Proc. Natl. Acad. Sci. USAVol. 83, pp. 6721-6725, September 1986Biochemistry

U1, U2, and U6 small nuclear ribonucleoproteins (snRNPs) areassociated with large nuclear RNP particles containingtranscripts of an amplified gene in vivo

(RNA processing/CAD RNA/small nuclear RNA/autoimmune antibodies/RNA blot)

RUTH SPERLING*t, PERAH SPANN*, DANIEL OFFENt, AND JOSEPH SPERLINGt*Department of Genetics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; and tDepartment of Organic Chemistry, The Weizmann Institute ofScience, Rehovot 76100, Israel

Communicated by Aaron KMug, May 27, 1986

ABSTRACT Nuclear ribonucleoprotein (RNP) complexesthat contain intact transcripts of the amplified gene for CAD,the multifunctional protein that initiates UMP synthesis inSyrian hamster cells, have been released from nuclei of Syrianhamster cells as large particulate structures that sediment atthe 200S region in a sucrose gradient. By the technique ofRNAhybridization, we have shown that Ul, U2, and U6 smallnuclear RNAs (snRNAs) cosediment with the large RNPparticles in the sucrose gradients. Autoimmune sera fromsystemic lupus erythematosus and mixed connective tissuedisease patients, characterized as anti-(Ul)RNP, have furtherbeen shown to immunoprecipitate CAD RNA along with Uland U2 snRNAs from the fractionated nuclear 200S RNPparticles. We conclude that Ul, U2, and U6 snRNPs areintegral constituents of the 200S RNP particles. The require-ment of snRNPs for RNA processing that evidently occurs onRNP particles has been recently demonstrated. Our resultsthus suggest that the 200S RNPs are structurally and function-ally close to the native particles on which RNA processingoccurs.

Heterogeneous nuclear RNA (hnRNA) of eukaryotic cells iscomplexed with a set of proteins to form ribonucleoprotein(RNP) complexes (1-5). It is thus reasonable to assume thatRNA processing (capping, splicing, and polyadenylylation)occurs on such complexes. Therefore, the identification andisolation of the native particles on which RNA processingoccurs is of major importance for understanding the mech-anism of the post-transcriptional regulation of gene expres-sion.

Cells of eukaryotic organisms also contain a number ofdiscrete small nuclear RNA (snRNA) species that have beenshown to exist in specific nucleoprotein complexes termedsnRNPs (6-8). In view of the sequence complementarityobserved between the 5' terminus ofU1 snRNA and the 5' and3' consensus sequences that surround the splice junctions, arole for U1 snRNA in splicing has been proposed (9, 10) andexperimentally supported (11-13). U2 snRNP has also beenimplicated in hnRNA splicing (14-16), and it has recently beenshown to be required for splicing in vitro (17, 18). On groundsof sequence complementarity, U4 snRNA has been proposed toplay a role in the processing ofthe 3' ends ofhnRNA (19). SinceU6 snRNA has been shown to be base-paired with U4 snRNA(20, 21), it seems likely that U6 snRNA also plays a role inprocessing of the 3' ends of hnRNA.

Associations between snRNPs and hnRNP have beenreported (9, 22-24). The observed RNP complexes composedeither of 30S particles that result from degradation of largerhnRNP particles, or ofhnRNP that sediment as a broad peak

in the sucrose gradients. Recent assembly experiments havedemonstrated the association of in vitro synthesized pre-mRNA-like molecules with proteins and snRNPs to formribonucleoprotein complexes that contain splicing interme-diates and sediment as 40S-60S particles in sucrose gradients(25-27). These in vitro assembled particles, however, containonly a restricted part of the native pre-mRNA molecules.For our studies of nuclear RNP, we have chosen to investi-

