Cytoplasmic assembly of snRNP particles from stored proteins and newly transcribed snRNA's in L929 mouse fibroblasts

Experimental

Cell Research

176 (1988) 344-359

Cytoplasmic Assembly of snRNP Particles from Stored Proteins and Newly Transcribed snRNA’s in L929 Mouse Fibroblasts ROGER

A. SAUTERER,

ROBERT

J. FEENEY,

and GARY

W. ZIEVE’

Department of Anatomical Sciences, and Program in Cellular and Developmental Biology, SUNY Stony Brook, Stony Brook, New York 11794

Newly synthesized snRNAs appear transiently in the cytoplasm where they assemble into ribonucleoprotein particles, the snRNP particles, before returning permanently to the interphase nucleus. In this report, bona fide cytoplasmic fractions, prepared by cell enucleation, are used for a quantitative analysis of snRNP assembly in growing mouse tibroblasts. The half-lives and abundances of the snRNP precursors in the cytoplasm and the rates of snRNP assembly are calculated in L929 cells. With the exception of U6, the major snRNAs are stable RNA species; UI is almost totally stable while U2 has a half-life of about two cell cycles. In contrast, the majority of newly synthesized U6 decays with a half-life of about 15 h. The relative abundances of the newly synthesized snRNA species LJI, U2, U3, U4 and U6 in the cytoplasm are determined by Northern hybridization using cloned probes and are approximately 2 % of their nuclear abundance. The half-lives of the two major snRNA precursors in the cytoplasm (Ul and U2) are approximately 20 min as determined by labeling to steady state. The relative abundance of the snRNP B protein in the cytoplasm is determined by Western blotting with the Sm class of autoantibodies and is approximately 25 % of the nuclear abundance. Kinetic studies, using the Sm antiserum to immunoprecipitate the methionine-labeled snRNP proteins, suggest that the B protein has a half-life of 90 to 120 min in the cytoplasm. These data are discussed and suggest that there is a large pool of more stable snRNP proteins in the cytoplasm available for assembly with the less abundant but more rapidly turning-over snRNAs. 0 1988 Academic Press. Inc.

The small nuclear RNAs are abundant components of the interphase nucleus where they function in the processing of preribosomal and premessenger RNA [25, 331. In mammalian cells, these RNAs comprise a family of six major species and several recently described less abundant species [32, 341. All the snRNAs except U6 are characterized by their unique trimethylated guanosine cap and their packaging into ribonucleoprotein (RNP) particles: the snRNPs (for review [5, 6, 271. All of the snRNPs with the exception of U3 and U6 share a common group of core proteins in addition to several species-specific proteins. The shared protein core includes the Sm antigen recognized by sera from patients with the autoimmune disease systemic lupus erythematosus [21, 301. Cell fractionation studies using several different techniques have demonstrated that snRNP assembly occurs in the cytoplasm, where newly transcribed snRNAs assemble into particles before returning permanently to the interphase nucleus [8, 14, 16, 40, 421. Kinetic studies have suggested that four of the shared snRNP proteins self-assemble into an 6S RNA free core and that this core, the fifth shared protein and species-specific proteins, assemble with the snRNA after it ’ To whom reprint requests should be addressed. Copyright @ 1988 by Academic Press. Inc. Ail rights of reproduction in any form reserved 0014-4827/S? $03.00

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enters the cytoplasm [ll, 121. However, the exact assembly pathway is not known and has been difficult to identify by traditional pulse and chase kinetics. In this report the kinetic behavior of the cytoplasmic and nuclear snRNAs and snRNP proteins are measured as a first step toward understanding the coordinate regulation of snRNP synthesis and assembly. Cytoplasts prepared by enucleation of L929 cells are used to measure the relative abundance and half-lives of both the snRNA and snRNP protein precursors in the cytoplasm of growing L929 fibroblasts. The cytoplasts provide bona fide cytoplasmic fractions uncontaminated by the more abundant stable nuclear snRNPs which leak from nuclei prepared by conventional procedures. The data demonstrate that in the cytoplasm there is a large pool of more stable snRNP proteins and a much smaller but more rapidly turning-over pool of newly synthesized snRNAs which return to the nucleus after binding the snRNP proteins. MATERIALS

