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Symplekin is a dual location protein that has been localized to the cytoplasmic plaques of tight junctions but also occurs in the form of interchromatin particles in the karyoplasm. Here we report the identification of two novel and major symplekin-containing protein complexes in both the karyo- and the cytoplasm of Xenopus laevis oocytes. Buffer-extractable fractions from the karyoplasm of stage IV–VI oocytes contain an 11S particle, prepared by immunoselection and sucrose gradient centrifugation, in which symplekin is associated with the subunits of the cleavage and polyadenylation specificity factor (CPSF). Moreover, in immunofluorescence microscopy nuclear symplekin colocalizes with protein CPSF-100 in the “Cajal bodies.” However, symplekin is also found in cytoplasmic extracts of enucleated oocytes and egg extracts, where it occurs in 11S as well as in ca. 65S particles, again in association with CPSF-100. This suggests that, in X. laevis oocytes, symplekin is possibly involved in both processes, 3′-end processing of pre-mRNA in the nucleus and regulated polyadenylation in the cytoplasm. We discuss the possible occurrence of similar symplekin-containing particles involved in mRNA metabolism in the nucleus and cytoplasm of other kinds of cells, also in comparison with the nuclear forms of other dual location proteins in nuclei and cell junctions.
Cell biologists had recently to recognize, much to their surprise, that certain proteins appear, often in the same cells, as “dual location proteins,” i.e., as general constituents of two rather distant and different structures: On the one hand, they occur as components of cytoskeletal plaques of a specific kind of intercellular junction, and on the other hand, they are located in karyoplasmic, interchromatinic granules, even in cells devoid of any junctions. Examples include the plakophilins PKP 1–3, typical of desmosomal plaques (Mertens et al., 1996 ; Schmidt et al., 1997 , 1999 ; Bonnéet al., 1999 ), the adherens junction proteins, ARVCF (Borrmann, 2000 ; Borrmann et al., 2000 ; for cDNA transfection experiments see also Mariner et al., 2000 ), afadin (Mandai et al., 1997 ) and protein 4.1 (Krauss et al., 1997 ; Lallena et al., 1998 ), and the tight junction plaque proteins ZO-1 (Gottardi et al., 1996 ), symplekin (Keon et al., 1996 ), Ash-1 (Nakamura et al., 2000 ), and ZONAB (Balda and Matter, 2000 ). Obviously, this constitutively dual location at junctions and in nuclei has to be distinguished from observations of transient nuclear accumulations of certain other junctional plaque proteins in special stages of the cell cycle or differentiation or upon expression of certain transfected cDNAs or genes (for examples see, e.g., Funayama et al., 1995 ; Karnovsky and Klymkowsky, 1995 ; Behrens et al., 1996 ; Huber et al. 1996 ; Molenaar et al., 1996 ; Schneider et al., 1996 ; Yost et al., 1996 ; Daniel and Reynolds, 1999 ; for reviews see Behrens, 2000 ; Hübner et al., 2001 ).
Such a constitutively dual localization has also been reported in many diverse cultured cells and tissues for the tight junction-associated Mr 150,000 protein, symplekin, which occurs in mostly granular-looking karyoplasmic structures, and on mitotic telophase, rapidly reacumulates, in the nucleus, like a typical nuclear protein (Keon et al., 1996 ; for review see Stevenson and Keon, 1998 ). Analysis of the amino acid sequence of this protein, however, has not revealed homologies—or at least similarities—to any known nuclear protein (cf. reviews of Cáceres and Krainer, 1997 ; de la Cruz et al., 1999 ).