gate cells containing amplified genes as a source of a specificabundant large nuclear RNP. A simian virus 40-transformedhamster cell line in which the gene for a multifunctional enzymedesignated CAD (for carbamoyl-phosphate synthetase,aspartate transcarbamylase, dihydroorotase) is amplified ""200-fold was used. The size of the CAD gene is 25 kilobases (kb), itcontains 37 introns or more, and its primary transcript is splicedto give a mature CAD mRNA of 7.9 kb (28, 29). We havepreviously shown that in growing cells the nuclear transcripts ofthe CAD gene are highly abundant and their size is apparentlysimilar to that of mature cytoplasmic CAD mRNA (30). Wehave further shown that CAD nuclear RNP can be releasedfrom the nuclei as particles that sediment in a band at ""200S ina sucrose gradient (30). These 200S CAD RNP particles containintact CAD RNA, and they can be converted to 30S RNPparticles by RNase digestion. If processing of hnRNA indeedoccurs on such particles and snRNPs are required for RNAprocessing, it follows that snRNPs should be an integral part ofthe 200S nuclear RNP particles. In the present study, we haveused anti-(Ul)RNP and anti-Sm antibodies from patients withautoimmune diseases in immunoprecipitation and immunoblot-ting experiments and in direct binding assays to demonstratethat U1, U2, and U6 snRNPs are associated with 200S nuclearCAD RNP particles.

MATERIALS AND METHODSCell Growth and RNP Fractionation. Syrian hamster cells

(line 165-28) were grown in culture as described (31). RNPwas released from purified nuclei and fractionated in sucrosegradients as described (30). Cytoplasmic RNP was preparedfrom the post-nuclear supernatant as described (30), and it isdesignated the cytoplasmic fraction.To purify the nuclear large particles, fractions correspond-

ing to the 200S region of the nuclear sucrose gradient werepooled together, dialyzed against 10 mM Tris HCl, pH 8.0/100 mM NaCl/2 mM MgC12/2 mM ribonucleoside vanadylcomplex, concentrated under reduced pressure in collodionbags (Sartorius), and loaded onto a 15-45% sucrose gradientin the buffer. Centrifugation was carried out in an SW41(Beckman) rotor for 90 min at 40,000 rpm and 4"C.

Abbreviations: hn, heterogeneous nuclear; sn, small nuclear; RNP,ribonucleoprotein; kb, kilobase(s); bp, base pair(s).tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Analysis of RNAs. RNA was recovered from sucrosegradient fractions as described (30). For slot blot analysis,RNA was resuspended in 6x SSC (lx SSC = 0.15 MNaCl/0.015 M sodium citrate), aqueous formaldehyde wasadded to 7.5%, and the mixture was incubated at 60TC for 15min. The salt concentration was raised to lOx SSC, and themixture was spotted onto nitrocellulose, heated at 80'C for 2hr, and hybridized according to Alwine et al. (32) with theappropriate 32P-labeled probes (see below). For blot analysesof snRNA, the ethanol-precipitated RNA samples weredenatured by heating for 2 min in 80% formamide, fraction-ated on 10% polyacrylamide gels (acrylamide/bismethyleneacrylamide, 27:1) containing 7M urea, 45mM Tris borate (pH8.3), 1.25 mM EDTA, and transferred electrophoretically toZetabind membrane [AMF Cuno, Meriden, CT (33)] in 25mM sodium phosphate (pH 6.4). Hybridizations to nick-translated probes were as described above. Densitometry ofautoradiograms that had been exposed to give a linearresponse of the film was carried out by a Beckman spectro-photometer (model DU-8).

Plasmids. pCAD41, a recombinant pBR322 plasmid con-taining a 2.3-kb insert of CAD cDNA (29), was provided byG. R. Stark. pHU1-1D, a recombinant pBR322 plasmidcontaining a 539-base-pair (bp) insert of human Ul DNA (34),was provided by J. E. Dahlberg. pU6.1, a recombinantpBR325 plasmid containing a 5-kb human genomic DNA, wasprovided by J. A. Steitz. A 360-bp Rsa I fragment of thisplasmid contains a U6 pseudogene. For U2 snRNA, we useda plasmid constructed by Nojima and Kornberg (35) thatcontains a U2 pseudogene in a 330-bp HindIII/Ava I frag-ment. For hybridization, the appropriate homologous frag-ments were separated from the remaining plasmids by elec-trophoresis through 0.8% agarose gels after digestion ofpCAD41 with Pst I and Pvu I; of pHU1-iD with BamHI; ofpU6. 1 with Rsa I; and ofthe U2 plasmid with HindIII and AvaI. The fragments were extracted from the gels and labeledwith [32P]dCTP by nick-translation (36).

