Newly synthesized small nuclear RNAs appear transiently in the cytoplasm

.J. Mol. N%ol. (lR88) 199. 2X- 267

Newly Synthesized Small Nuclear RNAs Appear Transiently in the Cytoplasm Gary W. Zieve, Roger A. Sauterer and Robert J. Feeney Department of Anatomical Sciences and Program in Cellular and Developmental Biology SUNY Stony Brook Stony Brook, NY 11794, U.S.A. (Received 9 January

1987, and in revised form

28 August 1987)

Newly synthesized small nuclear RNA (snRNA) species Ul and U2 are easily ident)ified in cytoplasmic fractions prepared by standard aqueous cell fractionation. However, because the mature stable snRNA species leak from isolated nuclei during cell fractionation, the possibility exists that these newly synthesized species also leak from the nucleus. To overcome the problems of nuclear leakage, mouse L929 cells were fractionated by cell enucleation. Enucleation extrudes the nuclei from cytochalasin-treated cells and produces cytoplasts that, by several criteria, are a bona Jide cytoplasmic fraction uncontaminated by nuclear material. All six of the major snRNAs are present in the cytoplasts (c-snRNAs) shortly after synthesis. The species are identified by immunoprecipitation with specific antisera against the ribonucleoproteins and by Northern blotting and hybrid selection using cloned probes. This confirms and extends similar studies that used non-aqueous cell fractionation and manual dissection to overcome nuclear leakage. Kinetic studies demonstrat’e that the c-snRNAs return to the interphase nucleus after approximately 20 minutes in the cytoplasm. The 172 precursor 172’ is processed to mat’ure-sized U2 in the cyt)oplast, fractions before returning to the nucleus. The c-snRNAs occur in ribonucleoprotein particles with similar antigenicity to the mature nuclear particles wit,hin six minutes of transcription. This suggests that in mammalian cells, important steps in the assembly of these ribonucleoproteins occur in the cytoplasm.

1. Introduction

participate in its processing. The snRNP particles function in both the splicing and the generation of the 3’ end of pre-messenger RNA (for reviews, see Sharp. 1987; Maniatis & Reed, 1987). Immunofluorescent staining with antisera, generat,ed by patients with the autoimmune disease systemic lupus erythematosus (SLE), that recognize protein determinants of the snRNPs identifies the stable snRNP particles in the interphase nucleus (Lerner & Steitz. 1981; Nyman et al., 1986). At the onset of mitosis, the snRNPs distribute throughout the cytoplasm following the breakdown of the nuclear envelope and then return rapidly and quantit’atively to the newly formed daughter nuclei (Deng et al., 1981: Reuter et al.. 1985: Spector & Smith, 1986; Zieve & Slitzky, 1986). However, during fractionation of interphase cells the mature snRNPs leak from nuclei prepared by conventional procedure (Zieve & Penman, 1976). The newly synthesized snRNA species are readily identified in cytoplasmic fractions almost immediately after transcription. Because of their short half-hves and high rate of synthesis, species 171 and 1:2 in the cytoplasm (c-snRNAs) are highly labeled in short’ pulse labels (for a review, see

The small nuclear RNAs are a family of low molecular weight RNA species in the cell nucleus characterized by their unusual tri-methyl G cap and their association wit’h a specific group of RNA binding proteins. There are six major species, designated Ul to U6, and several minor species (Strub et al., 1984: Reddy et al. 1985). The snRNAst are each complexed with a set of about eight proteins in ribonucleoprotein particles, the snR,NPs. Five of these proteins are shared by all of the species. except C3 and tJ6. The remaining proteins are species-specific. The two most abundant snRNA species, Ul and U2, are each present in approximately one million copies per cell (for reviews. see Busch et al., 1982; Mattaj, 1984; Krunel et al.. 1985). Recent studies demonstrate that’ several of the snRNAs base-pair with specific regulatory sequences in pre-messenger RNA and t Ahbrrviations used: sn, small nuclear; RSP. ribonuc~lroprotein: ST,E, systemic lupus erythematosus; c-snRSA. snR?jA in the cytoplasm; PBS, phosphatehfferrd saline; \‘R(‘. vanadyl ribonucleosidr complex.

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Eliceiri, 1981). However, the leakage of the mature particles from isolated nuclei raises t’he possibility that the newly synthesized species may also leak into the cytoplasmic fraction (Eliceiri. 1974; Madore et al.. 19846). Non-aqueous cell fractionation, which does not suffer from problems of nuclear leakage, has identified precursors of Ul and C2 in the cytoplasm shortly after transcription (Gurney & Eliceiri, 1980). This procedure avoids all aqueous buffers and mechanically removes the cytoplasm after flash freezing and lyophilization. However. non-aqueous cell fractionation is cumbersome for routine experimentation with tissue culture cells. In Xenopus oocytes, where the germinal vesicle is manually separated from the cytoplasm to avoid nuclear leakage, snRNPs were shown to assemble in the cytoplasm after the microinjection of snRNAs (De Robertis et al., 1982). This syst’em has been used t,o demonstrate that, newly synthesized snKNAs have a mono-methylated guanosine cap, and t’hat the characteristic tri-methyl guanosine cap is generated in the cytoplasm (Mattaj, 1986). These observations support data from conventional cell fractionation, which illustrate that newly synthesized species 111, 112 and IT4 appear transiently in t,he cytoplasm, where several of t,he species are processed (Eliceiri, 1980; Madore et al., 1984a,b). In this report, enucleation of 1,929 mouse fibroblasts is used to prepare bona fide cytoplasmic fractions. Thede cells enucleate with high efficiency and yield cytoplasts in biochemically useful quantities that show no evidence of contaminating nuclear material (Wigler & Weinstein, 1975: Zorn rf d., 1979). This procedure has been used to overcome problems of nuclear leakage during standard aqueous cell fract,ionation, and has demonstrated that the estrogen receptor actually resides in the nucleus (Welshons of al.. 1984). Karyoplasts and cytoplasts continue rnet’abolic activity for several hours after separation (Zorn et al., 1979). All six of the major snRNAs appear in t*hr cytoplasts immediately after transcription. Species U2 is processed while in the cytoplasm and all the species, with the possible exception of U6 and t,he nucleolar species U3, occur as ribonucleoprotein (RNP) particles in the cytoplasm. Pulse and chase experiments demonstrate that, after approximately 20 minutes in the cytoplasm, the newly matured species Ul and U2 move back into the nucleus. The transient appearance of all the snRNAs as c-snRNAs suggests that, important steps in the maturation of these RNAs occur in the cytoplasm.