To elucidate the nuclear function(s) of symplekin we have applied biochemical methods for isolating and characterizing the protein, using methods that recently have been successful in studies of the nuclear forms of plakophilin PKP2, which has been shown to be part of RNA polymerase III complexes (Mertens et al., 2001 ). For the sake of clarity we have further decided to start with the exceptionally large nuclei of Xenopus laevis oocytes (“germinal vesicles”) allowing both mass and manual isolations with minimal cytoplasmic contamination as well as the preparation of enucleated “ooplasms,” and thus the parallel analysis of both karyoplasm and cytoplasm (cf. e.g., Bonner, 1975a , 1975b ; De Robertis et al., 1978 ; Krohne and Franke, 1980a , 1980b , 1983 ; Kleinschmidt and Franke, 1982 ; Kleinschmidt et al., 1983 ; Peters et al., 1990 , 1994 ). Here we report the identification of the nuclear form of Xenopus oocyte symplekin as a distinct particle of about 11S in association with subunits of the CPSF complex known to be part of the 3′-end pre-mRNA processing machinery. In addition, symplekin occurs, together with protein CPSF-100, coilin and many other proteins known to function in RNA synthesis and processing, in the so-called “Cajal bodies,” which are also very large in these oocytes (cf. Gall et al., 1999 ; Gall, 2000 ; Morgan et al., 2000 ). Surprisingly, however, we have also found that symplekin-containing particles are not restricted to the nucleus but can also be detected in enucleated oocytes and in eggs.
Clawed toads (Xenopus laevis) were purchased from the African Xenopus Facility C.C. (Knysna, Republic of South Africa). Tissue samples from X. laevis (skin, heart, ovaries) were snap-frozen in isopentane cooled by liquid nitrogen to about −140°C and stored at −80°C. For X. laevis blood smear preparations, blood was obtained from larger vessels of decapitated toads. The blood was directly smeared on glass slides and air-dried for 3 h. Subsequently the smears were fixed for 10 min with freshly prepared 2% formaldehyde in PBS with 1 mM MgCl2 and permeabilized for 3 min with 0.3% Triton X-100 in PBS with 1 mM MgCl2.
Cell culture cell lines used included X. laevis kidney epithelial (XLKE) line A6, human colon carcinoma line CaCo2, and human SV-40 transformed fibroblasts line SV80 (for sources see American Tissue Culture Collection, Manassas, VA, and Cordes et al., 1996 ).
The monoclonal antibodies (mAbs) specific for symplekin used were mAb Sym-TJ-E150 (Keon et al., 1996 ) and mAb Sym-Nu (Becton Dickinson, Heidelberg, Germany). Guinea pig antibodies specific for symplekin (sym-CT) were obtained by immunization with a synthetic peptide representing the C-terminal sequence (AMKTPSPAAEDAREPEAKGNS, aa 1122–1144; cf. Keon et al., 1996 ; Ueki et al., 1997 ), coupled to keyhole limpet hemocyanin (Peptide Specialty Laboratories, Heidelberg, Germany). Rabbit sera specific for CPSF-100 (Jenny et al., 1994 ) or CPSF-73 (Jenny et al., 1996 ) have been described before. Rabbit antibodies specific for coilin (Bohmann et al., 1995 ) were generously provided by Dr. A. Lamond (University of Dundee, Scotland, United Kingdom). Mouse mAb H1 specific for coilin (Tuma et al., 1993 ) was obtained from Zytomed (Berlin, Germany), and mAb No-185 against a nucleolar protein (Schmidt-Zachmann et al., 1987 ) was kindly provided by M. Schmidt-Zachmann (German Cancer Research Center).
Secondary antibodies used for immunofluorescence microscopy were Texas Red-, Alexa 488-, Cy3-, Cy2-conjugated antibodies to immunoglobulins of mouse, guinea pig, or rabbit, respectively (Dianova, Hamburg, Germany).
Proteins of total cells and cell fractions were separated by SDS-PAGE (Laemmli, 1970 ). After electroblotting of the proteins to PVDF membranes, the filters were blocked for 1 h in Tris-HCl–buffered saline (TBS) containing 0.05% Tween (TBST) and 5% nonfat dry milk. The specific antibodies were incubated with the membranes for 1 h in TBST. Bound antibodies were detected by chemiluminescence using the ECL-system (NEN, Cologne, Germany) after incubation with horseradish peroxidase-coupled secondary antibodies.