Antibodies. Sera from patients with systemic lupuserythematosus and mixed connective tissue disease were pro-vided by D. Eilat (Hadassah University Hospital). The serawere first characterized as anti-(Ul)RNP or anti-Sm by counterimmunoelectrophoresis using RNase-treated and untreated ex-tractable nuclear antigens from rabbit thymus (Pel-Freez) asdescribed (37). Sera designated as anti-(Ul)RNP were furtherscreened by solid-phase radioimmunoassay (see below) usingsnRNPs as antigens. These were prepared by leakage from ratliver nuclei according to Martin et al. (4) in 0.1 M NaCl/2 mMMgCl2/10 mM Tris HCl, pH 8.0. The nuclear extracts wereapplied to a gradient of 15-30o sucrose in the same buffer andcentrifuged for 15 hr at 4°C and 25,000 rpm in an SW41 rotor.RNA analyses with pHU1-iD (see above) revealed a peak ofUl snRNA at the top of the gradient (1OS region), and fractionsfrom this region were used in the solid-phase radioimmunoas-says without further treatment. Sera that exhibited the highesttiter with these antigens were selected for the experimentsdescribed below. Sera designated as anti-(Ul)RNP were alsoshown to immunoprecipitate Ul snRNA from the lOS region ofthe sucrose gradient. The IgG fractions of these sera were usedin the immunoprecipitation experiments described below. Theywere prepared by precipitation in 50%' saturated ammoniumsulfate followed by column chromatography on protein A-Sepharose (Pharmacia). Loading buffer was 10 mM sodiumphosphate, pH 7.4/2.5 mM KCl/137 mM NaCl (PBS), andelution was with 3.5 M MgCl2. The IgG fraction was dialyzedagainst 0.15 M NaCl, followed by dialysis against PBS, andadjustment of the protein concentration to 1 mg/ml.

Solid-Phase Radioimmunoassay. U1 snRNP antigens wererevealed essentially as described (38). Antigens were at-tached to flexible microtiter plates (Falcon 3911, 96 wells) at40C for 10-16 hr. The plates were washed with PBS contain-

ing 0.05% Tween 20, and incubated for 1 hr in PBS containing1% bovine serum albumin. The wells were then washed threetimes each with PBS containing 0.05% Tween 20, incubatedwith anti-(U1)RNP antibodies at 1:10 or 1:50 dilution in PBSfor 40 min at 370C, and washed again as described above.Finally, the coated wells were incubated with 125I-labeledprotein A in PBS (5 x 104 cpm/well) for 1 hr at 370C, washedas described above, dried, and counted.

Analysis of Immunoprecipitated RNAs. Antigenic complex-es from sucrose gradient fractions of nuclear RNPs wereimmunoprecipitated according to Kessler (39). Nonspecificbinding material was removed from aliquots of each fraction(250 ,u1) by incubation with 2 ul of normal human serum and50 Fd of a 10% suspension of protein A-bearing staphylococcifor 30 min at 0C. The staphylococci were pelleted at 3000 xg for 10 min, and the supernatant was incubated with 10 ,ul ofpurified IgG for 1-3 hr at 370C. The immune complex wastreated with 100 ,1 of a 10% suspension of staphylococci andincubated for 10 min with end-over-end rotation. The solidphase was washed three times each with 50 mM Tris HCl, pH7.4/150 mM NaCl/5 mM EDTA/5 mM KI/0.02%NaN3/0.05% Nonidet P-40. For RNA analysis, the pellet wasresuspended in 10 mM Tris HCl, pH 7.5/1 mM EDTA/0.3 MNaOAc, pH 5.5, extracted with phenol/chloroform/isoamylalcohol (50:48:2), and the RNA was precipitated by additionof 2.5 vol of ethanol. Resuspended RNA pellets were ana-lyzed for the presence of CAD RNA and snRNAs by blothybridization as described above.

Immunoblotting. Proteins from pooled fractions corre-sponding to the 200S region of the sucrose gradient and fromaliquots of unfractionated nuclear supernatant were precip-itated with 5% trichloroacetic acid and analyzed on 12%polyacrylamide gels in the presence of NaDodSO4 (40).Protein transfer by electroblotting and probing with anti-(U1)RNP antibodies and "25I-labeled protein A were carriedout as described (41, 42).