2. Materials and Methods L929 cells were cultured in suspension in minimal essential medium (MEM; Gibco. Grand Island, NY) supplemented with ST/, (v/v) fetal calf serum. Cells were maintained at a density of between 30 x 104/ml and 60 x I04/ml.

et

al.

For pulse-labeling with r3H jnucsleosidrs, cells wrrt’ concentrated before the addit,ion of isotope. and for longterm labeling with [3H]nucleosides. cells wt~re maintained under normal growth conditions. For labeling with 32P~ cells were concentrated S-fold and resuspended ilr medium lacking phosphate. with 132Pjorthophosphat’~~ added to the indicated c:onc*entration, For studies with metabolic inhibitors, reagents were added direcatty to t,he culture medium at t,hr indicat,ed concrntration. (b) (‘rll j’t~rcfionatio,r 1, cells wtlrp mucleated in suspension by tnodilication of the procedure of Wiglrr 61 Weinstein (1975). Briefly. 1250b to 2,596 Ficoll stq) gradients in MEM with 10 pg c*ytochalasin K/ml (Sigma. St, T,ouis. MO) were’ pourt~~ in SW41 tubes and maintained at 37°C’ for 12 to 16 h hefore use. Appropriately iabeled cells werp trrat,ed with 10 pg cA.ytochalasin K/ml 10 min before runc~lration ;\ maximum of 3.0 X 10’ csrlls were then concent.ratrd tcb 3 ml of 12+4,, Fieoll and overlayered on each gradient. (iradients were centrifuged for 60 min at 33,000 rrvsinlilt in an SW41 rotor at 37°C. Cells separated into layrrs with a vesicular fraction at about 13O/;, Ficoll. cgt,oplasts at 16?& Ficoll, whole cells at 21 ?A Ficoll and karyoplast~s in the pellet. The enucleat,ed fractions were harvested from above with a Pasteur pipet. diluted with PBS antI fx~llrctpd by cent,rifugation of 1000 g for -4 miu. Thr presence of nuclei in t,he cytoplast fractions was identified hy staining a smear of the qtoplast prt’parations with 1 pg Hoechst. 33258/ml and ohsrrving with epifluorrscsencr optics. Routine enucleation rficienc+irs were greater than 9Soh. lt) is essential to avoid using clumpy cells. because this lowers the etliciencay of tht* enucleat,ion. RKA spec-irs in the cytoplasts and karyoplast,s were prepared and analyzed like normal cAyto plasmic and nuclear fractions. For thta conventional aqueous rxtra,ctiorl into nuc~lt~al~ and caytoplasmic fractions. RSH huffrr (10 mivl-Xa(‘l. I.5 m>~-Mg(‘12. 10 maf-Tris H(‘l (pH 7.1)) and Iron-iollica det,ergents were used as described (Zievtl & Penman. 1976). Vanadvl rihonuc>leoside complex (VRC) was afitlrti in concentrations of up to 6 rnM to inhibit, ribonu~lrasrs (ISerger 8r. Rirkenmeir. 1979). VRC also inhibits the acation of DBase 1 and was reduced to 1 mw in the extraction buff‘er used immediately before digest,ion with DBase. (c) Imm~LnflprPcir)itatic,n

lmmunopreripitation stufiirs used mouse mono&ma1 antibodies with the SLE srrotype of anti-Sm and antiRNP. The hybridomas werr generous gifts from l)r .I. Steitz (Lernrr sf al.. I!#]) and Dr S. Hoch (Billings el al., 1982). Tissuta cult)ure supernat’ants witah approximately 2 to 4 pg of immunoglobulin (:/ml were used fol all experiments. For the kinetic studies. 1 x 10’ L929 cells wert’ labeled wit,tr [ 3H luridinr ( 100 hc(‘i/ml) aud [ 3H]adenosine (100 &i/ml). (‘ells wtkrt‘ frnc+ ionat,rd iI> 1 ml of RSH buffer c~ontaining O..i*,, Tritorl S- 100. 6 m;n-VRC. and 0.5 ml of tissue culture suprrnatant from the Sm clones was added to eacah sampIt% and incwhatrd for 2 h with gent]? rocking. From 2 to IO mg of act.ivatr(l protein A-Sepharose (Sigma. St T,ouis, MO) was thru atlfiefl to ea(xh sample. Aft,rr 60 uiin ot’ infvt~ation. the prot,ein A-Sepharose was c~ollr~t~rd by crntrifugation ant1 washed extensively. Thr protrin A-Srpharosr pellt+s w<‘I’<’ resuspended in RhA sample buffer c*outaining 2’$, SIXi. Samples were heated t,o 60°C’ and t,ht, released material was analyzed directly on RX4 gels. Tmmunoprr~ipitat,ic~~~