Ovaries were surgically removed, and oocytes were defolliculated in 2 mg/ml collagenase (Sigma, Munich, Germany) in 87 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES (pH 7.8) with constant agitation at 28°C for 2–3 h. For separation of nuclear content and nuclear envelope, nuclei were isolated in “5:1 buffer” supplemented with 10 mM MgCl2 (for review see Krohne and Franke, 1983 ). This allowed stripping of the nuclear envelope and yielded a fraction containing only the somewhat gelified nuclear content. Manual isolation of nuclei from stage VI oocytes (Dumont, 1972 ) was in “5:1 buffer” as described (Krohne and Franke, 1983 ), followed by fixation in ethanol. Total oocytes or enucleated ooplasms were transferred into a microcentrifuge tube and excess buffer was removed. The oocytes were resuspended in a small volume of “5:1 buffer” supplemented with 1 × “complete protease inhibitors” (Roche Diagnostics, Mannheim, Germany) and homogenized by pipetting up and down in a narrow bore pipette. Homogenates were centrifuged at 13,000 × g for 10 min at 4°C. To examine the efficiency of the removal of follicle epithelial cells from the oocytes, the total cytoskeletal material of collagenase-treated and untreated oocytes was separated by SDS-PAGE and probed in Western blots with the mAb, Vim 3B4, which specifically recognized vimentin, a marker protein for follicle cells (Herrmann et al., 1989 ). Large-scale isolation of nuclei from mature (stages IV–VI) X. laevis oocytes was carried out as described by Scalenghe et al. (1978) with the modifications of Kleinschmidt and Franke (1982) . Subsequent fractionation of nuclear contents into low-speed pellet (LSP), high-speed pellet (HSP), and high-speed supernatant (HSS) was as described by Hügle et al. (1985) . LSP fractions were cleared from yolk proteins by Freon extraction (Evans and Kay, 1991 ). Egg extracts were prepared as described by Cordes et al. (1993) , and small scale preparations of nuclear extracts from A6 cells were done according to the method of Lee and Green (1990) .
The supernatant fractions were directly loaded on top of a 5–30% linear sucrose gradient buffered with “extraction buffer” (80 mM KCl, 20 mM NaCl, 15 mM HEPES, pH 7.5, 5 mM EDTA, 1.0 mM DTT, 250 mM sucrose). Centrifugation was performed in a SW40 rotor (Beckman Instruments, Munich, Germany) at 35,000 rpm for 19 h at 4°C. Sixteen fractions of 0.8 ml each were collected from top to bottom of the gradient and tested by immunoblotting. Extracts were also layered on linear 10–40% sucrose gradients in 5:1 buffer (80 mM KCl, 20 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1× Complete protease inhibitors). After centrifugation, fractions of 0.4 ml were collected from top to bottom. Marker proteins (bovine serum albumin [BSA], catalase, thyroglobulin; all from Sigma) or ribosomal subunits from X. laevis ovaries were used in parallel.
Immunoselection was performed with Dynabeads (Dynal, Hamburg, Germany) coated with antibodies specific to mouse IgG. Samples were cleared by addition of Dynabeads for 2 h on a rotating wheel at 4°C. The beads were then separated, and the supernatants were transferred to a tube containing Dynabeads preloaded with symplekin-specific mAb Sym-Nu. After incubation overnight at 4°C on a rotating wheel, the Dynabeads were washed four times in ice-cold buffer (140 mM NaCl, 5 mM EDTA, 20 mM HEPES, pH 7.5, 1% Nonidet-P40), then boiled in sample buffer, processed by SDS-PAGE, and either stained with Commassie Brilliant Blue or blotted to PVDF membranes. As a control, the Dynabeads used for preclearing were processed in parallel. Protein bands were excised from the gel and cut into 1 × 1-mm pieces that were washed twice with deionized water, 50% acetonitrile/water 1:1, and acetonitrile. Proteins were digested with sequencing grade modified trypsin (Promega, Mannheim, Germany) in 40 mM ammonium bicarbonate at 37°C overnight. The reaction was stopped by freezing.
MALDI mass spectra were recorded in the positive ion reflector mode with delayed extraction on a Reflex II time-of-flight instrument (Bruker-Daltonik GmbH, Bremen, Germany) equipped with a SCOUT multiprobe inlet and a 337-nm nitrogen laser. Ion acceleration voltage was set to 20.0 kV, the reflector voltage was set to 21.5 kV, and the first extraction plate was set to 15.4 kV. Mass spectra were obtained by averaging 50 to 200 individual laser shots. Calibration of the spectra was performed internally by a two-point linear fit using the autolysis products of trypsin at m/z 842.50 and m/z 2211.10. For the mass spectrometric analysis of tryptic digests MALDI samples were prepared on thin film spots (Jensen et al., 1996 ).