RESULTS

snRNAs Are Detected in the 200S Region of FractionatedNuclear RNPs. RNP particles were released from nuclei ofSyrian hamster cells and were fractionated in a sucrosegradient as described (30). RNA was prepared from fractionsalong the sucrose gradient and analyzed. Hybridization witha CAD cDNA probe revealed a peak of CAD RNA aroundfraction 8 (Fig. 1), where 200S CAD RNP has been previouslyshown to sediment (30). Hybridization with a U1 snRNA-specific probe (pHUl-iD) revealed a major peak of U1snRNA at the 200S region of the same gradient (Fig. 1). Inaddition, two more peaks of U1 snRNA were detected. Thefirst, at the 70S region (fractions 12-15), and the second at the1OS-30S region (fractions 17-19) where free U1 snRNP and30S nuclear RNP particles migrate (see below). The analysesfor U1 snRNA by either slot blot or RNA blot gave identicalresults.

Parallel hybridizations with U2 and U6 snRNA-specificprobes indicated that the major fraction of these RNAssedimented at the 200S region of the gradient, and their peakcoincided with that of CAD RNP (data not shown, see alsoFig. 5). Since free U snRNP complexes have a sedimentationcoefficient of 10S (7, 9), this result suggested association ofU1, U2, and U6 snRNPs with other nuclear components toform larger particles that sediment in the 200S region of thegradient. Further experiments were thus carried out to verifythis possibility.Anti-(Ul)RNP and Anti-Sm Antibodies Recognize Anti-

gen(s) Associated with 200S Nuclear Particles. The distributionof snRNPs along the sucrose gradient of nuclear RNPs wasdetermined by a direct solid-phase radioimmunoassay usingsera that had been characterized as anti-(Ul)RNP (see

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0 5 10 15 20bottom top

Fraction Number

FIG. 1. Distribution of U1 snRNA along a sucrose gradient ofnuclear RNP. RNP was released from nuclei of Syrian hamster cellsand fractionated on 15-45% sucrose gradient as described bySperling et al. (30). The gradient was collected in 20 fractions (0.55ml each), starting from the bottom. The RNA was analyzed by slotblot and RNA blot hybridization with pHU1-iD as described inMaterials and Methods, and the relative amounts of U1 snRNA (inarbitrary units) were estimated from densitometry of autoradi-ograms. Similar results were obtained for the distribution of U2 andU6 snRNAs along the gradient. Arrow indicates the peak of 200SCAD RNP, as determined by hybridization with a CAD cDNA probe(30). The gradient was calibrated with the following markers run onparallel gradients: 200S tobacco mosaic virus particles, fraction 8;70S bacterial ribosomes, fraction 14; lOS snRNP and 30S RNPparticles (not resolved under these conditions), fraction 18.

Materials and Methods). Fig. 2B demonstrates that the majorpeak of interaction with the antibodies was obtained in the200S region of the gradient. Identical experiments using asantigens lOS snRNPs and 30S nuclear RNPs that werereleased by leakage (1, 3, 4) from nuclei of 165-28 Syrianhamster cells, revealed a peak of U1 snRNP around fraction18 (data not shown), where free lOS snRNPs should migrate(7, 9). The use of an anti-Sm serum in parallel experimentsrevealed similar patterns to those obtained with the anti-(U1)RNP sera (data not shown).No interaction of the nuclear antigens with normal human

serum was observed (Fig. 2A). Furthermore, cytoplasmic

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RNPs that had been fractionated and treated in the samemanner as those of the nuclear origin did not react at all withthe antibodies (Fig. 2A). It should also be noted that althoughCAD mRNA sequences were found to be broadly and almostevenly distributed along sucrose gradient-fractionated cyto-plasmic RNPs (30), such particles were not recognized by theantibodies. This result ascertains the specificity of the inter-action with nuclear particles.200S Nuclear RNP Particles Contain a 68-kDa Protein That