Newly Synthesized of the cytoplast procedure.

fractions

was carried

(d) Northern

hybridization

snRNAs

out by a similar

Electrophoretic profiles of the snRNAs were transferred from the polyacrylamide gels to nitrobenzyloxymethyl paper (Transa-bind from Schleicher and Schuell, Keene, NH), which had been reduced and activated to DBM paper by standard procedures. Electrophoretic transfer was performed at 15 V, 0.4 to 0.5 A, for 4.5 h at 4°C in 0.2 M-sodium acetate (pH 4.0). After transfer, the paper can be stored in the cold in prehybridization solution for up to several months or processed directly for hybridization. Hybridization was carried out according to the procedure of Alwine et al. (1979). Briefly. the paper was prehybridized in 5 X SSC (SSC IS 0.15 M-NaCI, 0.015 M-trisodium citrate) containing 50% (v/v) formamide, 50 ITIM-NaPO, (pH 6.5), 1 y0 (w/v) SDS, 1 y& (w/v) glycine. 1 x Denhardt’s solution, and 0.5 mg denatured calf thymus DNA/ml 37°C overnight on a rotator. Following the prehybridization, the radioactive probe is denatured by boiling for 10 min and added to the hybridization buffer minus glycine with 5% (w/v) dextran sulfate replacing the calf DNA. The probe was added to a conrentration of 500,000 cts/min per ml of hybridization mixture. Incubation was carried out for 12 to 36 h on a rotator at 37°C. Following hybridization, the paper was washed for approximately 3 h in 4 1 of 2 x SS(‘. 0.1 y0 SDS at room temperature, then blot,ted until barelq moist. and analyzed by autoradiography. To reuse the blot, the hybridized probe was removed by 2 washes (30 min each) in 99% formamide at 45°C followed by 2 washes in prehybridization buffer lacking calf DNA. It can then be used again by sequential prehybridization and hybridization as discussed above. Radioactive probe capable of hybridizing to the snRNA was generated by copying the snRNAs cloned in the Ml3 vectors by primer extension (Hu & Messing. 1982). [cc-32P]ATP was included in the reaction mixture to label the probe. Synthesized duplex material was separated from t,he unincorporated nucleotides by chromatography on Sephadex G50, and t,he probe was concentrated by precipitation with ethanol. ITI. U2 and U3 cloned in the Ml3 vectors were generous gifts from Alan Weiner (Yuo et al., 1985). The c4 and U6 clone was a generous gift from Dr Thoru Pederson (Madore et al.. 19846). (P) Hybrid

&e&on

Hybrid selections were carried out by a slight modification of previously described procedures (Madon: et al., 39843). Antisense Ul, U2. U4 and U6 cloned in Ml3 vectors, gifts from E. Wieben, A. Weiner and T. Pederson, respectively. were grown and harvested as single-stranded DNA. From 50 to 70 pg of the M 13 DNA was spotted onto a 20 mm diameter Nytran filter (Schleicher and Schuell, Keene, NH), allowed to air dry and then baked at 80°C for 2 h. Filters were prehybridized for 2 h at 50”(~ in 50:/, formamidc. 0.6 M-NaCl, 0.1 M-Pipes (pH 6.6), 5 x Denhardt’s solution 0.1% SDS and 100 pg poly(A)/ml, then incubated with the appropriate RNA sample brought up to a volume of 1 ml in 509:, formamide, 0.6 M-NaCl, 0.1 M-Pipes (pH 6.6), 1.5 x Denhardt’s solution and 0.1% SDS in a siliconized scintillation vial and maintained with gentle shaking at 50°C. Filters were then washed extensively in 0.2 M-NaCl, 100 mM-Pipes (pH 6.6) and 0.5%) SDS at

261

Appear in the Cytoplasm

60°C. RNA was eluted from the filters by heating to 80°C for 5 min and then boiling for 1 min in distilled water. Filters were then removed, carrier tRNA added and the eluted snRNAs were precipitated with ethanol for further analysis. (f) HNA

preparation

and

electrophoresis

RNA was prepared from cell fractions by digestion with proteinase K followed by extraction wit,h phenol and chloroform in the presence of 0.5% SDS as previously described (Zieve & Penman, 1976). Fractions were precipitated with 2 vol. ethanol. Precipitated samples were collected by centrifugation at 10,000g for 15 min air-dried, and then resuspended in RNA sample buffer. The preparations were then analyzed on 6y’ to 157; gradient gels as described (Zieve & Penman, 1976). An advant,age of these non-denaturing gels is that the 5.5 S rRNA is not released from the 28 S rRNA. This species migrates with a mobility intermediate between 1Jl and U2 and interferes with the analysis of these 2 snRNA species. The mobility of species U3 is faster in these gels than in denaturing gels, most likely due to its extensive secondary structure. Also, the 7SL species migrates as bands of 2 different mobilities because of differences in secondary structure.

