MOLECULAR AND CELLULAR BIOLOGY, Jan. 1987, p. 111-120 Vol. 7, No. 10270-7306/87/010111-10$02.00/0

Pre-mRNA Splicing and the Nuclear MatrixSCOTT ZEITLIN,1 ANNETTE PARENT,' SAUL SILVERSTEIN,2 AND ARGIRIS EFSTRATIADISl*

Departments of Genetics and Development' and Microbiology,2 Columbia University, New York, New York 10032

Received 24 June 1986/Accepted 19 September 1986

We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo systemthat we have developed. Plasmids containing the inducible herpesvirus tk gene promoter linked to anintron-containing segment of the rabbit j(-globin gene were transfected into HeLa cells, and then the promoterwas transactivated by infection with a TK- virus. Northern analysis revealed that the globin pre-mRNA andall its splicing intermediates and products are associated with the nuclear matrix prepared from suchtransfected cells. When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in thepresence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag inthe reaction, with a corresponding increase in free intron lariat. Thus, most of the events of the splicing process(endonucleolytic cuts and branching) occur in this in vitro complementation reaction. However, ligation ofexons cannot be monitored in this system because of the abundance of preexisting mature mRNA. Since thematrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinizedmatrix RNA, we conclude from our in vitro complementation results (which can be reproduced by usingmicrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preas-sembled ribonucleoprotein complexes that have the potential to function when supplemented with solublefactors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing)present in the HeLa cell extract.

The nuclear matrix is operationally defined as the insolu-ble structural framework (10% of the total nuclear protein)remaining after high-salt and detergent extraction ofinterphase nuclei that have been treated with DNase andRNase (see reference 2 for a review). Morphologically, thestructure consists of residual elements of the nuclear enve-lope (pore complexes and lamina), remnants of nucleoli, andan internal network of thin proteinaceous fibers. It has beensuggested that the matrix is involved in DNA replication,transcription, and RNA processing (see references 2 and 17for reviews).The observation that several pre-mRNAs and mature

mRNAs are tightly associated with the nuclear matrix isconsistent with the notion that this structure might be thesite of nuclear pre-mRNA splicing (6, 14, 27, 28, 33). Arelated observation is that omission of the RNase step duringmatrix preparation yields a structure associated with heter-ogeneous nuclear ribonucleoproteins (HnRNPs), which canbe cross-linked with the matrix in situ (37). In contrast, freeHnRNP particles (see reference 21 for a review) are isolatedfrom nuclei by other means, for example, by the use of shearforce (sonication). However, in the absence of a directfunctional test, it can still be argued that the association ofRNP complexes with the matrix is the artifactual conse-quence of aggregation and precipitation in high salt of RNAand soluble proteins onto a subnuclear fraction.To examine the possible involvement of the matrix in

nuclear pre-mRNA splicing, we used a model system whichwe have developed that can be used to study splicing eventsin vivo. This system utilizes the inducible promoter of theherpesvirus (HSV) thymidine kinase (tk) gene linked to anintron-containing segment of the rabbit P-globin gene. Plas-mids containing this chimeric gene are transfected into theHeLa cell transient transcription system, and the promoteris activated in trans (transactivated) by infecting the cells

* Corresponding author.

with a TK- HSV (10). Transcription products and splicingintermediates are then assayed by high-resolution Northernblotting (41).

According to recent results (see reference 31 for a review),the mechanism of nuclear pre-mRNA splicing is a two-stepprocess. In the first step, a cut is introduced at the 5' splicesite with concomitant formation of an intron lariat structure.Thus, two products appear after this endonucleolytic step: afree exon 1 and a lariat exon (lariat form of the intron stillconnected to its downstream exon). In the second step,another endonucleolytic cut at the 3' splice site of the lariatexon releases free intron lariat, while concomitant ligation ofexons 1 and 2 generates mature mRNA. These events occurafter assembly of the pre-mRNA into a multicomponentsplicing complex, the spliceosome (3, 12, 16). In vivo (and,under certain conditions, in vitro), free linear intron alsoappears (8, 24, 41) that is thought to be a product of lariatdebranching (34, 41). Thus, Northern analysis of in vivosteady-state total cell RNA, using intron and exon 1 probes,reveals the presence of pre-mRNA and five splicing inter-mediates and products: exon 1, lariat exon, free intron lariat,free linear intron, and mature mRNA.