Is a Specific U1 snRNP Antigen. To identify polypeptides thatare directly recognized by the anti-(Ul)RNP antibodies usedin these experiments, protein transfer methods were used.Total proteins from pooled fractions of the 200S nuclear RNPpeak were precipitated by trichloroacetic acid, run on apolyacrylamide gel in the presence of NaDodSO4, andtransferred electrophoretically to nitrocellulose. For compar-ison, total proteins from the nuclear supernatant prior tofractionation were prepared and processed in the samemanner. The immobilized proteins were probed with anti-(U1)RNP antibodies and with 125I-labeled protein A.Autoradiography (Fig. 3) revealed a single band of a poly-peptide of 68 kDa both in the pooled 200S RNP peak (lane 1)and in the nuclear supernatant (lane 2). Such a 68-kDapolypeptide has been identified as a U1 snRNP-specificprotein (43-46).Anti-(Ul)RNP Antibodies Precipitate Simultaneously Nucle-

ar CAD RNP with U1 and U2 snRNPs. The presence ofsnRNAs in the 200S region of fractionated nuclear RNPraised the possibility that snRNPs are an integral part oflarger nuclear complexes that sediment as 200S particles. Itthus follows that RNA species other than U1 snRNA shouldbe indirectly recognized by anti-(Ul)RNP antibodies, byvirtue of their association with U1 snRNP. To test thisprediction we treated sucrose gradient fractions of nuclearRNP with anti-(Ul)RNP antibodies and precipitated theimmune complexes with protein A-bearing staphylococci.RNA was prepared from the immunoprecipitates, blottedonto nitrocellulose, and hybridized with 32P-labeled U1, U2,and CAD probes. The profiles of immunoprecipitated U1snRNA and CAD RNA along the sucrose gradient are similar(Fig. 4A). Furthermore, as expected from our previousresults, a major peak of both CAD RNA and U1 snRNA can

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FIG. 2. Distribution of U1 snRNP along sucrose gradients ofcytoplasmic (A) and nuclear (B) RNPs (30) as determined bysolid-phase radioimmunoassay with anti-(U1)RNP antibodies. Sam-ples from each fraction were attached to flexible microtiter plates andunderwent reaction with anti-(U1)RNP sera (e), or with normalhuman sera (o) as described. The peak of 200S nuclear CAD RNP isin fractions 5-7.

FIG. 3. Identification of a 68-kDa protein in fractionated RNPsthat is directly recognized by anti-(Ul)RNP antiserum. Total pro-teins from pooled sucrose gradient fractions corresponding to the200S peak (lane 1) and from the nuclear supernatant prior fraction-ation (lane 2) were analyzed by immunoblotting as described. LaneM, 1251-labeled size markers: phosphorylase B (94 kDa), bovineserum albumin (68 kDa), and carbonic anhydrase (30 kDa).

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FIG. 4. Simultaneous immunoprecipitation of CAD RNA withU1 snRNA (A), and with U2 snRNA (B) from sucrose gradientfractions of nuclear RNP. Samples from each fraction were treatedwith anti-(Ul)RNP antibodies, precipitated with protein-A bearingstaphylococci, and their RNA content was analyzed by slot blothybridization with the following probes: (A) pCAD41 (e) andpHUl-lD (o); (B) pCAD41 (e) and cloned U2 mouse DNA (A). Therelative amounts (in arbitrary units) of each respective RNA wereestimated from densitometry of the autoradiograms.

be observed in the 200S region. Peaks of both RNA speciesat other positions of the gradient are probably due to partiallydissociated or degraded forms of the 200S particles that stillretain U1 snRNP. Similar results were obtained for U2snRNA, with a major peak ofimmunoprecipitated U2 snRNPin the 200S region (Fig. 4B). It should be pointed out againthat cytoplasmic RNPs did not react at all with the antibodies.

U1, U2, and U6 snRNPs Are Associated with Purified 200SRNP. To support our supposition that snRNPs are associatedwith a defined larger form of nuclear RNP, we refractionatedthe 200S nuclear RNP peak region. The pooled fractionscorresponding to the 200S region of the sucrose gradient(fractions 6-10 of Fig. 1) were concentrated and centrifugedthrough a 15-45% sucrose gradient. RNA was extracted fromeach fraction across the gradient and analyzed by gel elec-trophoresis under denaturing conditions and by hybridizationwith specific probes on RNA blots.