3. Results of L929 cells and identijkation of snRNAs

(a) Enucleation

Enucleation of mammalian tissue culture cells is an alternative method of cell fract,ionation that prepares a cytoplasmic fraction uncontaminated by nuclear components. Centrifugation of cytochalasin-treated L929 cells in Ficoll gradients allows the enucleation of 3 x 10’ cells per gradient with efficiencies of over 98% (Wigler & Weinstein, 1975; Fig. I). L929 cells enucleate with a higher efficiency than HeLa cells, most likely because of the absence of keratin-type intermediate filaments in the cytoplasm. The cytoplasts are free of any observable nuclear material; however, the extruded nuclei are surrounded by a thin rim of cytoplasm. When assayed by measuring the distribution of the mature tRNA and 18 S rRNA, 75 to 85% of the cytoplasm fractionates in the cytoplasts and the remainder stays associated with the nuclei in the 18 S rRNA exits the nucleus karyoplasts:

L929

Cell

enucleation

/

1

cells

kG>

Whole

cells

Figure 1. Enucleation of suspension cultures of L929 cells. L929 cells were pretreated with 10 pg cytochalasin B (CCB)/ml and enucleated by centrifugation at 33,000 revs/min for 60 min at 37 “C in 12.5:/, to 25% Ficoll gradients as described in Materials and Met’hods.

-u5 -U6

US-;, tRNA-(

1

A

I3

C

D

E

G

F

H

I

J

K

Figure 2. Identification of the snRN.As. The snRSA species it1 1,929 culls \crre identified I,y c-rll ti’acxtionaiiott and Northern blotjtinp. The stable snKX;I spccirs were combined with metabolic labeling. immunoprecipitation tttc~tabolically labeled with L3H]uridine for I6 h and the species in the cytoplasmic (lane -L\) and nuclear (lane 13) fractiotls were analyzed by gel electrophoresis. The nuclear fractions were then itnmunoJ)re(~i~)itated with the Sm (lane (‘) and KNF’ (lane I>) antisera. IVon-immune immunoprecipitatrs show no low molecular weight species. Identification of nr\bl> synthesized species in the cytoplasts were carried out) by labeling I%!9 cells wit#h 32f (750 ~(‘i/ml) for 2 h. prqtaring cytoplasts and then immunoprecipitating t,he cytoplastjs with the Sm antiserum (lane F) and with nott-immune serum (lane E). Identification of t,he individual sprc-irs in t,hr cvtoplasts was also carried out by preparing c,,vtoplastn from unlabeled cells and analyzing the same gel by sequential iorthern blotting with cloned probes of 1!2 (lane (i). I-3 (lattt H). VI (lane I), U4 (lane ?I) and lI6 (lane K). For some unknown reason t,hr I’4 probe. which was the first prohr us~tl. did not strip romplet,el;v and this facilitat’ed in lining up the aut,oradioprams of thr diftPrent blot,s.

immediately after processing, unlike 28 S rRr\‘A. which remains in the nucleolus for some t’ime. The exact distribution can vary between different experiments (data not shown). In this work, cell fractionat,ion by enucleation is used to investigate the intracellular distribution of the newly synthesized snRNAs. The individual snRSA species are identified by their characteristic m’obilities in polyacrylamide gels (Fig. 2, lanes A and K). The snRNAs are the major stable low molecular weight RNAs in the nucleus (lane K) and they have electrophoretic mobilities between tR?r’A and 7SL. Significant amounts also appear in the by aqueous cytoplasmic fraction (lane A) prepared cell fractionation because of nuclear leakage. The polymerase III transcripts tRNA, 5 S RNA and 7S1, RXA are the other highly labeled low molecular weight RSA species. and are found quantitatively in the cyyplasmic fractions. The identification of the individual snRh’A species can be confirmed by immunoprecipitation of the snRNI’ pa,rticles with specific antisera, and by Northern hybridization and hybrid selection with specific cloned probes. The 8m and RNI’ antisera derived from patients with systemic lupus erythematosus recognize protein components of the snRNPs. These antisera

now available as monoclonal antibodies derived mice with a lupus-like disease (I,erner it 01.. I981 : Billings ~‘f rrl., 1982). The Sm ant~iserum recognizes common proteins in t,he nucleoplasmic. snRST’ particles and precipitatcas ITI. I’?. (‘4. 175 and (‘6. whilst REP immunopreripit,ates only 1‘1 Fig. 2. I,anrs(’ ilrld I)). (Lerner & Steit,s. 1981; Other dat,a suggest that l’fi is not associa,ted with the same RNP binding proteins and only irnmuno precipitates because it is c~omplt~xrd on to a sinpk~ particle with 1’1 (Hashirnoto Cy- Stritz. I!)%: Kringmann Pf al.. 1984). Immunoprecipit’ation of solubilized extracts of the I, cell nuclei with t hescb antisera confirms these results and helps t,o su I)stantiate the identification of these species in this system (Fig. 2. lanes C’ and I)). rlectrophoretica Several of the snRSA species are no\v cloned. and Sorthern blott)ing with t’he appropriat,e probes (WI be used to identify the individual species (MadortA rf al.. 19846). Figure 2, in lanes G to Ii. a single gel of’ a rytoplast preparation is probed secluentiall~ wil’tr 111, 112. (3. 1’4 and Uci. Each clone identifies t\ unique species with t#hr expect)ed elt,c~tro1)tlorrt,i(. mobilit)y, and helps tJo confirm thr identity of these species. The I::! (*lone recognizes maturt, 62 antI its precursor Y2’. which is approximately t,cn nuc*lootides larger (Yuo r’t crl.. 198.5). Mat,ur(‘ IT% oft.cbri are