Using our in vivo system, we show here that the pre-mRNA and all its splicing intermediates and products areassociated with the nuclear matrix after a high-salt wash(final step of the preparation procedure). However, thelow-salt-wash fractions (intermediate steps of the prepara-tion protocol) are relatively enriched in free linear intron andmature mRNA, suggesting the existence of two RNP com-partments with differential stability to salt. When the nuclearmatrix preparation is incubated in an in vitro splicing nuclearextract, either intact or treated with micrococcal nuclease,reconstitution of most of the splicing steps can be detectedwithout a lag in the reaction time. This result indicates thatthe nuclear matrix preparation retains parts of preassembled"splicing complexes" that can function when supplementedwith salt-labile factors present in the splicing extract.

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112 ZEITLIN ET AL.

MATERIALS AND METHODS

Materials. Restriction enzymes were purchased from NewEngland BioLabs, Inc. (Beverly, Mass.) or Bethesda Re-search Laboratories, Inc. (Gaithersburg, Md.). T4 DNAligase, HindIII linkers, and oligo(dT) were from New En-gland BioLabs. Reverse transcriptase, Escherichia coliRNase H, and RNase-free DNase I were from BethesdaResearch Laboratories. Placental RNase inhibitor (RNasin)was from Promega Biotech (Madison, Wis.). Proteinase Kwas from Boehringer Mannheim Biochemicals (Indianapolis,Ind.). Micrococcal nuclease was from Worthington Diagnos-tics (Freehold, N.J.). P--y-Methylene ATP was from SigmaChemical Co. (St. Louis, Mo.). Nylon membranes (GeneScreen Plus) and [a-32P]deoxynucleoside triphosphates (800or 3,000 Ci/mmol) were from New England Nuclear Corp.(Boston, Mass.). As source of rabbit ,B1-globin gene se-

quences we used various subclones of clone pPst5.6 (25). Assource of HSV tk gene sequences we used clone pBiM6 (29).DNA fragments were subcloned into pUC9 or M13 vectors(38). The constructions used in this work are described in thelegend to Fig. 2.

Cells and viruses. The viruses used in this work were D2,a TK- deletion mutant of HSV type 1 (HSV-1) (36), and F(9). They were grown and titrated in Vero cells (30). HeLacells were grown in Dulbecco modified Eagle medium sup-plemented with 10% calf serum and were seeded at a densityof 5 x 106 cells per 10-cm dish for infection or at a density of2 x 106 for transfection. Cells were infected with HSV-1(F)at 10 PFU per cell in the presence of 80 ,ug of cycloheximideper ml. After adsorption for 1 h at 37°C, the virus wasremoved and the cells were overlaid with Dulbecco modifiedEagle medium containing 10% calf serum and 80 ,ug ofcycloheximide per ml. They were then incubated for varioustimes at 37°C. Cells were transfected as described previously(40) with a calcium phosphate precipitate containing 10 ,ug ofplasmid DNA and 10 jig of carrier salmon sperm DNA per

plate. At 16 to 24 h after transfection the cells were infectedwith 5 PFU of D2 per cell for 1 h in the presence of 80 ,ug ofcycloheximide per ml or in the absence of drug. The viruswas then removed, and the cells were incubated as describedabove for various times in the presence or absence ofcycloheximide. The drug was removed by four washes withmedium and then incubated in fresh medium for varioustimes.RNA analysis. Total cell RNA was purified by the