Hybridization with cloned U1 DNA (Fig. 5A) revealed apeak of U1 snRNA around fraction 9 that corresponds to the200S region of the sucrose gradient and to the peak of CADRNA (data not shown). Hybridization of the same blot witha cloned U6 DNA probe (Fig. 5B) and of another blot with a

3 ,5i79 3 '5 17 !9 NI 3 5 7

*J2 -

U2 probe (Fig. 5C) revealed peaks of U6 and U2 snRNAs inthe same region of the gradient.

DISCUSSIONBased on the assumption that hnRNA processing occurs onnuclear RNP complexes and is possibly controlled byprotein-RNA interactions, and on various observations thatindicate the involvement of snRNPs in processing of nuclearRNA (9-21, 25-27, 47), the native RNP particles on whichRNA processing occurs are expected to be a multicomponentcomplex ofhnRNA, proteins, and snRNPs. We have recentlydescribed, the isolation of an abundant form of nuclear RNPparticles that contain CAD RNA. Over 80%o of nuclear CADRNA can thus be released in compact RNP particles thatsediment in the 200S region of a sucrose gradient and havebeen shown to contain undegraded CAD RNA of 7.9 kb (30).Preliminary experiments using subcloned probes for CADintervening sequences show that nuclear CAD RNA doescontain introns. These probes also reveal a major fraction ofCAD RNP in the 200S region that might represent putativeprocessing intermediates (data not shown). Thus, in compli-ance with the predicted composition and function of thenuclear RNP, we sought to demonstrate the association ofsnRNPs with the 200S CAD RNP particles. In this study, wepresent evidence that such is the case for U1, U2, and U6snRNPs. First, U1, U2, and U6 snRNAs have been found tobe abundant in the 200S region of sucrose gradients of CADRNP. Second, direct immunobinding assays of fractionsacross the sucrose gradient with anti-(Ul)RNP or anti-Smantibodies revealed a major peak of antigens in the 200Sregion of the gradient, whereas free snRNPs are expected tosediment as 10S particles. Third, the antibodies were able toprecipitate CAD RNA, along with U1 and U2 snRNAs, fromnuclear 200S CAD RNP particles, while they failed to do sowith the cytoplasmic form of CAD RNA. These resultsdemonstrate that U1, U2, and U6 snRNPs are integral partsof the 200S nuclear CAD RNP particles. The RNP particlesdescribed here differ from the previously described lOS-150SsnRNP-containing hnRNP particles (9, 22-24) and from the40S-60S pre-mRNA splicing complexes (25-27) in two re-spects. First, they seem to retain the structural integrity oftheir RNA components, as judged by the persistence ofundegraded 7.9-kb CAD RNA in the particles. Second, theysediment in a relatively sharp band in a sucrose gradient andappear to have a defined ard homogeneous size.U1, U2, and U6 snRNPs were found associated with CAD

RNP at three different regions of the sucrose gradient: (i) atthe 200S region, where they are associated with CAD RNPand presumably with other large RNP particles; (ii) at the topof the gradient, where free 10S snRNP particles and 30Sdegraded CAD RNP particles migrate; and (iii) at the 70S

7 9 ,7 15 7 9 5 7 9 5) 17 '

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FIG. 5. The persistence of U1, U2, and U6 snRNAs in refractionated 200S RNP particles. Sucrose gradient fractions corresponding to the200S peak were pooled and refractionated. RNA was extracted from each fraction across the gradient and analyzed by electrophoresis on 10opolyacrylamide gels in the presence of 7 M urea, followed by RNA blot hybridization with pHU1-1D (A); pHUl-lD followed by pU6.1 (B) (laneM, DNA size markers ofpBR322 cut with Msp I); cloned U2 DNA probe (C). The gradients were calibrated with tobacco mosaic virus particles(200S, fraction 9) and with bacterial ribosomes (70S, fraction 15).