from

Newly

Xynthesized

snRNAs

Appear

in

663

the Cytoplasm

appears as a closely spaced dimer, which may represent heterogeneity on the 3’ end. For some unknown reason there is a small amount of crosshybridization of several of the probes with the U4 species. This is useful for helping to line up the different autoradiograms. (b) Newly

synthesized in

snRNAs

K

appear

7SL

the cytoplasm

Newly synthesized species of the two most abundant snRNAs, Ul and U2, are readily identifiable in oytoplasmic fractions prepared from pulse-labeled cells (Eliceiri, 1974; Frederiksen & Hellung-Larsen, 1975; Zieve & Penman, 1976; et al., 1984a). After the tRNA, 5 S rRNA Madore and 781, RNA, they are the most highly labeled low molecular weight RNA species in cytoplasmic fractions. To localize the snRNA species in the cytoplasm and nucleus unambiguously, newly synthesized and mat)ure stable snRNA species were selectively labeled with [3H]uridine by short and long-term labeling, respectively. The cells were then enucleated, and cytoplasts and karyoplasts were analyzed by gel electrophoresis. Figure 3 illustrates the distribution of pulse-labeled and stable snRNAs in enucleated fractions from 1,929 cells. The six snRNA species IJl to U6 are readily identifiable in cytoplasts from L929 cells pulselabeled for 20 min (Fig. 3, lane A). Because of their abundance and short half-lives in the cytoplasm, these species are highly labeled in a short pulse. All six of the major snRNAs (Ul to U6) can be identified by their characteristic mobilities. The identity of these species was also confirmed by immunoprecipitating pulse-labeled cytoplasts with the Sm antiserum (Fig. 2, lane F) and by Northern blots (Fig. 2. lanes G to J) of the cytoplast preparat,ions. The immunoprecipitation with the Sm ant,iserum demonstrates that Ul, U2. U2’, U4 and UF, are in the form of RNPs and are precipitated. The snRNAs in the cytoplasm are designated as c-snRNAs to distinguish them from the mature nuclear species. U6 is precipitated from nuclear ext,racts but is not precipitated from thn cgtoplast, fractions. This suggests that the association between U4 and U6, responsible for the immunoprec~ipitation of CT6 by the anti-Sm antiserum, forms aft,er the species return to the nucleus. In lanes (: to K of Figure 2, a cytoplast preparation is hybridized with the Cl, U2 U3, U4 and IT6 probes. The U2 probe recognized both mature-sized II2 and its larger precursor, U2’. The precursor-product relationship of these two species has been est,ablished by kinetic studies and sequence analysis (Eliceiri, 1981; Yuo et al., 3985). The I’l. U3, 114 and U6 probes recognize species with mobilities similar to those of the mature forms. Because the cloned probes recognize the unlabeled RNA, st’able material from contaminating nuclei will also be recognized. However, as described above, t,he cytoplasts contain less than So/,, cbontaminating nuclei. Also, t,he clear recognition of

u2 u3 UI

u4 SSRNA 5s

U5 U6

tRNA A

B

C

D

E

F

Figure 3. Localization of snRiVAs in cytoplasts and karyoplasts: 2.5 x IO’ C3H]uridine labeled JS29 cells were pretreated for 10 min with 1Opg cytochalasin B/ml and enucleated on 12.5% to 257; Ficoll gradients as described in Materials and Methods. Cytoplasts and karyoplasts were harvested and the small RPu’A species were analyzed on 67; to 15% gradient gels. J>ane A illustrates the small RXA species present’ in cytoplasts prepared from cells pulse-labeled for 20 min with [3H]uridine (80&i/ml). Lane B shows the small RKAs present in an identical preparation of cytoplasts cultured for 60 min after enucleation and before harvesting. Lanes C and D display the stable RNAs in cytoplasts and karyoplasts, respectively, from cells labeled for 16 h wit’h [3H]uridine (5 pCi/ml). Lanes E and F illustrate the stable RXAs in cells fractionated into cytoplasm and nucleus, respectively, using standard a,queous procedures. All lanes are from the same gel, although lanes A and B are from a longer exposure.

species U2’ in these profiles indicates that the newly synt,hesized snRNAs are present. In our electrophoretic system we do not see significant amounts of larger Ul and U4 precursors. as reported by other investigators (Madore et al., 1984a,b; Patton & Wieben 1987). In the pulse-labeled cyt,oplast preparation (Fig. 3, lane A), an unidentified species appears with mobility intermediate between those of U-4 and 5 S rRNA (*). No species of this mobility is found in the nucleus, and it does not immunoprecipitate with the anti-Sm antibodies. There is very little prein the caytoplasts. This is consistent, with the tRNA observation. from non-aqueous fractionation and manual dissections of oocytes, that these species are nuclear and only leak into cytoplasmic fractions during aqueous cell fractionation (MeltSon et al., 1980). The absence of pre-tRNA, which migrates with a mobility between 5 S ribosomal RNA and mature-sized tRNA, facilitates the identification of V75 and C6 in the pulse-labeled species. Tn cell