AND

METHODS

Cell culture. L929 cells were cultured in suspension in minimum essential medium (GIBCO, Grand Island, NY) supplemented with 5 % fetal calf serum and maintained between 30X lo4 and 60x lo4 per milliliter. For pulse labeling with ‘H-labeled nucleosides cells were concentrated in normal medium and for long-term labeling cells were maintained under normal culture conditions. For the kinetic studies requiring labeling with [“Slmethionine, cells were concentrated threefold and resuspended in medium with a reduced methionine concentration, supplemented with 25 mM Hepes and 2.5 m&f glutamine. Cdfractionation. L929 cells were enucleated in suspension by isopycnic centrifugation of cytochalasin-treated cells using a modification of the procedure of Wigler and Weinstein [36, 421. For the conventional aqueous extraction into nuclear and cytoplasmic fractions, RSB buffer (10 tr&f NaCl, 1.5 mM MgClr, 10 mM ‘Iris, ph 7.4) and nonionic detergents were used as previously described [40]. Vanadyl ribonucleoside complex (VRC) was added at a concentration of up to 5 m&f to inhibit ribonucleases [3]. To solubilize the snRNP components in the cytoplasm, cytoplasts were extracted in CSK buffer (10 mM Pipes, pH 6.9, 100 n&f NaCl, 3 n&f MgClJ with 0.5% Triton X-100, 5 mM VRC, and 1% aprotinin. Insoluble material was sedimented at 2000 g for 3 min and the supematant was saved. Zmmunoprecipitation. Immunoprecipitation studies used the mouse monoclonal antibody Y 12 with the systemic lupus erythematosus Sm serotype 1221.The clones were grown in cell culture, and tissue culture supematants containing approximately 2-4 pglml IgG were used for all experiments. The Sm patient antiserum was the gift of E. DeRobertis and has been characterized previously [15]. Western blots. Proteins were transferred to 0.2 pm pore sized nitrocellulose by a capillary transfer procedure using a transfer buffer of 20% methanol, 75 m&4 Tris, and 576 mM glycine. The capillary transfers proceeded for 3 days, sufficient to transfer 60-75% of the 28 kD B protein and nearly 100% of the smaller snRNP proteins. The nitrocellulose sheets were blocked for 1 h in Blotto [19] (PBS with 5 % nonfat dry milk, 0.02 % NaNr, and 0.1% anti-foam A emulsion (Sigma)), washed three times for 10 min in PBS-O.5% Tween 20, and incubated for 2 h at 37°C with the appropriate antibody. Blots used either the tissue culture supematant from the Y12 monoclonal antibody or a 1 to 100 dilution of the patient serum. After incubation, the nitrocellulose sheet was rinsed three times for 10 min with PBS-Tween and probed with 3 &ml alkaline phosphatase conjugated to either goat antimouse or goat anti-human antibodies in PBS-Tween for 2 h at 37°C 121.The blot was then washed four times in PBS-Tween, rinsed in 50 mM glycine-NaOH, pH 9.6, and then developed in 50 n-&f glycine-NaOH, pH 9.6, with 0.1 mg/ml p-nitro blue tetrazolium chloride and 0.05 mg/ml 5-bromo-4chloro-3-indoyl phosphate, p-toluidine salt. For quantitative analysis, the blots were photographed and then printed on Kodak fine grain positive film (7302). These films were treated similary to normal autoradiograms and were scanned using an E-C Apparatus Corp. (St. Petersburg, FL) densitometer coupled to a IBM XT equipped with a data translation DT2801 analog to digital converter. Scans were then displayed and appropriate peaks were selected and the areas were determined by numerical integration. Adjustments were made to account for band width.

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Fig. I. Stable low-molecular-weight RNAs in L929 cells. L929 cells in normal medium were labeled with either 1.5 uCi/ml of “‘P-orthophosphate (lanes A and B) or 8 uCi/ml [‘HI-uridine (lanes C-H). Cells labeled with “P-orthophosphate were grown for 5 days by dilution with medium containing 1.5 @i/ml ‘*P. Cells (2x 10’) were harvested at 24-h intervals and fractionated into cytoplasm and nucleus by standard aqueous cell fractionation. Cytoplasmic (lane A) and nuclear (lane B) RNA species after 5 days of labeling are illustrated. Cells labeled with [‘HI-uridine were transferred to fresh unlabeled medium after 8 h and maintained in culture by standard techniques of daily dilution with fresh medium. Cells (1.5~ 10’) were harvested at 16 h (lanes C and D), 3 days (lanes E and F’) and 5 days (lanes G and I$ after the initiation of labeling, and cytoplasmic and nuclear fractions, respectively, were analyzed by gel electrophoresis.

Northern hybridization. Profiles of the snRNAs were electrophoretically transferred from the polyacrylamide gels to nitrobenzyloxymethyl paper (NBM) (Transa-bind from Schleicher & Schuell, Keene, NH) which had been reduced and activated to DBM paper by standard procedures. Electrophoretic transfer was performed at 15 V, 0.4-0.5 A for 4.5 h at 4°C in 0.2 M sodium acetate, pH 4.0. After transfer the paper could be stored at 4°C in prehybridization solution for up to several months or processed directly for hybridization. Hybridization was carried out according to the procedure of Alwine ef al. [l]. Ul, U2, and U3 cloned in the Ml3 vectors were generous gifts from A. Weiner [31]. The U4 and U6 clones were a generous gift from T. Pederson [20, 241. RNA preparation and electrophoresis. RNA was prepared from cell fractions by proteinase K digestion followed by phenol and chloroform extraction in the presence of 0.5% SDS and analyzed on 6-15% gradient gels as previously described [40]. An advantage of these nondenaturing gels is that the 5.8 S rRNA is not released from the 28 S rRNA. This species migrates with a mobility intermediate between that of Ul and U4 and interferes with the analysis of these two snRNA species. Also, U3 migrates faster than in denaturing gels where it can comigrate with the U2 precursor, U2’.

