particles were windowed into 40- by 40-pixel images and were subjected to 10 rounds of multireference alignment and K-means classification specifying 20 output classes.
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RESULTS |
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Because Cef1p binds directly to Prp19p, we asked whether it, too, was present in more than a single copy in protein complexes in vivo. We constructed a diploid strain containing both Cef1p-HA3 and Cef1p-myc13 and did immunoprecipitations using either anti-HA or anti-myc antibodies that were then blotted with either anti-HA or anti-myc antibodies. In these experiments, Cef1p-HA3 was not able to coimmunoprecipitate Cef1p-myc13 and Cef1p-myc13 could not precipitate Cef1p-HA3 from the diploid strain (Fig. 2C). Therefore Cef1p, unlike Prp19p, does not appear to be present in multiple copies within the NTC in vivo. This situation is conserved in S. pombe, since different epitope-tagged versions of the S. pombe homolog of S. cerevisiae Cef1p, Cdc5p, also failed to interact with one another in the appropriate diploid strain (Fig. 2D).
The analyses performed above left open the possibility that oligomers of Prp19p/Cwf8p exist only outside of the Cef1p/Cdc5p-containing spliceosomal complex. To examine this possibility, an S. pombe diploid strain was constructed with the relevant genotype cdc5-TAP/cdc5-TAP cwf8-HA3/cwf8-myc13. After tandem affinity purification was performed on this strain, the eluate was found to contain both tagged proteins as expected (Fig. 2E, left panels). To determine whether each Cdc5p molecule bound to one molecule of Cwf8p or whether a single molecule of Cdc5p could bind multiple molecules of Cwf8p, the eluate was diluted and subjected to immunoprecipitation with anti-myc or anti-HA antibodies, and the immunoprecipitates were blotted for the presence of Cwf8p-myc13. As would be predicted if each Cdc5p-TAP complex contained multiple copies of Cwf8p, both anti-myc and anti-HA immunoprecipitates contained Cwf8p-myc13 (Fig. 2E, right panel). We could not examine the reciprocal immunoprecipitate by immunoblotting because Cwf8p-HA3 comigrates with the immunoglobulin G heavy chain. However, because Cdc5p is present in single copy in the NTC (Fig. 2D), this result could only be explained if at least two molecules of Cwf8p interacted within the same Cdc5p-containing complex.
Prp19p binds to itself. To further delineate protein-protein interactions involving Prp19p, we performed a stringent two-hybrid screen using an N-terminal fragment of Prp19p containing both its U-box and predicted coiled-coil region (Prp19p 1-131) as bait. The ability of Prp19p 1-131 to fold correctly was confirmed by CD (data not shown). In this screen PRP19 76-134, which encompasses a large portion of the predicted coiled-coil domain, was the only clone isolated multiple times (Fig. 3A), suggesting that this region of Prp19p is capable of interacting directly with itself and Prp19p self-association might be mediated by this domain. To test this we used the entire predicted coiled-coil region of Prp19p 57-144 as both bait and prey and asked if they could interact in the two-hybrid system. As we predicted, Prp19p 57-144 interacts with itself (Fig. 3B). These results are consistent with previous gel filtration chromatography experiments that showed recombinant Prp19p exists in an oligomeric state (data not shown) (33).
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Prp19p forms a stable tetramer.
To define what type of oligomer Prp19p formed, recombinant His6-Prp19p (mass, 58.6 kDa) was purified and analyzed using sedimentation velocity analytical ultracentrifugation. Analysis of the velocity data with the program Sedfit (31) showed that 78% of His6-Prp19p exhibited a sedimentation value (s) of 5.8 (Fig. 4A; root mean square deviation of the fit, 0.0073). This value is consistent with a complex of 230 kDa (Fig. 4B), the same molecular mass as a Prp19p tetramer. The rest of the protein sedimented either as a monomer (s = 2.3; 2.5%) or as a nonspecific aggregate (20%). His6-Prp19p has a frictional ratio value of 1.7, indicating that the tetramer is elongated in shape.
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Structural analysis of the Prp19p tetramer by electron microscopy. In order to understand the architecture of Prp19p tetramers, full-length protein was purified and examined using electron microscopy. Micrographs of negatively stained His6-Prp19p revealed a particle of variable shape, but with each containing an elongated central stalk that was flanked by two globular domains on each side (Fig. 5). A total of 9,725 particles were selected from digitized electron micrographs and subjected to multireference alignment and classification specifying 200 output classes. A representative subset of these two-dimensional projection averages (Fig. 5, panels 1 through 12) emphasizes the variability in the shape of Prp19p. The two globular domains that flank the central stalk on either side show central stain accumulations, giving them a donut-like appearance. These domains appear to be tethered to the central stalk by flexible linkers, as they can either be close together or spread wide apart (Fig. 5, compare panels 8 and 2).