0. IV. Zieve et al.

264

fractions prepared by aqueous fractionation, the highly labeled pre-tRNA leaks from the nuclei and obscures these species. The 7SL small RNA is also easily identified in the cytoplasts; however, there is no appreciable amount of the 7SK species. This supports the results from non-aqueous cell fractionation that suggest that this species is nuclear (Gurney & Eliceiri, 1980). The distribution of the mature stable low molecular weight RNA species in cytoplasts and karyoplast’s is also illustrated (Fig. 3, lanes C and D). The enucleation of L929 cells labeled with 13H]uridine for 16 hours demonstrates that the mature stable snRNA species are nearly quantitatively localized in the karyoplast fraction (Fig. 3. lane I)). [3H]nucleosides are rapidly taken up from the medium, and an incubation of this length is actually a short pulse-label with an extended chase. The low levels of stable snRNAs detectable in the cytoplast fraction are likely the result of the dividing cells in the population and the few whole cells contaminating the cytoplast preparations. In dividing cells, where the snRNPs disperse throughout the cytoplasm, the majority of snRNAs remain in the “mitoplast”, which is formed when the chromosomes are extruded from the cell by the enucleation procedure (Zieve & Slitzky. 1986). The localization of nearly all the snRNAs in the karyoplasts is consistent with t,hr nuclear localization of the snRNY particles observed by fluorescent, antibody staining of the particles in fixed cells. The only stable small RNAs found in the cytoplasts are the tRNA, ribosomal5 S and the 7SL RNA. Tn contrast t’o the localization observed in cytoplasts and by fluorescent staining of fixed cells, all the ma,ture snRNAs are found in bot’h cytoplasmic and nuclear fractions prepared l)y conventional aqueous cell fractionation (Fig. 2. lanes A and B, and Fig. 3, lanes E and F). The extent of the nuclear leakage is a function of the specific buffers used to extract the nuclei. Increased ionic strength and elevated pH promote t.he leakage of the snRNPs from isolated nuclei (Zieve & Penman. 1981).

(c) Maturation of c-snRNAs Several experiments were carried out to assay for the maturation of the snRNAs in the cytoplasm. Tn the first experiment, a preparation of pulse-labeled cytoplasts identical with that illustrated in Figure 3 (lane A) was cultured for 60 minutes after enucleation and before harvesting (Fig. 3, lane H). Species U2 shows post-transcriptional modifications during this interval. The U2 precursor, U2’, diminishes and the amount of mature 112 increases proportionally during the incubation. Quantitative analysis by densitometry indicates that 65% of the radioactivit,y in U2 plus U2’ is in the precursor form immediately after enucleation. After the subsequent’ culture, only 25% is in this precursor form and the remainder has been converted to mature-sized LJ2. Control experiments indicate that, there is convrr-

7SL U2’ u2 UI SSRNA tRNA A

B

C

D

E

F

G

H

I


K

!.

Figure 4. Processing of xnRNAs in tllc* c*ytoplasm during a pulse and chase. TB29 cells WI’C pulse-lahrlrci fhr 10 min and t,hen cahasrd for 30 min with IO pg art,inorngcin D/ml. Following the ~mlse and chase, c*rlls were fractionated by h&h st,andard aqueous wII fractionation and enucleation of Ficoll gradients. ‘I’hf, snRXAs in the cyt~oplasm and nucleus wcrr analyzrti t)>aqueous cell fractionation after the pulse (lanes A and K) and after the chase (lanes (1 and 1)). Species 1‘1 (Iant’s 14: and F). I-2 (lanes (: and H). U4 (lanes I >Irld .i) ant1 I’ti (lanes K a.nd I,) were hybrid-selrcatrtf front (A~Iol&st t’rac+ons I)reI)ared after a 30 min label with [3H]uridinr (lanes E. (:. 1 and K) and after a X) min label anti I;? rnin chase with acatinomyc%in 1) (lanrs F. H. ‘1 and 1,). sion of 178’ t)o ly2 during the 60 minutes needed to enucleate the labeled cells. There is very lit)tlr (‘2 present in the initial pulse-labeled c.ells. Howrvcr, the caonvrrsion is only WC& of thtb amounl SWII in unfract’ionated controls. The st,ress of the centrifugation in the high concentrations of Fic*oll apparentl?; slows down the normal processing. It then contmues in the cytoplastx after they WV returned to normal medium. To assay for t,he movement of the newly synthesized snRNAs out of the cytoplasm. cells were pulse-labeled and t)hen chased wit,h actinomycin 1). ITsing aqueous cell fra&onation, InlIst,labeled [:I and 712 cb-snRNAs are observed t#o move back into t’he nucleus during an act~inomgcin I) cahase and 1’2’ is processed to its mature size (Fig. 1 lanes A to II: Zicve. 1987). The effectivrness of the chase is also evident in the processing of the pulse labeled prc-t,RIVA to mature-sized tRNA. The maturation of species IT1 , 112. I-4 and I’6 in the labeled cyt~oplast fract,ion was analyzrd hy hybrid-selectming these species from cytoplast I’rac.t’ion prepared after a short pulse-label and after a pulse-label and short chase (Fig. 4, lanes E to 1,). Anti-sense clones of t’hese four snRNAs ident if) pulse-labeled snRNAs in t’he cyt,oplast, fractions and processing of U2. 111 and lJ4 is observed during the short chase. UP is processed from itIs discrete highclr molecular weight precursor 1‘2’ to mat,ure ITI, Both LJl and 1’4 show a small family of slightly largcar species in the pulse-labeled fractions that WY processed to discrete-sized mature species during the cnhasr. The abundance of t#hr I’6 species increases after the chase and suggests there ma.>- hi a delayrd export of this species int,o I ht. c~ytoplasrrl.