Post-source decay (PSD) analysis was performed in the positive ion reflector mode with delayed extraction by setting an ion gate width of 40 Da around the ion of interest. Data were acquired in 14 segments by decreasing the reflector voltage in a stepwise manner. For each segment 100–200 individual laser shots were accumulated. The fragment ion spectrum was obtained by pasting together all segments to a single spectrum using the FAST software provided by Bruker. Fragment ion calibration was performed externally with the fragment masses of the adrenocorticotropic hormone (ACTH) 18–39 clip. Sample preparation for PSD analysis was achieved by cocrystallization of matrix with ZipTip C18 (Millipore, Bedford, MA) concentrated samples (Regula et al., 2000 ).
Singly charged monoisotopic peptide masses were used as inputs for database searching. Searches were performed against the NCBInr database using the ProFound search algorithm (http://188.8.131.52/prowl-cgi/ProFound.exe) and the Protein prospector software developed at the University of California, San Francisco, (http://prospector.ucsf.edu). Isoelectric points were allowed to range from 0 to 14, and the oxidation of methionine was included as possible modification. Up to one missed tryptic cleavage was considered, and the mass tolerance for the monoisotopic peptide masses was set to ±100 ppm or ±0.1 Da.
Searches with fragment masses from PSD experiments were performed against the NCBInr database using the MS-Tag search algorithm provided by the Protein prospector software package. Parent mass tolerance was set to ±0.1 Da and fragment ion tolerance was set to ±0.7 Da.
For immunofluorescence microscopy studies of cultured cells, cells grown on coverslips were fixed in methanol (5 min, −20°C), followed by acetone (30 s, −20°C), washed twice in PBS, and incubated with antibodies for 20 min at room temperature. After several PBS washes, cells were incubated for 20 min with the appropriate secondary antibodies, washed in PBS, dehydrated in ethanol, air-dried, and mounted in Fluoromount (Biozol, Eching, Germany).
Cryosections (5 μm) of frozen tissues were fixed either in acetone (−20°C) for 10 min or in PBS with 1 mM MgCl2 (PBS-MgCl2) containing 2% formaldehyde for 10 min at room temperature. Formaldehyde-fixed samples were washed in PBS-MgCl2 containing 50 mM NH4Cl for 5–10 min and twice for 5 min in PBS-MgCl2 before incubation with antibodies. In some experiments cells were stained with 4′,6-diaminidino-2-phenylindole (DAPI, 0.1 μg/ml; Serva, Heidelberg, Germany) for 5 min during incubation with the secondary antibodies. Micrographs were taken with an Axiophot microscope (Zeiss, Jena, Germany).
Confocal laser scanning immunofluorescence microscopy was done on a Zeiss LSM 410 UV instrument (Zeiss). For simultaneous double-label fluorescence, an argon ion laser operating at 488 nm and a helium-neon laser operating at 543 nm were used together with a band-pass filter combination of 510–525 nm and 590–610 nm for visualization of Cy-2 and Cy-3 fluorescence.
Symplekin antibody mAb Sym-TJ-E150 reacted with cell–cell junctions of cultures of human CaCo2 cells, corresponding to tight junction markers such as occludin and protein ZO-1 (Keon et al., 1996 ) as well as throughout the karyoplasm (Figure (Figure1A),1A), whereas mAb Sym-Nu specifically recognized only the nuclear form of symplekin (Figure (Figure1B).1B). Gradual focusing through such nuclei allowed the resolution of individual granular structures, leaving the nucleoli negative. In dividing cells, symplekin staining with mAb Sym-TJ-E150 was still positive at the tight junction plaques (Figure (Figure1A,1A, inset), whereas the nuclear form was dispersed throughout the cytoplasm.
A similarly intense karyoplasmic immunofluorescence was observed in cultured Xenopus kidney epithelial cells (Figure (Figure1,1, C and C′). On frozen sections of Xenopus tissues, intense immunofluorescence was seen in nuclei of all the different cell types examined, including epidermal keratinocytes of all layers of the skin (Figure (Figure1,1, D–D"), epithelial cells of glands and ducts, fibroblasts, and other dermal cells in the skin (our unpublished results), cardiomyocytes, endothelial cells, and erythrocytes of heart tissue (Figure (Figure1,1, E and E′). Because of the unexpected reaction in Xenopus erythrocytes, in which transcriptional and replicational activities are notoriously low or absent, we further examined whole mount preparations of erythrocytes in blood smears. As shown in Figure Figure1F,1F, the nuclei of erythrocytes were clearly positive for symplekin, indicating that this protein is a general nuclear constitutent and that its presence is not directly depending on ongoing nuclear RNA synthesis activities.