RESULTS Stability

and Relative Abundance

of the Mature

snRNAs in L929 Cells

SnRNP particle assembly provides for the maintenance of the existing pool of snRNP particles and for the demands of cell growth. Therefore, as an approach toward calculating the rate of assembly of the snRNPs in growing L929 mouse fibroblasts, cell growth rates and the turnover of the mature snRNP particles were determined. Standard growth curves indicated that the L929 ceils maintained in suspension under our conditions grow with a generation time of 30 h. The rate of turnover of the mature particles is a function of their stability and

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abundance. The relative abundance of the different snRNAs in the nucleus was determined by labeling cellular RNA to constant specfic activity with low levels of 3’P. Under these conditions the amount of radioactivity in the individual RNA species is directly proportional to the mass of RNA and when corrected for the size of the RNA it is proportional to the number of copies present. The gel in Fig. (lanes A and B) illustrates the distribution of radioactive low-molecular-weight RNAs from cytoplasmic and nuclear fractions prepared by aqueous cell fractionation from L929 cells after 5 days of labeling in the presence of 1.5 &i/ml of 32P. The identity of the small RNA species in our gel system has been established by immunological analysis of the snRNP particles, hybridization with cloned probes, and sequence ana,lysis. The mobilities of the Ul-U6 snRNA species are indicated. This nondenaturing gel system was chosen for this analysis because it does not release the 5.8 S rRNA that is hydrogen bonded to the ribosomal 28 S RNA. This species migrales with a mobility similar to that of Ul and U4 and the small amount of mature ribosomal RNA contaminating the nuclear fractions can release sufficient 5.8 S rRNA to mask the snRNAs. The snRNAs are found in both the cytoplasmic and nuclear fractions in this aqueous cell fractionation. The snRNAs in the cytoplasmic fraction are the result of nuclear leakage during cell fractionation and from the few percentage of dividing cells in which the nuclear envelope has dispersed [39]. Under these conditions up to 20 % of the snRNAs leak from the isolated nuclei. U2 often leaks to a greater extent than IJl , and U3, localized in the nucleolus, leaks to the least extent. As determined by the level of labeling, the different snRNAs have a wide range of concentrations. Assuming the 5 S rRNA is present in 4~ lo6 copies per cell, Ul is present in 1 X lo6 copies, U2 in 0.9X lo6 copies, U3 in 0.2X 10” copies, U4 in 0.2~ IO6 copies, U5 in 0.25~ lo6 copies, and U6 in 0.5~ lo6 copies. To measure the stability of the mature snRNAs, L929 cells were pulse labeled with low levels of [3H]-uridine and then chased for 4 days to monitor the decay of the labeled snRNAs. After the addition of [3H]-uridine to the medium, cells continue to synthesize labeled snRNAs for up to 8 h, after which very little radioactivity is incorporated into newly synthesized snRNAs. The disappearance of labeled snRNAs from the growing cells is then a function of the half-lives of the individual species and dilution by cell growth. Figure 1 illustrates the profiles of the snRNAs 16 h (lanes C and D), 3 days (lanes E and fl, and 5 days (lanes G and H) after labeling. A major difference in the pattern of the snRNAs after the 4-day chase is the lower amount of radioactivity in species U6 after the chase period. In the shortterm label this species is far more abundant than U4 and after the chase its abundance is about equal. Analysis at 24-h intervals suggests a biphasic behavior of U6. There is a rapid decay of species U6 with a half-life of about 15 h and a small fraction of U6 behaves as a nearly stable species. However, sequence analysis will be required to determine if this band represents a single species of RNA or possibly several species with different behaviors.

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Close inspection of the gels also suggests that species U2 decays more rapidly than Ul. This can be seen because the radioactivity remaining in species U2 is reduced relative to Ul during the several-day chase. After 1 day of labeling there is 30% more label in species U2 than in Ul, however, after 5 days of chase there is almost twice as much label in Ul than in U2. Careful calculations suggest that Ul is nearly stable under these conditions and that U2 has a half-life of approximately 40 h. Short-Lived

snRNAs in the Cytoplasm

Newly synthesized snRNAs appear in the cytoplasm almost immediately after synthesis. The precursors of the two abundant snRNA species Ul and U2 are easily identified in cytoplasmic fractions from pulse-labeled cells. Nonaqueous cell fractionation, cell enucleation, and manual dissection of oocytes all contirm that these are bona tide cytoplasmic species and have not leaked from the nucleus during cell fractionation [16, 27, 421. Pulse and chase experiments demonstrate that there is a quantitative maturation of all the snRNAs that appear in the cytoplasm [39]. Therefore, the rate of synthesis of the cytoplasmic species equals the rate of synthesis of the mature nuclear snRNPs and should equal the sum of the turnover rate of the stable nuclear species and the rate of cell growth. In our gel system, Ul in the cytoplasm is of nearly identical size to mature Ul; however, precursors a few nucleotides larger than mature Ul can be detected in hybrid-selected preparations [29, 421. U2 appears as a precursor, U2’, approximately eight nucleotides larger than mature sized U2. In an actinomycin D chase, new snRNA synthesis is halted and there is a quantitative movement of the newly synthesized cytoplasmic precursors into the nucleus [29]. U2’ is cleaved to U2 in the cytoplasm before returning to the nucleus. The half-lives of the cytoplasmic precursors of Ul and U2 snRNAs were examined using two different kinetic approaches. In the first procedure, the incorporation of radioactivity into the cytoplasmic snRNA precursors was monitored under conditions of constant specific activity. From an analysis of the approach to maximum labeling it is possible to calculate a half-life. In a second procedure the decay of label during an actinomycin D chase was recorded and also allows a calculation of a half-life. The data from both approaches are illustrated in Fig. 2. In the first procedure of monitoring the accumulation of label, cells are labeled with 80 @ml of [3H]-uridine and aqueous cytoplasmic fractions were prepared at 15min intervals. This nucleoside is rapidly taken up by the cells and enters the large internal pool of uridine. The specific activity of this pool stays relatively constant for several hours. This is monitored by following the incorporation of label into stable low-molecular-weight species such as 7 SL RNA or 5 S rRNA. Under the conditions used both 7 SL and 5 S show a linear increase in radioactivity for the entire 75-min labeling period. In contrast, the accumulation of radioactivity in U2’ and Ul plateaus after about 45 min. Quantitative analysis of the data, as described in the figure legend, suggests that these two species have half-lives in the cytoplasm of approximately 20 min.