Newly

Synthesized snRNAs

u2’. u2. UI’ u4 U5 ABCDEFG Figure 5. Development of' the SLE antigen Sm on newly synthesized p-snRBAs: 2 x 10’ 7,929 cells were concentrated IO-fold and labeled with [3HJuridinr (lOO~Ci/ml) and L3H]adenosine (lOO#J/ml). At, 6, 9, 12 and 15 min aft,er the addition of label, cells were chilled to 4°C and a cytoplasmic extract was prepared by lysis in RSR buffer containing 030/,, Triton X-100 and 6 mM-VR(‘. The cytoplasmic extract was then immunoIJrrripitatrd with the mono&ma1 anti-Sm antibodies. and the precipitated snRNAs were analyzed by gel electrophoresis. Lanes A, R. C and I) are the precipitations of snRBAs from veils labeled for 6, 9, 12 and 15 min with the anti-Sm antibody. Lanes E, F and G are hybrid srlect~ions of immunoprecipitations of cells labeled fi,r 20 min with the (:I. 1’2 and 114 probes. Tn kinet’ic studies using cytoplasmic fractions prepared hy standard aqueous extraction, species ITI and I:2’ appear in the cytoplasm as early as 45 seconds after the addition of 13H]uridine to the culture medium. The processing of species U2’ to the mature sized Ir2 is first observed after about 15 minutes, which is about) the same time that the mature-sized species LTl and LJ2 begin to appear in the nucleus. Although the enucleation studies do not allow similarly precise studies, they confirm that t,he newly synthesized precursors, including the short-lived 112’ species, appear in the cytoplasm. k~oth approaches suggest that all Takrn together, the snRXAs appear transiently in the cytoplasm immediately after transcription, and that at, least the two major species, Lrl and U2, return to the interphase nucleus af%er approximately 15 minutes in the c~yt.oplasm. (tl) l)rrwloprr~rnt

of thr

The immunoprecipitation species from c,vtoplast,s

ISLE

antigen

r‘+n

of 32P-labeled snRNA illustrat’es t,hat these

Appear

in

the

Cytoplasm

265

determinants are present on the snRNAs Ul, U2, U4 and U5 in the cytoplasm (Fig. 2, lane D) as has been reported elsewhere (Madore et al., 1984a; Chandrasekharappa et al., 1983). However, because of the large internal pools of phosphate and the lag in incorporation of exogenous phosphate into ribonucleic acid, it is difficult to do precise kinetic studies with phosphate label. To determine more accurately the time at which the Sm antigen develops on the c-snRNA species, 1,929 cells were pulse-labeled with [3H]uridine and [ 3H]adenosine, and conventional aqueous cytoplasmic fractions were immunoprecipitated with the Sm antisera. These nucleosides enter newly synthesized RNA within one minute of addition to the cells. As early as six minutes after the initiation of labeling, species Ul, U2’, U4 and C5 are immunoprecipitable by the Sm antiserum (Fig. 5, lanes A to D). The identity of Ul, U2 and U4 is confirmed bv hybrid-selecting immunoprecipitates with the antisense Ul , 1J2 and U4 clones (lanes E, F and G, respectively) and the U5 is tentatively identified by was its characteristic mobility. Hybrid selection performed on immunoprecipitates of cells pulselabeled for 20 minutes in order to have sufficient radioactivity incorporated into the c-snRNAs. LJ2 is precipitated as the U2’ precursor in pulse-labels of 15 minutes and less, and the mat’ure form only begins to appear after about 15 minutes. These data illustrate that the assembly of the snRNAs into the antigenic snRNPs occurs very soon after transcription, and that the c-snRNAs occur as RNT’s. However. they do not allow us to distinguish whether this occurs in the nucleus immediately after transcription, or soon after their appearance in the cytoplasm.

4. Discussion The use of cell enucleation to prepare bona jide cytoplasmic fractions uncontaminated by nuclear material demonstrates that newly synthesized snRNA species Ul t’o U6 appear transiently in the cytoplasm shortly after transcription. This confirms and extends a number of previous studies that utilized both aqueous and non-aqueous cell fracbtionation, and manual dissection to identify several of the newlv synthesized snRNA species and snRNP pro&ins in the cytoplasm (Gurney & et al.. 1984h; Zeller rt al.. Eliceiri. 1980: Madore 1983).

Enucleation of suspension cult’urrs is easy to perform and allows the preparation of bona jdc cytoplasmic fractions in quantities arnenable to biochemical analysis (Wigler & Weinstein, 1975). Intact nuclei are extruded from cytochalasintreated cells to yield cytoplasts t,hat include approximately 75O/b of thr original cdytoplasm surrounded by an intact plasma membrane. Ultrast’ructural and biochemical studies indicaate that the nuclei remain intact during the extrusion process, with no evidence of nuclear leakage. Enualeation has previously localized other molecules in the