guanidine thiocyanate-CsCl procedure (5). Northern analy-sis was performed as described previously (41), with uni-formly labeled single-stranded DNA probes synthesized onM13 templates by a modification of a published procedure(20). The sizes and positions of globin sequence probes areshown in Fig. 2. The probe for the 78-nucleotide (nt) intronof the HSV-1(F) immediate-early (IE) 5 pre-mRNA was anHpaII to TaqI fragment containing the intron flanked by 20nt of exon at the 5' side and 15 nt of exon at the 3' side. TheIE5 mRNA is a 1.8-kilobase species whose precursor con-tains two introns of 75 and 107 nt (11). In the HSV-1 strain Fthat we are using the second intervening sequence (IVS) isonly 78 nt long presumably because of deletion of some ofthe tandem repeats that are present in this sequence (39).

Matrix preparation. Nuclear matrix was prepared by ei-ther of two procedures, which we call here protocol 1 (6) andprotocol 2 (14), essentially as described previously. Briefly,washed crude nuclei were incubated with 0.5 mg of DNase Iper ml in low-salt buffer (10 mM NaCl, 1.5 mM MgCl2, 10mM Tris, pH 7.5) and then washed successively with 1 and

2 M NaCl (protocol 1). Alternatively, the crude nuclei werefirst washed with a buffer containing 0.1 M KCI and thenincubated with 100 ,ug of DNase I per ml, followed by twowashes, one in 0.1 M KCl and the second in 0.4 M KCI(protocol 2). Protocol 1 is more complicated than protocol 2because it entails ultracentrifugation and steps carried out at-20°C. For complementation experiments the matrix prep-aration from 107 to 108 cells was further washed with 2 ml ofbuffer D (7) and pelleted by centrifugation for 15 min at 600x g. The pellet was resuspended in 200 ,ul of buffer D, and10- or 30-,ul aliquots were quick-frozen in liquid N2 andstored at -80°C.

In vitro reactions. RNA was deadenylated with E. coliRNase H in the presence of oligo(dT) as described previ-ously (41). Debranching reactions were performed as de-scribed previously (34). For in vitro reactions withdeproteinized matrix RNA, a matrix sample was treated withproteinase K in the presence of sodium dodecyl sulfate asdescribed previously (23), followed by phenol extraction andethanol precipitation. Such an RNA preparation (derivedfrom the insoluble matrix pellet) can be readily dissolved.For complementation, a 30-,ul matrix sample was incubatedin a 150-,ul reaction mixture containing 80 mM KCl, 2 mMMgCl2, 1.5 mM ATP, 5 mM creatine phosphate, 0.9%polyvinyl alcohol, 150 units of RNasin, and 90 ,u1 of HeLacell nuclear splicing extract (23). Samples (25 pul each) weretaken from this reaction mixture at various times and imme-diately deproteinzed with proteinase K as described above.For better electrophoretic resolution, ethanol-precipitatedRNA was deadenylated. Samples were then treated withRNase-free DNase I before electrophoresis. In certain reac-tions (see text) ATP was omitted or replaced with 1.5 mMP--y-methylene ATP (AMP.PCP). Incubation of matrix aloneunder splicing conditions was performed as described for thecomplementation reaction, except that the splicing extractwas replaced with buffer D. Certain complementation reac-tions were fractionated by centrifuging a sample taken atvarious time points for 15 s in an Eppendorf microcentrifuge.For complementation with nuclease-treated splicing extract,a sample of extract was digested with 40 U of micrococcalnuclease per ml in the presence of 1 mM CaCl2 for 30 min at30°C, and then EGTA was added at 3 mM final concentra-tion.

Densitometric scanning. Autoradiograms were scannedwith the Beckmann DU-8 densitometer, and the areas underthe peaks were calculated (analysis of the data in Fig. 3 and4d; see legends to Fig. 3 and 5 for details).