To study the nuclear structures containing symplekin, we decided to examine Xenopus oocytes as its large nucleus (“germinal vesicle”) can be isolated with minimal cytoplasmic contamination. We prepared extracts of total Xenopus oocytes, manually isolated their nuclei or ooplasmic material from enucleated oocytes, and analyzed the fractions obtained side-by-side by SDS-PAGE, followed by immunoblotting with symplekin-specific mAbs (Figure (Figure2).2). A protein band of the typical size (Mr 150,000 as introduced for mammalian cells; cf. Keon et al., 1996 , or slightly lower corresponding to ca. 140,000) was selectively reactive in total cell lysates from human CaCo2 and Xenopus A6 cells and in extracts from total Xenopus oocytes (Figure (Figure2,2, compare CaCo, T, and A6), indicating that this band contains the Xenopus homologue to human symplekin. This band was also detected in samples from manually isolated nuclei and in extracts of enucleated oocytes. The protein reacting with the symplekin-specific antibodies seemed to be present in rather large amounts in Xenopus oocytes, as the material from one or two hand-isolated nuclei and one ooplasm was sufficient to yield a strong signal. Moreover, this protein was enriched in the nuclear content, as comparable amounts were detected in hand-isolated nuclei and in a fraction of nuclear contents from which the nuclear envelopes had been removed (compare Figure Figure2,2, NU5 and NC).
The fractionation of particles from isolated Xenopus oocyte nuclei by differential centrifugation leads to well-defined subnuclear fractions (Figure (Figure3,3, left part; cf. Hügle et al., 1985 ; Schmidt-Zachmann et al., 1998 ). When the distribution of symplekin in such fractions from oocyte nuclei, egg extracts and somatic cells was analyzed by immunoblotting (Figure (Figure3,3, A and B), symplekin was detected in total oocyte nuclei, in the HSP and HSS fractions and in egg extracts. For SDS-PAGE equal volumes of LSP, HSP, and HSS fractions were loaded. As the two pellets (LSP and HSP; Figure Figure3)3) were solubilized in small volumes their material was consequently concentrated, with respect to the HSS fraction. Thus, we concluded that the major proportion of symplekin was recovered in the HSS fraction containing nuclear proteins in soluble form or in small particles. In addition, some symplekin was bound to larger nucleoplasmic particles, as indicated by the reaction in the HSP fraction. The fractions were also characterized by immunoblotting with mAb No-185 against nucleolar protein NO38, known to be enriched in the LSP and HSP but absent from the HSS fraction (Schmidt-Zachmann et al., 1998 ), and by immunoblotting with mAb H1 against coilin. The protein coilin was enriched in the HSP fraction, minor amounts were also detected in the LSP and HSS fractions (our unpublished results).
The HSS fraction obtained from Xenopus oocyte nuclei was used for immunoselection to obtain symplekin-containing protein complexes. Material bound to Dynabeads loaded with symplekin antibodies was solubilized with sample buffer and separated by 10% SDS-PAGE. After staining with Coomassie Brilliant Blue, five major protein bands, ranging from 70 to 160 kDa, were visible among the immunoselected proteins (Figure (Figure4A),4A), whereas no such enrichment was seen after preclearing of the lysate (unpublished results). By mass spectrometric sequencing using PSD analysis, two amino acid sequences were identified in the protein band of ca. 140 kDa: RRPEPIIPVTQGR and DPLLAHVR. From database search these sequences were identified as homologous to the tryptic peptides 521–533 and 1073–1080 of human symplekin (Keon et al., 1996 ; Ueki et al., 1997 ), with exchanges of amino acid P532 to G and I1073 to D, respectively. The other proteins present in these immunoprecipitates were identified as the Xenopus proteins CPSF-160, CPSF-100, and CPSF-73, respectively, and these identifications were verified by immunoblotting with specific antibodies. Interestingly, the two protein bands of ~95 and 100 kDa were both identified as CPSF-100, corresponding to a recent report showing the existence of two forms of CPSF-100 in X. laevis oocytes (Dickson et al., 1999 ).