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Fig. 2. Half-lives of newly synthesized snRNAs, L929 cells (1 x 10’) were labeled with 80 uCiim1 of [3H]-uridine. At 15min intervals 2x10’ cells were harvested and fractionated into cytoplasm and nucleus and the low-molecular-weight RNAs in the cytoplasm were analyzed by gel electrophoresis (lane A, 15 min; lane B, 30 min; lane C, 45 min; lane D, 60 min; lane E 75 min). Radioactivity in the individual species was determined by quantitative densitometry. L929 cells (6x 10’) were labeled with 80 @/ml of [3H]-uridine for 45 min and then cells were treated with 10 Kg/ml actinomycin D. At the time of actinomycin D addition and at 15min intervals cells were harvested and fractionated into cytoplasm and nucleus and the low-molecular-weight RNA species in the cytoplasm were analyzed by gel electrophoresis (lane F, time of addition of actinomycin D, lane G, 15 min after addition, lane H 30 min after addition).

After the cytoplasmic snRNA precursors have been labeled to their maximum, the ratio of radioactivity in the two species, corrected for the number of uridines in the sequence, is proportional to the relative abundance of the two species. U2’ incorporates about 2.5 times as much 13H]-uridine as the cytoplasmic precursor of Ul snRNA. However, U 2’ also has approximately 50% more uridines. Correcting for this, U2’ is 1.7 times more abundant than Ul in these cytoplasmic fractions. In a second approach to measuring the half-lives of the cytoplasmic snRNA precursors, a pulse and chase protocol using actinomycin D was used. After a pulse label of the snRNAs, further RNA synthesis is halted by treating the cells with 10 ug/ml actinomycin D. This allows the snRNAs already synthesized to mature and move into the nucleus. In lanes F, G, and H it can be seen that species U2’ chases to mature-sized U2 and then out of the cytoplasm very rapidly after an actinomycin D chase. Calculations suggest a half-life of about 7 min. The disappearance of species Ul from the cytoplasm in the presence of actinomycin D suggests a half-life of approximately 15 min. The discrepancy between the halflives of U2’ and Ul from labeling to equilibrium and from a pulse and chase experiment is not understood. However, because the pulse and chase involves the use of actinomycin I>, the data from labeling to steady state are more reliable. These data demonstrate an approximately equal stability in the cytoplasm of Ul and U2 but a greater abundance of U2. This suggests that U2 is synthesized at a greater rate than Ul. To determine the abundance of the cytoplasmic snRNA

350 Sauterer, Feeney, and Zieve

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Fig. 3. Abundance of snRNAs in cytoplasts and karyoplasts L929 cells (2.5~ IO’) were enucleated and the cytoplast and karyoplast fractions were harvested. The cytoplasts were then detergent extracted in CSK buffer and the detergent-soluble species were saved. 1, 2, 5. and 10% of the karyoplast fraction (lanes 1, 2, 3, and 4 respectively) and 50% of the detergent-soluble cytoplast fraction (lane 5) were analyzed by gel electrophoresis. The gel was then processed for Northern hybridization and hybridized sequentially with the U2 (A), U3 (B), U1 (C). U4 (D), and U6 (E) probes. The abundance of the snRNAs in the cytoplasts was then determined relative to the quantity of the species in the nucleus.

precursors by an alternative approach, the cytoplasm was determined directly Relative Abundance

of Cytoplasmic

the relative abundance of the snRNAs in by Northern hybridization.