As noted above, Prp19p contains three structural domains, an N-terminal U-box, a central coiled-coil, and C-terminal WD40 repeats that likely adopt a typical-bladed ß-propeller structure (10) (Fig. 1B). The donut-shaped domains, with dimensions of 6.5 by 6.5 nm, are consistent in size and shape with the expected ß-propeller structure, while the central stalk, with its rope-like appearance, is consistent with the expected projection structure of a coiled-coil domain. The small size of the U-box (7.8 kDa) would make it difficult to identify by negative stain electron microscopy.
To confirm our prediction regarding the identity of the donut-shaped domains, we analyzed the C terminus of Prp19p by negative stain electron microscopy. Micrographs of His6-Prp19p 165-503 revealed well-dispersed donut-shaped particles (Fig. 6, upper panel). These monomeric particles looked identical to the domains seen tethered to the central stalk of the full-length Prp19p tetramers (Fig. 5). We selected 4,572 particles from digitized micrographs and used multireference alignment and classification to group them into 20 classes. The resulting projection averages (Fig. 6, lower panel) confirmed that the four donut-like domains seen in the full-length Prp19p tetramers correspond to its WD40 repeats.
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(Fig. 7F). These results indicate that the ability of Prp19p to tetramerize through its central coiled-coil stalk is required for its in vivo function. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise shape and relative orientation of the domains in Prp19p tetramers vary with the four globular WD40 domains flexibly tethered to a central coiled-coil tetramer. Class averages of Prp19p tetramers illustrated the myriad positions and angles that the WD40 repeats can adopt in relation to the stalk-like coiled-coil. It is likely that the flexibility of the WD40 repeats will decrease when other NTC binding partners bind. However, the variability of the Prp19p tetramer structure may be important for numerous transitions that occur during the formation of the active spliceosome. Such flexibility could allow Prp19p to accommodate substantial structural rearrangements during dynamic reorientation of spliceosome components without compromising strong binding interactions with other components of the NTC.
Indeed, structural characterization of the Prp19p tetramer by electron microscopy has provided significant clues as to how Prp19p might act as a central organizing unit within the NTC. Taking into account the plethora of described NTC protein-protein interactions, a model of NTC organization can be constructed highlighting the many protein-protein interdependencies within the NTC (2, 7, 8, 27, 30) (Fig. 8). In addition to creating a binding platform for Snt309p and Cef1p, the distinct spatial environment sampled by each of the four WD40 domains creates four independent protein-protein interaction modules surrounding the coiled-coil domains. As yet, only Cwc2p, an essential RNA recognition motif containing splicing factor, has been shown to interact directly with the C-terminal region of Prp19p (Fig. 8) (27). It is likely that other NTC components also interact with the WD40 repeats, although these interactions could depend on the presence of the full complement of complex members. The ability of Prp19p to interact with numerous NTC components combined with its essential role in NTC stability suggest that the Prp19p tetramer acts as an essential scaffold for the organization of the entire NTC and perhaps other associated factors. It may be for this reason that human PRP19 was identified as the nuclear matrix protein hNMP200 (12). Extension of our structural analysis to include other NTC components should be revealing in this regard.
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Interestingly, one common feature of the otherwise-diverse family of RING/U-box-containing proteins is that they are often integral components of macromolecular assemblies (18, 29). It has been postulated that the spatial organization of the RING structure within these E3 ligase complexes may be essential for positioning the substrate in the correct orientation to optimize the transfer of ubiquitin from the E2 (36, 37). Although highly speculative, proteins that interact with either the Prp19p WD40 repeats or the coiled-coil region might also contact the U-box and be modified to promote changes in NTC organization and/or function during the splicing reaction. If, in fact, Prp19p exhibits E3 ligase activity within the context of the spliceosome, the flexible Prp19p tetramer might be well suited for facilitating the correct orientation of substrates for U-box-directed modification.
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ACKNOWLEDGMENTS |
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This work was supported by the Giovanni Armenise-Harvard Foundation (T.W.), the Howard Hughes Medical Institute (K.L.G.), and National Institutes of Health grant RO1 GM62112 (W.J.C). J.A.R. received training grant support from T32CA09385. C.W.V.K. received training grant support from T32GM08320. M.D.O. is an Agouron Institute Fellow of the Jane Coffin-Childs Foundation, and K.L.G. is an Investigator of the Howard Hughes Medical Institute. The molecular EM facility at Harvard Medical School was established by a generous donation from the Giovanni Armenise Harvard Center for Structural Biology and is maintained by funds from the National Institutes of Health, GM62580 (to T.W.).
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FOOTNOTES |
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