Because all three proteins were known as constituents of the protein complex named CPSF (Bienroth et al., 1991 ; Murthy and Manley, 1992 ; for review see Wahle and Rüegsegger, 1999 ), we concluded that symplekin is also a component of this complex. A fourth polypeptide of 30 kDa that had also been described for this complex (Bienroth et al., 1991 ) was not identified in these immunoprecipitates, perhaps because it was obscured by the large amount of immunoglobulin light chains.
We also wondered to which proteins symplekin was associated in eggs, i.e., after the breakdown of the nuclear envelope, and examined egg extracts obtained by immunoselection with symplekin antibodies. After staining with Coomassie Brilliant Blue, four major polypeptide bands, ranging from 70–160 kDa, were visible (Figure (Figure4B).4B). By mass spectrometric analysis the proteins of 160, 100, and 70 kDa were identified as CPSF-160, CPSF-100, and CPSF-73, respectively. The protein band of −140 kDa showed a spectrometric pattern of fragments similar to that of the protein identified as symplekin. Moreover, by mass spectrometric sequencing using PSD analysis, the same amino acid sequence RRPEPIIPVTQGR was determined as mentioned for the tryptic fragment of nuclear symplekin (peptide 521–533; see above). The 160-kDa protein band showed an unexpectedly intense dye staining, indicative of either suprastochiometric amounts of the CPSF-160 subunit or the coincidence of an additional yet unidentified protein.
To analyze the physical state of symplekin-containing material in the HSS fraction of fractionated Xenopus oocyte nuclei, this fraction was further subjected to centrifugation in 5–30% sucrose gradients, and the resulting fractions were analyzed by SDS-PAGE and immunoblotting using PVDF membranes (Figure (Figure5A).5A). Symplekin was recovered in fractions 4–8, with a maximum in fractions 6 and 7, corresponding to −11S. When the PVDF membranes were reprobed with antibodies against protein CPSF-100, two protein bands of −100 and 95 kDa were decorated. The 100-kDa form of CPSF-100 was found in fractions 6–8, with a maximum between fractions 6 and 7, corresponding to the distribution of symplekin. The 95-kDa version of CPSF-100 was detected in fractions 7–9, with a maximum in fractions 7 and 8 (Figure 5A′ ). An identical distribution was found with CPSF-73 antibodies (unpublished results).
As a smaller proportion of the nuclear symplekin was recovered in the HSP fraction, the protein material of the pellet was resuspended and separated by sucrose gradient centrifugation. Some symplekin was again recovered in fractions corresponding to 10–14S, whereas the main portion was detected in the pellet, indicative of its association with relatively large structures (for Cajal bodies see below).
As the Xenopus oocyte is a highly specialized cell, we also used nuclear extracts from cultured Xenopus cells of line A6 (cf. Figure Figure1C)1C) for sucrose gradient centrifugation. Here most of the symplekin present could also be detected in fractions corresponding to 10–11S (unpublished results).
To examine the intranuclear localization of symplekin, we performed immunocytochemistry on cryosections through Xenopus laevis ovaries. Using symplekin-specific antibodies, distinct intranuclear, nonnucleolar bodies of diameters of 5–10 μm were intensely stained (Figure (Figure6A).6A). Double-label experiments with CPSF-100 antibodies revealed a clear colocalization of both proteins (Figure (Figure6C).6C). Moreover, double-labeling with coilin-specific antibodies also showed colocalization, thus indicating that symplekin is concentrated in Cajal bodies, as has also been described for CPSF-100 and other factors of the 3′-end pre-mRNA processing complex by Gall and coworkers (Gall et al., 1999 ; Gall, 2000 ; Morgan et al., 2000 ). Using standard protocols, a weak immunofluorescence with symplekin antibodies was also seen throughout the karyoplasm. However, the intense immunolocalization of symplekin in the Cajal bodies seems to represent only a minor portion of the total nuclear symplekin, as indicated by our estimations from recovery experiments that only a small proportion of the nuclear symplekin is contained in HS pelletable structures (for general difficulties of demonstrating even most abundant extractable karyoplasmic proteins and particles such as actin, histones, nucleoplasmin, and their complexes see, e.g., Krohne and Franke, 1980b ; Ankenbauer et al., 1989 ; Hofmann et al., 2001 ).