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region of the gradient. Recent studies in our laboratory haveshown that the 200S CAD RNP particles are sensitive tochanges in the ionic environment and can be converted to 70Sparticles upon such changes (unpublished data). This transi-tion probably occurs because of conformational changes ordissociation of some components of the complex. Althoughthe distribution of snRNAs in the above mentioned threeregions of the gradient is variable, a major fraction (30-60%)of the snRNPs was reproducibly found associated with the200S region of the gradient. In this context, it should bepointed out that upon refractionation of the 200S peak region,the 200S CAD RNP particles were further purified, and thesnRNAs were almost exclusively found in that region (Fig.5). This indicates that the observed association of the snRNPswith the large nuclear CAD RNP is mainly due to specificinteractions and most likely does not result from nonspecificaggregation. Furthermore, our protocol for preparing CADRNP uses tRNA at 2 mg/ml that should be sufficient tocompete for the snRNAs involved in nonspecific interac-tions. One such specific interaction might be attributed tobase-pairing interactions that have been predicted to occurbetween complementary sequences in the 5' end of U1snRNA and splice junctions in hnRNA (9, 10). We haveshown that such interaction exists in vivo between U1snRNA and nuclear precursors of CAD mRNA by psoralen-mediated UV-induced crosslinking (unpublished observa-tions). However, since both nuclear CAD RNA and snRNAsare complexed with proteins, the possibility that protein-protein interactions between the protein components ofsnRNPs and CAD RNP modulate the interactions betweentheir RNA components should be considered and tested.

Association with CAD RNA alone cannot account for thehigh abundance of snRNPs in the 200S region of the sucrose

gradient. Our results suggest, therefore, that nuclear RNPsincorporating other species of the hnRNA population areassociated with snRNPs and also migrate as 200S particles.This may reflect the possible general pattern of organizationof nuclear RNA in large particulate structures and is con-sistent with our observation that nuclear RNAs of smallersizes than CAD are also packaged in 200S particles (data notshown). Finally, recent studies of splicing and RNP assemblyin vitro provide evidence for the involvement of nuclear RNPparticles in the formation of a multifunctional RNA process-ing complex (25-27). Ul and U2 snRNPs have been shownto be required for splicing of pre-mRNA-like precursors (11,12, 17, 18), and U4 and U6 snRNPs have been proposed toplay a role in processing the 3' end of hnRNA (19-21). Since200S nuclear CAD RNPs have been shown to contain U1,U2, U6, and, presumably, U4 snRNPs [by virtue of its strongassociation with U6 snRNP (20, 21)] as integral parts of thecomplex, we suggest that the 200S nuclear RNPs are struc-turally and functionally close to the native particles on whichRNA processing occurs.

We thank Dr. Dan Eilat for samples of sera and for his mostvaluable help and advice. The skillful technical assistance of Mrs. S.Tel-Or is gratefully acknowledged. The study was aided by grantsfrom the Israeli Commission for Basic Research and from theMuscular Dystrophy Association to R.S., by grants from the UnitedStates-Israel Binational Science Foundation, and from the Leo andJulia Forchheimer Center for Molecular Genetics, The WeizmannInstitute of Science to J.S.

1. Samarina, 0. P., Lukanidin, E. M., Molnar, J. & Georgiev,G. P. (1968) J. Mol. Biol. 33, 251-263.

2. Miller, 0. L., Jr., & Hamkalo, B. A. (1972) lnt. Rev. Cytol. 33,1-25.

3. LeStrougeon, W. M., Beyer, A. L., Christensen, M. E.,Walker, B. W., Poupore, S. M. & Daniels, L. P. (1977) ColdSpring Harbor Symp. Quant. Biol. 42, 885-898.

4. Martin, T., Billings, P., Pullman, J., Stevens, B. &

Kinniburgh, A. (1977) Cold Spring Harbor Symp. Quant. Biol.42, 899-909.

5. Pederson, T. & Davis, N. G. (1980) J. Cell Biol. 87, 47-54.6. Busch, H., Reddy, R., Rothblum, L. & Choi, Y. C. (1982)

Annu. Rev. Biochem. 51, 617-654.7. Lerner, M. R. & Steitz, J. A. (1979) Proc. Natil. Acad. Sci.

USA 76, 5495-5499.8. Lerner, M. R. & Steitz, J. A. (1981) Cell 25, 298-300.9. Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin, S. J. &

Steitz, J. A. (1980) Nature (London) 283, 220-224.10. Rogers, J. & Wall, R. (1980) Proc. Natl. Acad. Sci. USA 77,

1877-1879.11. Padgett, R. A., Mount, S. M., Steitz, J. A. & Sharp, P. A.