snRNA Precursors

To investigate the relative abundance of the snRNAs in the cytoplasm and nucleus, cell fractions prepared by cell enucleation were analyzed by gel electrophoresis and hybridization with cloned probes for the snRNAs. A single gel containing an increasing amount of the karyoplast fraction and half of the detergent-soluble snRNAs in the cytoplast fraction was blotted to DBM paper and hybridized sequentially with probes for Ul, U2, U3, LJ4, and U6 (Fig. 3). Enucleation prepares a cytoplast fraction that is contaminated with less than 2 % interphase cells and the approximately 3 % of the cells that are in mitosis. The newly synthesized snRNAs in the cytoplasm are soluble when cells are detergent extracted in a hyperosmotic buffer designed to stabilize the fibrous components of the cytoskeleton [39]. Using only this detergent-soluble fraction dramatically reduces the contamination of stable snRNAs. Any contaminating nuclei and about half the stable snRNAs in the dividing cells remain in the insoluble fraction. Therefore the nuclear contamination in the detergent-soluble cytoplast fraction is no more than 2%. The extruded karyoplasts generated by cell enucleation include the intact nucleus and a rim of cytoplasm. Greater than 95 % of the stable snRNAs fractionate in the karyoplast fraction with the nuclei [42]. Analysis of the distribution of mature ribosomes suggests that approximately 75 % of the cell cytoplasm is in the cytoplast fraction and 25% remains with the karyoplast (data not shown). To analyze the relative abundance of the snRNAs in the cytoplasts and karyoplasts, the gels were loaded with 1, 2, 5, and 10% of the karyoplast fraction and half of the soluble snRNAs from the cytoplast fractions. The resulting blot was then hybridized sequentially with the cloned probes for Ul, U2, U3, U4, and U6. The Northern hybridizations with the probes show a linear increase in radioactivity

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proportional to the amount of karyoplast material. The abundance of the snRNAs in the cytoplasts is then determined by direct comparison to the karyoplast samples with correction for the maximum 2 % contamination of mature snRNAs and the 25% of the cytoplasm left with the karyoplasts. In hybridization analysis the U2 probe recognizes both the cytoplasmic precursor U2’ and mature-sized U2 in the cytoplast fraction. The U2’ precursor is 0.8 % of the nuclear abundance and the mature-sized U2 is approximately 3%. When corrected for the possible contribution from nuclear leakage and dividing cells the cytoplasmic concentration of mature-sized U2 is about 1% and the total of U2’ plus U2 is about 1.8%. The U3 probe recognizes only 1% of the total cellular U3 in the cytoplasm. This species behaves differently than the others because it is localized quantitatively in the nucleolus. It does not usually leak from isolated nuclei and is not soluble in dividing cells so that the 1% value is probably correct. The Ul probe recognizes 3 % of the nuclear quantity in the cytoplasm and this should probably be corrected down to 1%. The U4 and U6 probes recognize approximately 4 % of the total in the cytoplasm and again this should probably be corrected down to near 2%. These data then suggest a small pool of newly synthesized snRNAs in the cytoplasm of approximately 1 to 2% of the nuclear abundance of snRNAs. Abundance

of snRNP

B Protein

in the Cytoplasm

To complete the analysis of the cytoplasmic assembly of the snRNPs, the halflives and abundances of the snRNP proteins in the cytoplasm were also determined. These analyses use Sm autoimmune antisera developed in association with the autoimmune disease systemic lupus erythematosus (SLE). A mouse monoclonal antibody derived from inbred mice with SLE and a human patient antiserum were both used. In L929 cells the Y12 anti-Sm monoclonal antibody recognizes only the B protein in Western blots, although in some other systems it also recognizes additional snRNP proteins [30, 351 (Fig. 4). The patient antiserum also recognizes primarily the B and B’ protein; however, limited reactivity is seen with other snRNP proteins [15]. The Y 12 anti-Sm monoclonal antibody immunoprecipitates the five snRNAs, Ul, U2, U4, U5, and U6. In Fig. 4 it is shown that this antibody also selectively immunoprecipitates a family of proteins previously identified as components of the snRNP particles. Newly synthesized snRNP proteins are immunoprecipitated from pulse-labeled cytoplast preparations and identical stable snRNP proteins are precipitated from nuclear fractions. The identities of the proteins are assigned by molecular weight and by comparison to published reports. We consistently see both the B and B’ in the immunoprecipitates of mature snRNP particles but primarily the B protein in immunoprecipitates of pulse-labeled cytoplastic fractions. The immunoprecipitations are less than 10% effective in precipitating the stable snRNPs. However, under similar conditions the efficiency is constant between duplicate samples. To determine the concentration of the snRNP protein in the cytoplasm and nucleus, L929 cells were fractionated by enucleation and the relative abundance of the B protein was determined by Western blotting with the Sm antisera using

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Fig. 4. Recognition of polypeptides by the Sm antibodies in L929 cells. Tissue culture medium from the Y12 hybridoma containing approximately l-2 &ml of antibody and a patient serum obtained from E. DeRobertis [15] were used as indicated. L929 cells (1 X 10’) were labeled with 50 pCi/ml [?S]methionine for 1 h and enucleated and cytoplast preparations were immunoprecipitated with the Y 12 Sm antiserum (lane A). Cells (1 x 107) were labeled for 16 h with 5 &i/ml [3SS]-methionine and whole cell preparations were immunoprecipitated with the Y12 Sm antiserum (lane B). Immunoprecipitation with the patient antiserum gives similar results. Whole cell extracts were separated by gel electrophoresis and then analyzed by Western hybridization with the Y12 Sm antiserum (lane C) and the Sm patient serum (lane D).