RESULTS AND DISCUSSION

System to study in vivo splicing. We have previously shownthat splicing intermediates of the rabbit ,-globin pre-mRNApresent in the steady-state RNA population of fetal liver canbe detected by high-resolution Northern analysis, despitetheir exceedingly low concentrations (41). However, use ofcell culture systems is necessary to study in vivo splicingkinetics or other aspects of the splicing mechanism (byfollowing, for example, the fate of "pre-mRNAs" generatedby transient transcription of in vitro-mutagenized tem-plates). In this respect, viral pre-mRNAs synthesized ininfected cells are potentially useful, because their splicingintermediates usually occur in much higher concentrationsthan their counterparts from cellular transcription units (see,for example, reference 35). However, processing of suchviral transcripts often involves events of differential splicingthat could complicate the discrimination between various

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VOL.7,1987~~~~~~~~SPLICINGAND THE NUCLEAR MATRIX 113

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FIG. 1. (a). Time study of the appearance of free lariat (La) and linear (Li) intron forms of a 78-nt intron excised from the HSV-1(F) IE5pre-mRNA after infection of HeLa cells with wild-type HSV-1(F) in the presence of cycloheximide. Total cell RNA (30 j±g per lane) was

electrophoresed on a denaturing 5% urea-polyacrylamide gel. The probe used for Northern analysis was an IE5 gene fragment containing the

78-nt intron (see Materials and Methods). Only the region of the gel transfer containing the intron forms is shown. In this particular gel, the

lariat species migrates faster than its linear counterpart. The size markers in lane M are labeled HpaII DNA fragments of pBR322 (in

nucleotides). (b) HeLa cells transfected with the ptkRl construction (see Fig. 2) were infected with virus D2 in the absence (A series) or

presence (B series) of cycloheximide. RNA was isolated from the A series of cells at 0, 3, and 6 h postinfection. The B series of cells were

washed free of drug at 6 h postinfection (BO sample), and two other samples were cultured for an additional 2 or 6 h. Total cell RNA (20 p.g)

from each sample was used for Northern analysis. The probe was rabbit 0-globin IVS1 sequence (see Fig. 2). The areas above and below the

arrow are from different exposures of the same blot (12 and 48 h, respectively). The species indicated are the precursor (P), the lariat intron

(La), and the linear intron (Li). 0 is the origin. The size markers are as in panel a. (c) Comparison of protocols 1 and 2 used for nuclear matrix

preparation (see text). The experiment was as in panel a, lane 6, except that nuclear matrix was prepared at 6 h postinfection, instead of RNA.

Deproteinized matrix RNA (10 p.g) was analyzed by Northern blotting. The abbreviations are as in panel b. In this gel (8%) the order of

migration of the lariat and linear intron species is inverted in comparison with panel a. The lengths (nucleotides) of size markers (Hinf

fragments of pBR322) that were electrophoresed in parallel are indicated.

intermediates and splicing products. Thus, we sought to

develop an in vivo system that could generate relatively highamounts of splicing intermediates by utilizing a segment

from any cellular gene containing two exons (or exon parts)

separated by an intron. For this purpose, we exploited the

ability to transactivate the HSV tk promoter in a transient

transcription assay system (10).

HSV gene expression in infected cells is temporally regu-

lated in a cascade fashion (18). Viral mRNAs from genes

which are transcribed from parental templates shortly after

infection (through the involvement of virion polypeptides)are translated into gene products that positively activate in

trans the promoters of a second (early) group of genes (19,

32), which includes the gene encoding thymidine kinase (15,

26). We have previously shown (10) that tk plasmids

transfected into a HeLa cell transient transcription system

are also transactivated after infection with a TK- virus and

accumulate 10,000-fold more transcripts than the basal level

(transfection without infection). Thus, we thought that this

transfection-infection protocol could be useful to generate

assayable amounts of splicing intermediates from any

promoterless gene or gene segment that would be ligated

downstream from the tk promoter.