(1983) Cell 35, 101-107.12. Kramer, A., Keller, W., Appel, B. & Luhrmann, R. (1984) Cell

38, 299-307.13. Yang, V. W., Lerner, M. R., Steitz, J. A. & Flint, S. J. (1981)

Proc. Natl. Acad. Sci. USA 78, 1371-1375.14. Ohshima, Y., Itoh, M., Okada, N. & Miyata, T. (1981) Proc.

Natl. Acad. Sci. USA 78, 4471-4474.15. Fradin, A., Jove, R., Hemenway, C., Keiser, H. D., Manley,

J. C. & Prives, C. (1984) Cell 37, 927-936.16. Keller, E. B. & Noon, W. A. (1984) Proc. Natl. Acad. Sci.

USA 81, 7417-7420.17. Krainer, A. R. & Maniatis, T. (1985) Cell 42, 725-736.18. Black, D. L., Chabot, B. & Steitz, J. A. (1985) Cell 42,

737-750.19. Berget, S. M. (1984) Nature (London) 309, 179-182.20. Hashimoto, C. & Steitz, J. A. (1984) Nucleic Acids Res. 12,

3283-3293.21. Rinke, J., Appel, B., Digweed, N. & Luhrmann, R. (1985) J.

Mol. Biol. 185, 721-731.22. Zieve, G. & Penman, S. (1981) J. Mol. Biol. 145, 501-523.23. Calvet, J. P. & Pederson, T. (1981) Cell 26, 363-370.24. Calvet, J. P., Meyer, L. M. & Pederson, T. (1982) Science

217, 456-458.25. Brody, E. & Abelson, J. (1985) Science 228, 963-967.26. Frendewey, D. & Keller, W. (1985) Cell 42, 355-367.27. Grabowski, P. J., Seiler, S. R. & Sharp, P. A. (1985) Cell 42,

345-353.28. Wahl, G. M., Padgett, R. A. & Stark, G. R. (1979) J. Biol.

Chem. 254, 8679-8689.29. Padgett, R. A., Wahl, G. M. & Stark, G. R. (1982) Mol. Cell.

Biol. 2, 293-301.30. Sperling, R., Sperling, J., Levine, A. D., Spann, P., Stark,

G. R. & Kornberg, R. D. (1985) Mol. Cell. Biol. 5, 569-575.31. Swyryd, E. A., Seaver, S. S. & Stark, G. R. (1974) J. Biol.

Chem. 249, 6945-6950.32. Alwine, J. C., Kemp, D. J. & Stark, G. R. (1977) Proc. Natl.

Acad. Sci. USA 74, 5350-5354.33. Gershoni, J. M. & Palade, G. E. (1982) Anal. Biochem. 124,

396-405.34. Murphy, J. T., Burgess, R. R., Dahlberg, J. E. & Lund, E.

(1982) Cell 29, 265-274.35. Nojima, H. & Kornberg, R. D. (1983) J. Biol. Chem. 258,

8151-8155.36. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977)

J. Mol. Biol. 113, 237-251.37. Kurata, N. & Tan, E. M. (1976) Arthritis Rheum. 19, 574-580.38. Heusser, C. H., Stocker, J. W. & Gisler, R. H. (1981) Meth-

ods Enzymol. 73, 406-418.39. Kessler, S. W. (1981) Methods Enzymol. 73, 442-459.40. Laemmli, U. K. (1970) Nature (London) 227, 680-685.41. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl.

Acad. Sci. USA 76, 4350-4354.42. Renart, J., Reiser, J. & Stark, G. R. (1979) Proc. Natl. Acad.

Sci. USA 76, 3116-3120.43. Hinterberger, M., Pettersson, I. & Steitz, J. A. (1983) J. Biol.

Chem. 258, 2604-2613.44. Guldner, H. H., Lakomek, H. J. & Bautz, F. A. (1983) J.

Immunol. Methods 64, 45-59.45. Wooley, J. C., Zukerberg, L. R. & Chung, S. Y. (1983) Proc.

Natl. Acad. Sci. USA 80, 5208-5212.46. Kinlaw, C. S., Robberson, B. L. & Berget, S. M. (1983) J.

Biol. Chem. 258, 7181-7189.47. Chabot, B., Black, D. L., LeMaster, D. M. & Steitz, J. A.

(1985) Science 230, 1344-1349.

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