an approach analogous to that used in the Northern hybridization analysis of the snRNAs (Fig. 5). The soluble proteins from the cytoplasts were concentrated and analyzed relative to dilutions of the karyoplast fractions. Again, the cytoplasts were detergent extracted to minimize the contribution from contaminating nuclei and dividing cells. The detergent-soluble cytoplasmic fractions contain greater than 90% of the total B protein in the cytoplasm (data not shown). The Western blots provide a signal proportional to the amount of B protein loaded on the gel. Comparison of the detergent-soluble cytoplast fraction and the karyoplast fractions from several blots using the Y 12 monoclonal antibody suggests a relative abundance of the B protein in the cytoplasts of approximately 20% of the nuclear abundance. After compensation for the 2% nuclear leakage and the 25 % loss of cytoplasm from the cytoplasts this corrects to approximately a 25 % relative abundance of the B protein in the cytoplasm. Western blots using the patient serum routinely suggested a relative abundance of 20% of the B protein. The patient serum also recognized the D protein in the nuclear fractions, although the intensity of the signal was far less than that for the B protein. A small amount of D protein was also recognized in the cytoplasm, although the relative abundance was less than 4% (Fig. 5, lane M). The relative concentration of the B protein in the cytoplasm is much greater than that of the snRNAs. As will be discussed later, neither the Yi2 monoclonal antiserum nor the patient serum which recognize 20% of the B protein in the cytoplasm by Western blotting of cell fractions show an equivalent level of

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Fig. 5. Abundance of the B protein in the cytoplast and karyoplast fractions. L929 cells (5x 10’) were enucleated and the cytoplast and karyoplast fractions were harvested. The cytoplasts were then detergent extracted in CSK buffer and the soluble fraction was TCA precipitated. All of the cytoplast fraction and 5, 10, and 20% of the karyoplast fraction were analyzed by gel electrophoresis. The gel was then processed for Western hybridization and hybridized with the Sm monoclonal antiserum and patient serum. The abundance of the B protein in the cytoplasts was then quantitated relative to the quantity of the species in the nucleus by photographing the blot, printing it on positive transparency film, and performing quantitative densitometry. Lanes A, B, C, and D are 5, 10, and 20% of the karyoplast fraction and 100% of the cytoplast fraction in experiment I and lanes E, F, G, and H are similar samples from experiment 2 blotted with the Y 12 monoclonal antibody. Lanes I, J, K, L, and M are 5, 10, 20, and 30% of the nuclear fraction and 100% of the cytoplast fraction from an experiment probed with the Sm patient serum.

fluorescent staining in the cytoplasm. This suggest that the proteins recognized in the blots are not available for fluorescent staining in fixed cells. Half-Life of the snRNP Proteins in the Cytoplasm

To complete the analysis of the B protein, the stability of the protein in the cytoplasm was determined using the same kinetic approaches used previously to analyze the cytoplasmic snRNA precursors. The half-life of the B protein in the cytoplasm was determined by analyzing both the accumulation of radioactive protein under labeling conditions of constant specific activity and the decay of labeled protein following a pulse label and chase. All experiments were carried out using cytoplast fractions to eliminate problems of nuclear leakage and the snRNP proteins were identified by immunoprecipitations with the Y12 anti-Sm monoclonal antiserum. In the first approach, cells were labeled under conditions of constant specific activity and the accumulation of the radioactivity in the snRNP proteins was

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ABCDEF” Fig. 6. Half-lives of the snRNP proteins in the cytoplasm as determined by labeling to steady state. For label to equilibrium, 2.5~ 10’ L cells are labeled with 15 @/ml [?S]-methionine in 30 ml 80% methionine-free SMEM supplemented with 5 % fetal calf serum, 25 mM Hepes, pH 7.1, and 2.5 m&f L-glutamine (GIBCO) for the indicated intervals with staggered starts so that all samples were harvested and enucleated simultaneously. The addition of glutamine and Hepes was necessary to allow linear incorporation of labeled methionine into cellular protein. Cytoplasts were collected after the enucleation, rinsed in ice-cold PBS, and extracted in 1 ml CSK buffer (100 mM NaCl, 10 mM Pipes, pH 6.9, 3 mJ4 MgQ, 5% VRC, and 1% aprotinin. The extract was divided into two 500~ul aliquots, immunoprecipitated with 500 ul of the Sm Y 12 tissue culture supernatant as described under Materials and Methods, and run on duplicate 12.5% SDS-polyacrylamide gels (bottom panel, intervals of 1, 2, 3, 4, 6, and 8 h). Autoradiograms were scanned with an E-C Apparatus Corp. densitometer coupled to an IBM XT as described under Materials and Methods. Areas of the peaks were calculated and the data were plotted. Data for the B protein from duplicate immunoprecipitations are indicated by the open and closed circles. Data were fitted to a curve of the form R(t)=&,, (1 -e-‘? where R(t) is intensity at time t, R,,, is the maximum intensity and k=log,/half-life. The curve illustrated is for a half-life of 2 h (top panel). To monitor the incorporation of radioactivity into whole cells, triplicate 50-ul aliquots of cells (equal to 180,000 cells each) were taken from each time point immediately prior to processing the cells for enucleation and added to 500 ~1 0.5 % SDS buffer (100 mM NaCl, 10 mM Tris-HCL, pH 7.4, 5 mM EDTA, and 0.5 % SDS) and then trichloroacetic acid was added to 6.7%. The samples were incubated from 30 min to overnight at 4°C. and the acid-insoluble protein fraction was collected on glass fiber filters (Whatman) for scintillation counting (top panel, inset).