Unexpectedly, a large proportion of the oocyte's symplekin was found in extracts from “ooplasms,” i.e., enucleated oocytes (see Figure Figure2).2). To prove that hand-isolated, enucleated oocytes were not contaminated with follicle cells or remnants thereof, we performed immunoblotting with antibodies specific for vimentin as a marker of follicle cells (cf. Herrmann et al., 1989 ). These controls revealed that cytoskeletal preparations of collagenase-treated oocytes contained only traces if any vimentin (our unpublished results). Cytoplasmic extracts from enucleated oocytes were used for immunoselection (Figure (Figure7).7). When the material bound to Dynabeads loaded with antibodies to symplekin was solubilized and separated by SDS-PAGE, enrichment of symplekin (Figure (Figure7A)7A) and protein CPSF-100 (Figure (Figure7B)7B) was detected by immunoblotting. This again showed that the immunoselected symplekin complexes also contained protein CPSF-100 (compare lane 3 in Figure Figure7,7, A and B).
To determine the physical state of this symplekin-containing cytoplasmic material, sucrose gradient centrifugation was applied, and the resulting fractions were analyzed by SDS-PAGE, followed by immunoblotting for symplekin (Figure (Figure8).8). Symplekin was distributed in three classes, one in fractions 5–8 with a peak in fraction 6, corresponding to ca. 11S, and the other in fractions 24 and 25, corresponding to particles larger than 65S, and some material accumulated in the last gradient fraction and the pellet.
In oocytes and eggs of X. laevis we have discovered and characterized distinct karyo- and cytoplasmic particles containing symplekin, a protein also described as a component of the tight junction plaque. A major result of our study is that a large part of the nuclear symplekin occurs in particles with an approximate mean peak value of 11S (“11S particles”), where it is complexed with proteins involved in mRNA biogenesis, notably 3′-end processing. Symplekin particles have also been found in egg extracts and, most surprisingly, in cytoplasmic particles of enucleated oocytes. In addition, a notable proportion of the nuclear symplekin is associated with much larger, i.e., readily pelletable structures, and this seems to include the Cajal body symplekin.
Clearly, the major part of the symplekin-containing nuclear particles can be precipitated together with CPSF subunits such as proteins CPSF-160, CPSF-100, and CPSF-73. Here, the identification of CPSF-73 presents further evidence that we have identified a nuclear protein complex, because this subunit has not been found in cytoplasmic CPSF complexes involved in regulated polyadenylation of mRNAs in Xenopus oocytes (Dickson et al., 1999 , 2001 ). Furthermore, the relative staining intensities of the separated polypeptides of the immunoprecipitated particles indicate that symplekin is an iso-stoichiometric component with respect to the other CPSF proteins. The relative amount of total nuclear CPSF particle-bound symplekin may actually be even higher because we cannot exclude that pelleted material, including Cajal bodies, contains similar −11S particles, although in a state associated with—or integrated into—larger structures (Gall et al., 1999 ; Gall, 2000 ).
The finding of symplekin as a constituent of CPSF particles is not restricted to X. laevis oocytes and cultured A6 kidney-derived cells, as shown in the present study. In similar experiments using cell culture lines from various tissues and species, including human HeLa cells, we have also identified symplekin in association with such particles. Moreover, by immunodepletion the functional importance of symplekin for 3′-end cleavage and polyadenlation has recently been demonstrated in in vitro assays using extracts from HeLa cell nuclei (I. Hofmann, I. Kaufmann, W. Keller, and W.W. Franke, unpublished results). Therefore, we think it is a sensible working hypothesis that symplekin is a widespread, if not ubiquitous CPSF protein.