Before attempting to use this approach to ask specific

questions about the involvement of the nuclear matrix in

splicing, we performed control experiments to characterize

the system.

We first examined whether cycloheximide, a protein syn-

thesis inhibitor which is used in the general transfection-

infection scheme, could affect splicing. Because of negative

feedback, proteins encoded by IE genes switch off their own

templates early in the infectious cycle. Cycloheximide inhib-

its translation of the IE mRNAs, resulting in the accumula-

tion of these mRNA species in high amounts. Thus, after

release from the block, early gene promoters are transactiv-

ated in a synchronous fashion in all infected cells (18).

To document that cycloheximide does not affect splicing

of IE transcripts, we first followed the appearance of lariat

and linear intron forms released from the pre-mRNA of the

viral gene which contains a 78-nt intron (see reference 11

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114 ZEITLIN ET AL.

B cap S R* p~A H,I L L-4 13

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' ptkR2FIG. 2. Restriction maps of constructions ptkRl and ptkR2. ptkRl was constructed from parts of the HSV-1 tk gene (top) and the rabbit

,-globin gene (third line). Lines represent introns and flanking sequences, and boxes represent exons (open boxes for tk, and filled boxes forglobin). In the 5'-to-3' direction, ptkRl consists of tk flanking sequence (beginning with a Bam linker at position -418), the beginning of thetk 5'-noncoding region from the capping site to a BglIl site (56 base pairs), rabbit 0-globin gene sequence beginning at a PvuII site (9 base pairsupstream from the capping site) and including exon 1, IVS1, and part of exon 2 (ending at a Bam site at position +475), and a tail of tksequence containing the tk polyadenylation (pA) signal [from a Sma site at position +1213, which is present at a distance of 104 base pairsupstream from the poly(A) addition site, to a HindIII linker attached to position + 1598]. This construct was inserted into the Bam and HindIlIsites of pUC9 to generate ptkRl. ptkR2 contains the same 5' tk sequences as ptkRl and rabbit ,-globin sequence (in which the IVS1 has beeneliminated) from an SfaNI site at position +68 to a PvuII site in the 3'-flanking region that is present at a distance of 367 base pairs downstreamfrom the poly(A) addition site. This construct was inserted into the Bam and (filled-in) HindIlI sites of pUC9. The exon 1 and 2 fusion andthe exon 3 of globin sequence in this construction are referred to in the text as exons 1 and 2, respectively, of the corresponding pre-mRNAtranscript. Restriction sites are: B, BamHI; Bg, BglII; S, Sma; H3, Hindlll; P, PstI; Pv, PvuII; H, HaeIII; D, DdeI; Sf, SfaNI; A, AIuI; R,EcoRI. Wavy lines indicate the probes (PR) used for Northern analysis.

and Materials and Methods). After infection of HeLa cellswith wild-type HSV in the presence of cycloheximide, we

purified RNA from cells harvested at various times postin-fection and examined the two free intron forms by Northernanalysis with an intron probe.The amounts of free lariat and free linear intron increased

progressively between 0.5 and 6 h postinfection (the amountof the lariat form is greater than that of the linear) (Fig. la).Although we did not examine this blot further with an exonprobe, we concluded from the appearance of free lariat,which reflects both events during the second splicing step(endonucleolytic cut at the 3' splice site and ligation ofexons), that at least qualitatively, cycloheximide did notimpair the splicing process.