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determined by immunoprecipitation of solubilized cytoplast fractions (Fig. 6). Cells were labeled for up to 8 h in medium containing only 20% the normal methionine concentration and 15 @/ml [35S]-methionine. The initiation of labeling for the different time points was staggered so that all samples were harvested simultaneously for enucleation. After the indicated times of labeling, cells were enucleated and the snRNP proteins in the detergent-soluble fraction of the cytoplasts were immunoprecipitated with the Y12 monoclonal antibody and analyzed by gel electrop’horesis (Fig. 6, bottom panel). TCA precipitations of whole cells taken at the time of harvesting indicate that there is a nearly linear incorporation of radioactivity under these conditions (Fig. 6, top panel, inset). The B and D proteins are the most highly labeled snRNP proteins under our conditions with [35S]-methionine, and in the immunoprecipitates they show a nearly parallel accumulation of radioactivity. The accumulation saturates at around 4 to 6 h of labeling and suggests a half-life in the cytoplasm of approximately 2 h for these proteins (Fig. 6, top panel). A similar result is observed when the decay of radioactivity is monitored after a pulse and chase. For the pulse and chase protocol, cells were labeled with 100 uCi/ml [“‘S]methionine for 45 min in medium containing only 5% the normal methionine concentration. Cells were then removed from this medium and resuspended in normal medium to halt further labeling. At hourly intervals for up to 8 h after the initiation of the chase, the radioactivity in cytoplasmic snRNP proteins was monitored by immunoprecipitation of the cytoplast fractions as described above (Fig. 7, bottom panel). Again, labeling and chasing were staggered so that all samples were harvested simultaneously. TCA precipitations of whole cells harvested immediately before enucleation suggest that the nonradioactive chase was effective and that further labeling was halted (Fig. 7, top panel, inset). Radioactivity in the EI and D proteins in the cytoplasm show a rapid decline during the chase protocol (Fig. 7, top panel). Presumably this is because they are assembling into snRNPs and moving into the nucleus. However, formally it can not be ruled out that some are turning-over in the cytoplasm. Quantitative analysis of the decay of radioactivity suggests a half-life of approximately 1.5 to 2 h for these proteins which is similar to that determined by the labeling to saturation protocol discussed above. DISCUSSION This report systematically investigates the abundance and half-lives of the snRNAs and snRNP proteins in the nucleus and their precursors in the cytoplasm of mouse L929 cells. This information is the necessary first step in our investigation of the regulation Iof snRNP particle assembly. Cytoplasmic fractions prepared by cell enucleation are used for this analysis to avoid the nuclear leakage encountered during aqueous cell fractionation. These experiments identify a large pool of snRNP proteins in the cytoplasm that are available for assembly with the less abundant and more rapidly turning-over snRNA precursors. Because five of the major snRNAs, each with different abundances and half-lives, share five

356 Sauterer, Feeney, and Zieue

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8

6

D A

I3

C

D

E

F

Fig. 7. Stability of the cytoplasmic snRNP proteins following a pulse label and chase. For the label and chase protocols cells were labeled in staggered intervals so that all the samples were harvested simultaneously for enucleation. Six aliquots of 25x10’ L cells were labeled for 45 min with 100 uCi/ml [“S]-methionine in 10 ml 9.5% methionine-free SMEM supplemented by 3 % fetal calf serum, 12 m&f Hepes, pH 7.1, and 2.5 mM L-glutamine, and chased by resuspension of the cells in 30 ml of normal unlabeled growth medium. All samples were then harvested after the indicated time of chase and enucleated. The cytoplasts were immunoprecipitated with the anti-Sm Y12 antiserum as described in the legend to Fig. 6 (bottom panel). The autoradiogram was scanned and the data were plotted. The open circles are for the B protein and the closed circles for the D protein. Data were fitted to a curve of the form A(r)=A&-“) where A(f) is intensity at time f, A0 is intensity at time 0. and k=log,/half-life. The top curve illustrated is for the D protein and illustrates a half-life of 1.5 h. The bottom curve is for the B protein and illustrates a half-life of 1.5 h (top panel). The incorporation of label into the cells following the pulse and chase protocol was determined by TCA precipitations of whole cells as indicated in the legend to Fig. 6 (top panel, inset).

snRNP proteins in common, in addition to species-specific proteins, this suggests that snRNP synthesis and assembly is regulated at multiple levels. In growing mouse L929 cells, the assembly of new snRNP particles provides for both the demands of cell growth and for replacement of mature nuclear snRNP particles. The mature nuclear snRNAs have relative abundances and stabilities similar to those observed in other cell types. Ul and U2 are 2- to 5-