It is perhaps somewhat astonishing that in the numerous previous studies symplekin has been overlooked as a component involved in 3′-end processing of pre-mRNA. So far six transacting factors have been listed that are required for the in vitro reconstitution of mammalian 3′-end processing (reviewed by Wahle and Rüegsegger, 1999 ). Besides the symplekin-containing factor CPSF, composed of the five subunits CPSF-160, CPSF-100, CPSF-73, CPSF-30, and symplekin (cf. Bienroth et al., 1991 ; Murthy and Manley, 1992 ; this study), the trimeric cleavage stimulation factor (CstF), with the subunits CstF-77, CstF-64, and CstF-50, recognizes sequence elements on the pre-mRNA (for references see Wahle and Rüegsegger, 1999 ). In addition, cleavage factors I (CFI) and II (CFII), poly(A)-binding protein II and poly(A) polymerase are needed for 3′-end processing in vertebrates (Raabe et al., 1991 ; Wahle, 1991 ; Wahle et al., 1991 ; Bienroth et al., 1993 ; Rüegsegger et al., 1996 ; de Vries et al., 2000 ; Kim et al., 2001 ).
The concept of symplekin as a constituent of the larger 3′-end cleavage and polyadenylation complex is also supported by the observation of Takagaki and Manley (2000) who, in “far Western” screens with the protein CstF-64, have identified HeLa cell symplekin as a strongly interacting protein that in solid phase and pull down assays competes for binding to CstF-64. Moreover, using immuno-affinity column chromatography on CstF-64, these authors have copurified subunits of CstF, CPSF, and symplekin from nuclear extracts, indicating that CstF, CPSF, and symplekin are part of a large complex. That symplekin serves an important role in fundamental cellular processes is also suggested from the existence of homologues in the genomes of very distantly related species such as Caenorhabditis elegans (AF022973), Drosophila melanogaster (AE003601) and Arabidopsis thaliana (AL161746; these authors, unpublished results). The involvement of symplekin in 3′-end processing of premRNA is also supported by the notion of a homologue in Saccharomyces cerevisiae, protein PTA1p, described to be associated with the proteins of the yeast equivalent of CPSF (YHH1, YDH1, YSH1, YTH1) and to be important for cleavage and polyadenylation of pre-mRNA (Preker et al., 1997 ; Zhao et al., 1999 ).
Certainly, the most unexpected finding of our study is the discovery of ca. 11S complexes containing symplekin together with CPSF proteins, notably CPSF-100, in the cytoplasm. As these particles have also been obtained from manually enucleated ooplasms, a contamination by nuclear particles appears to be excluded. This finding indicates that reactions known to be involved in cytoplasmic polyadenylation (Hake and Richter, 1994 ; Dickson et al., 1999 , 2001 ; for review see also Wickens et al., 2000 ) and in the regulation of translation (Stebbins-Boaz et al., 1999 ; Mendez et al., 2000 ; for reviews see Macdonald, 2001 ; Mendez and Richter, 2001 ) are located in symplekin-containing complexes.
Interestingly, all so far characterized nuclear forms of junctional plaque proteins have in common that they are somehow involved in processes of transcription, splicing, or 3′-end processing: Plakophilin 2 has been detected in RNA polymerase III complexes, p120ctn and β-catenin are involved in regulations of RNA polymerase II transcription, and protein 4.1 has been found in splicing factors (Behrens et al., 1996 ; Huber et al., 1996 ; Molenaar et al., 1996 ; Krauss et al., 1997 ; Lallena et al., 1998 ; Daniel and Reynolds, 1999 ; Mertens et al., 2001 ). Symplekin is the first protein associated with factors involved in 3′-end processing of premRNA in the nucleus as well as in cytoplasmic translational control. Future studies will help understanding the biological significance of the interactions between CPSF and their regulation and determining the functions of symplekin in oocytes as well as in somatic cells. In addition, it will be important to clarify whether the nuclear and the plaque-bound forms exist in a regulated exchange equilibrium.
The authors are indebted to Dr. Walter Keller (Biozentrum, University of Basel, Switzerland) for continuous support and advice as well as to Drs. Reimer Stick (University of Bremen, Germany) and Marion Schmidt-Zachmann (German Cancer Research Center, Heidelberg, Germany) for stimulating discussions. They also thank Jutta Osterholt for preparing the photographs and Eva Ouis for arranging the manuscript. The technical assistance of Sonja Reidenbach and the expert help of Dr. Herbert Spring with the laser scanning confocal microscopy is also gratefully acknowledged. This study has been supported in part by the Deutsche Forschungsgemeinschaft.
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.01–12–0567. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.01–12–0567.