In a second control experiment we used the plasmidconstruction ptkRl (Fig. 2) that carries the tk promoterconnected to a rabbit P-globin gene segment (exon 1-IVS1-exon 2), and after applying the transfection-infection proto-col described above, we compared the levels of splicingintermediates in the presence or absence of drugs.HeLa cells transfected with plasmid ptkRl were cultured

for 16 to 24 h and then infected with a TK- deletion mutantof HSV-1 (D2) in the presence or absence of cycloheximide.Samples of untreated cells were harvested 3 and 6 h postin-fection for RNA isolation. Treated cells were washed free ofdrug at 6 h postinfection, and samples were cultured for anadditional 2 or 6 h before harvesting. The level ofpre-mRNAand the two intron forms that accumulated in the untreatedcells was not significantly different between 3 and 6 hpostinfection (Fig. lb, lanes A3 and A6). In the treatedcultures, the level of hybridizing species during a 2-h incu-bation period after drug removal was significantly higherthan the level at 6 h after drug release (Fig. lb, lanes B2 andB6) and slightly higher than at 3 h postinfection in theuntreated cells (compare lanes B2 and A3). Comparison ofthe autoradiographic profiles between treated and untreatedcultures confirmed that the protein synthesis inhibitor didnot impair splicing of primary transcripts from transfectedtemplates.To establish the time point at which the hybridizing

species are most abundant, we repeated the second part ofthis experiment (treated cultures) in a more narrow time

study (40, 80, and 120 min). The peak was at 80 min afterdrug release (data not shown). Thus, we fixed this time in ourscheme for all subsequent experiments.An additional conclusion from these results was that our

approach was viable because the level of splicing intermedi-ates we could detect by transfection of a chimeric gene was

comparable to the level of the correspondng products from atranscript of the wild-type virus.

In a final control experiment we compared establishedprotocols for nuclear matrix isolation. For this purpose, we

followed the precursor and its intron products from the HSVgene IE5 and compared two protocols (6, 14) for preparationof nuclear matrix (which we call here protocols 1 and 2).Protocol 2 is much simpler than protocol 1 (see Materials andMethods for details) and yields a matrix preparation after a

final 0.4 M KCl wash. The corresponding step of protocol 1is a 2 M NaCl wash. The lariat and linear forms of the 78-ntIE5 intron were associated with the nuclear matrix (Fig. lc).Moreover, the result indicated that the two protocols yieldidentical results. Thus, we chose the simpler protocol 2 forall subsequent experiments.

Differential salt resistance of RNP complexes of splicingintermediates during nuclear matrix preparation. To examinein detail the presence of pre-mRNA and the five splicingintermediates and products in the various fractions gener-ated during the matrix preparation procedure, we used our

transfection-infection scheme and the construction ptkR2(Fig. 2) consisting of the tk promoter linked to a segment ofthe rabbit 3-globin gene that includes an exon 1 and 2 fusion,IVS2, and exon 3. For simplicity, we call the two exons inthe construction exon 1 and exon 2, respectively. Wedesigned this construction to resolve all the IVS2-hybridizing species by Northern analysis with gel electro-phoresis in only one dimension.

Figure 3 (lane MT) displays the Northern profile of theRNA species that are present in the nuclear matrix (pelletobtained after a final 0.4 M KCl wash). The correspondingprofiles of the RNAs in the supernatants of the precedingsuccessive salt washes (two low-salt washes and the final 0.4M KCI wash; see Materials and Methods) are displayed inlanes 1, 2, and 3, respectively. Four of the six RNA specieswe monitored in the various fractions (precursor, lariat

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SPLICING AND THE NUCLEAR MATRIX 115