G. IV. Zieae et al. nucleus t.hat, are prone to leak int,o the cytoplasmic fraction during aqueous cell fractionation. including the estrogen receptor (Welshons et al.. 1984). Enucleation is a useful tool when contamination with the more abundant and stable nuclear snRNAs will interfere with the analysis of the cytoplasmic fraction However, enucieation suffers from thr possible effects of treating cells with cytochalasin and the long time needed to complete t,he procedure. The two most abundant snRNA species, 1-l and 1’2, are readily identifiable in cyt,oplasmic fractions immediately after synt,hesis. The? are the most highl? labeled low molecular weight species in addltlon to the RNA polymerase I11 transcripts tR?c’A. 5 S RNA and 7SL RNA in pulse-labeled fractions. The other less abundant snR1\;As do not incorporate as much radioactivit,y, and they are not easily detectable until after 15 minutes of labeling. However. it is likely they also appear in t)he cytoplasm immediately after tra.nscription. (:I. 1’1 and. by analogy. t,he other tri-met’hyl (: rapped snRNAs are t’ranscribed by RNA polymerasr I1 (Murphy et nl.. 1982). Tn X’ULO~IUS oocyt,es, Mat’taj (1986) has shown that, the init.ial snRNAs trarlscribed by polymerase Tl havp a typical gllanosint~ cap, and that’ the additiona,] methylations arr added post-transcriptionally in the cyt~oplasrn. These dat)a suggest that a similar cytoplasmica methylation may occur in t.he Gssur culturtx c~rlls. I’6 has an unidentified 5’ end and is transcribed by polymrrase TTT (Kunkel rf 01.. 1986). Mature I’6 precipitates with the Sm antiserum along wit,h t hc, other snRNAs because it is complexed in a singlr particle with LJ4 (Hashimot,o & Stflit.z, 1!#4: Kringtnann rf nl., 1984). However. t!S in tht> c~ytoplasm does not imrnuno~)rc~~ipitatc~ with I-4. This suggest,s that the associat)ion between I’6 and I’4 must form or become st)abilized in the nucleus, or itnmrdiat,ely brforca t.hc> spG~~s Itaavta t,trl, csytoplasm. Sequence analysis has identified that I’:! appears in the cytoplasm as a precursor. V%‘. approximat,el) ten nucleot~ides larger than the tnat,ure form, and that 1!1 appears in a form that is. a.t most. a f&v nuc*leotides larger than the mature form (Eliceciri c1 Sayavedra, 1976: Tani e:f distinguished by having base> rnet hyla-

t ions on the t.wo nucleotides internal to t hc~ (*a11 struct,urr (Glory & Adams, 1975). These differ from the type I caps, which have only a single rnethylated nucleotide. Studies on mRX:A suggest that, the second base methylation is a posltranscriptional methylation that, occurs in the cyt,oplasm (Perry Rs Kelly, 1976). This suggests that the snRNAs acsquire the additional second bask met~hylation during their transient appcaranc.c in t,he cyt,oplasm. Previous studies have demonstrated that I’ I ;LII~ I’% have half-lives of approximately trbrt to 15 minutes in the cytoplasm (Eliceiri. 1974: %ievth cf 01.. unpublished results). Because of t,heir Iont-r abundance. it is more difficult to det,ermine thr half-lives of species 113. 114, IJ5 and (16 in thra c~ytoplasm. Tn normal aqueous frac%ions of f)ulsc:labeled material, (‘6 and C6 are obscured by the pre-tRNA leaking from the nucleus. and I’:3 and 1.1 are often difficult t,o identify. Howcvc~r. a I~/OW inspection of c+ytoplasniic fractions prepared aftclr an ac%inomyc:in 1) chase (Fig. 4) suggests i hat t II~W may have similar half-lives in the spec+s Vytoplasm. Species I’ I I’?. I’4 and I’5 are imtlllllropr,rcil,l. table in the cytoplasm I)!, antibodicss \\.it,tl t hts SIII sprcificity (Lernr~r 8 Steit,z., 1981 ). 1n kVrst,ern blot5 of 1,929 wIIs, t.his monoc~lonal antiserum t-e<-ogniz(hs thrl ti protein t,hat is a component of t htl snRN t’s (IWtersson rt crl.. 1984: Zipve et rrl.. unpublisheti results). The snKNAs apparently caotnplrs with thts fivcb identic~al snKNP proteins in ;\dtlit,iotl to several prot)eins unique t)o racah species. Tiiis confirms sirnilar observations by ot hclr investigators and pinpoints t ht. drvt~lopmrnt~ of thpsc2 ant,igells t I) ivithin a few minutes of transcsription. Tht*scb sprc+ic~it~ic~s. originally charac~terizrti on 1hr, mat IIW nucalear l)artic~lrs. arc\ developed ilS tAi*rlJ- iL:: hi.\ rriinut’c3 aftclr synthesis for spec+s 1’1. I’?. I-4 ant1 1’5. I::! is ~)~‘c~c~i~)ilat,c~cl in both it.?: f)~‘(‘(‘ui’sor ;~tltl more mat ur
Newly

Synthesized

snRNAs

Fritz et al., 1984). Large pools of snRNP proteins are stored in the ooplasm of Xenopus oocytes. At the mid-blastula transition, snRNAs are one of the earliest and most abundant transcription products. The newly transcribed snRNAs move into the cytoplasm, assemble with the stored snRNP proteins, and then return to the nucleus. Although the mammalian cells are too small to allow the sophisticated manipulations possible with oocytes, advantages for biochemical they offer several studies. The opportunity for precise kinetic studies and cell fractionation combined with the availabilit,y of specific probes for the RNA and protein components will allow a precise determination of the int,racellular traffic of the snRNA and snRNP prot>eins

in mammalian

cells.

This work was supported by grants from the National Science Foundation (DCB-8511112) and the Lupus Foundation of America. We thank Elizabeth Roemer for rxcrllent tjechnicaal assistance.

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h?y I’. Ch~a~mbwrr