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fold more abundant than the other species and while Ul is totally stable, U2 turns over with a half-life of about 40 h. However, the U6 species, identified as a single band of characteristic mobility on a polyacrylamide gel, has a kinetic behavior unlike the other snRNAs. U6 has a steady-state concentration about threefold greater than that of U4 and a large fraction of this species decays with a half-life of approximately 15 h. However, after an extended chase the abundance of the species is almost identical to that of U4. U6 is complexed in a particle with the U4 species 14, 171 and recently was shown to be transcribed by RNA polymerase III, unlike the other snRNAs transcribed by polymerase II [20]. This suggests that a subpopulation of the species may be stabilized by its association with U4, although, sequence analysis will be required to determine if the pulse-labeled U6 band may represent different species with unique behaviors. The bulk of the snRNAs, however, behave as stable species. Pulse and chase experiments have previously demonstrated a quantitative maturation of the Ul and U2 cytoplasmic snRNA precursors into mature nuclear species, and presumably the other cytoplasmic snRNA precursors also have a similar quantitative maturation. Therefore the rate of synthesis of the cytoplasmic precursors, which is a function of their abundance and half-life, equals the rate of synthesis of the mature nuclear species [39]. Kinetic studies employing labeling to steady state suggest that Ul and U2 have half-lives in the cytoplasm of about 20 min, consistent with previous determinations [8, 401. The Northern blotting experiments suggest a cytoplasmic abundance of 2%. Over 30 h of cell generation, the turnover of the small cytoplasmic pool with a 20-min halflife should almost double the quantity of stable snRNAs as is appropriate for cell growth. The second set of experiments in this study calculates the relative abundances and half-lives of the snRNP proteins in the cytoplasm. SnRNP proteins have been detected in the cytoplasm of Xenopus oocytes and in mammalian cells fractionated by nonaqueous procedures but the analyses were not quantitative [ 15, 351. Due to the available antisera, our analysis was limited primarily to the B protein. Quantitative Western blotting demonstrated that the B protein was present in the cytoplasm in an abundance of about 20-25 % of the nuclear abundance using both a mouse monoclonal and human polyclonal anti-Sm sera. All cell fractions were boiled in a high concentration of SDS and reducing agents to ensure a quantitative recovery of the snRNP proteins. The human polyclonal antiserum also shows limited reactivity with the D protein in nuclear samples. However, in the cytoplasmic samples there is only a trace of reactivity with the D protein. This suggests that the antigenic determinant recognized is not present on the D protein in the cytoplasm or that the cytoplasmic abundance of the D protein is much lower than that of the B protein. Fluorescent staining with the same antisera that detected 25 % of the B protein in the cytoplasm by Western blotting detects only a low level of staining in the cytoplasm [21, 28, 411 It does not identify a cytoplasmic pool of B protein approaching 25 % of the nuclear abundance. This suggests that in formaldehydefixed cells the unassembled B protein in the cytoplasm does not display the same 23t-888336

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antigen as the nuclear B protein or alternatively the dispersed distribution of this antigen makes it difficult to identify in the cytoplasm. However, the antigen is clearly present on the cytoplasmic B proteins after electrophoresis and Western blotting. We are currently using alternative non-autoimmune antisera and microinjection to test the hypothesis that the antigenic determinant is available for fluorescent staining only after assembly into mature snRNP particles. The presence of a large pool of snRNP proteins in the cytoplasm is consistent with the observation that snRNA synthesis and snRNP assembly occur normally for over 1 h after the inhibition of protein synthesis [9, 391. The kinetic studies on the cytoplasmic snRNP proteins suggest that these proteins have half-lives of approximately 90 to 120 min in the cytoplasm. Due to the multiple manipulations required for each data point, including cell fractionation, immunoprecipitation, gel electrophoresis, and densitometry of the autoradiograms, there is scatter in the data points. However, the general conclusion that the half-lives of the snRNP proteins in the cytoplasm are about 2 h is supported by over eight different sets of experiments using both labeling to steady state and pulse and chase protocols followed by enucleation and immunoprecipitation. Control experiments indicate that because the cells used in each experiment differ only in the exact protocols of labeling, the efficiencies of cell fractionation and immunoprecipitation are identical. It is currently not possible to determine if there is a quantitative movement of the cytoplasmic B protein into the nucleus. The 2-h half-life of the B protein in the cytoplasm and the 25% relative abundance suggest that every 8 h there is sufficient B protein synthesized to completely replace the B protein in the nucleus. This compares to the doubling of the snRNA each cell generation as discussed above. The rate of synthesis of the B protein is greater than that needed for synthesis of new snRNP particles and suggests either that not all of the cytoplasmic B protein enters the nucleus or that the snRNP B protein may be less stable than other components of the snRNP particles and may turn over independently. Alternatively, the high abundance of this protein suggests that it may have an independent function in the cytoplasm. The situation in the cytoplasm of the fibroblast that we describe is similar to that previously described for the Xenopus oocyte [7. 13, 15, 381. The snRNP proteins are stored in the oocyte cytoplasm during oogenesis and are available for assembly with the snRNAs that are transcribed during early development. Like the situation in the mammalian cells, the large amounts of antigen in the cytoplasm of the oocytes stain only weakly in indirect immunofluorescence [38]. In the fibroblast cytoplasm there is a large pool of snRNP proteins that are available to assemble with the newly transcribed snRNAs that appear transiently in the cytoplasm before returning permanently to the interphase nucleus. Experiments are under way to determine if the cytoplasmic precursors are in storage forms unavailable for indirect immunofluorescent staining. This work was supported by grants from the National Science Foundation (DCB-X51 I I I?) and the Lupus Foundation of America. We thank Elizabeth Roemer for excellent technical assistance.

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