IVS2 probeM MT 1 2 3

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FIG. 3. Northern analysis of the pre-mRNA and its splicing intermediates and products present in the nuclear matrix and in the salt washesof preparation protocol 2. Construction ptkR2 (Fig. 2) was transfected into HeLa cells, which were then infected with D2 in the presence ofcycloheximide for 6 h. At 80 min after drug removal, the cells were harvested, and matrix was prepared according to protocol 2 (see Materialsand Methods and text). RNA was then extracted from the matrix (pellet) and the three consecutive salt-wash supernatants (1, 0.1 M KCI; 2,0.1 M KCI after DNase I treatment; and 3, 0.4 M KCI). The RNA yields were 122 ,ug (matrix), 3 ,ug (wash 1), 10 p.g (wash 2), and 55 ,ug (wash3). The Northern profile of 10 ,ug of RNA from the matrix (MT) is compared with the profiles of the RNA present in the washes (1, 3 jig; 2,10 jig; and 3, 10 ,ug). Lane T is total cell RNA (10 p.g) displayed for comparison. The blot was hybridized with IVS2 probe (a) and thenrehybridized with an exon 1 probe without intermediate washing (a'). The position of the probes is shown in Fig. 2. Abbreviations: P,pre-mRNA; m, mature mRNA; El, exon 1; La, IVS2 lariat; Li, linear IVS2; and Y, nicked lariat (Y-form). The lariat exon has penetratedthe gel only in lanes 2 and 3 (arrowheads). 0 is the origin. The size markers (nucleotides) in lane M are end-labeled Hinf fragments of pBR322.To calculate the percentage of each RNA species that is associated with the matrix (see text), we first scanned lanes MT, 1, 2, and 3 of panelsa (for P, La plus Y, and Li) and a' (for m and El) with a densitometer. The intensity of each species was then adjusted for the total amountof RNA recovered from each fraction (see yields, above). For each species, the intensity values in lanes MT, 1, 2, and 3 were added, and theintensity in lane MT was divided by this sum.

exon, free lariat, and linear intron) were detected by hybrid-ization with an IVS2 probe (Fig. 3a), whereas an overlappingsubset (precursor, exon 1, and mature mRNA) was detectedby subsequent hybridization of the same blot with an exon 1probe, without washing off the IVS2 signal (Fig. 3a'). Theintron probe also hybridized to nicked free lariat (the Y-formwe described previously [41]) which we consider to be anartifact of the in vitro manipulation of the RNA. Thus, thisRNA species will not be discussed further.

Figure 3 (a and a') reveal that all six RNA species arepresent in the nuclear matrix preparation and in the super-natants of the salt washes but that they are differentiallydistributed (ratios estimated after densitometric scanning;see the legend to Fig. 3 for details). Thus, 95 to 99% of eitherthe precursor or exon 1 (present in both the matrix and thewashes) is associated with the matrix. The correspondingvalue for either the mature mRNA or the free intron lariat isabout 80%. However, the linear intron, which has beenpostulated to represent another end product of the reaction,generated by debranching of the lariat before turnover (34),is almost equally distributed between the matrix and thewashes. These results imply the existence of two compart-ments in the preparation with differential resistance to salt.(We cannot comment about the distribution of the lariat exonbecause it penetrated the gel only in lanes 2 and 3 of Fig. 3a[arrowheads]).The identity of the RNA species we discussed above was

verified by gel electrophoresis in two dimensions with adifferent acrylamide concentration in each dimension (datanot shown); by deadenylation of the RNA in the matrixpreparation to resolve better the lariat exon (which occasion-ally does not penetrate the gel); and by a debranchingreaction which established the identity of the lariat forms(Fig. 4a).

In vitro splicing of matrix-associated pre-mRNA. We thenexamined whether the salt-resistant compartment of the

preparation retained preassembled splicing complexes with apotential to function if supplemented with soluble splicingfactors. We inferred that such factors were removed fromthe putative complexes because of their presence in thenuclear in vitro splicing extract (7, 23), which, according tothe protocol of its preparation, is a concentrated supematantanalogous to the matrix salt washes.For these experiments, we first prepared nuclear matrix

and performed an additional wash step (after the 0.4 M KClwash) with the same buffer (buffer D [7]) used for dialysis ofthe nuclear splicing extract during preparation and also for invitro splicing reactions. We then performed a time studyunder splicing conditions using this matrix preparation. Theprofile of the hybridizing RNA species did not changesubstantially in 2 h of incubation, except for a decline in theamount of the precursor (which is discussed below) (Fig.4b). This negative result indicated that the matrix is not aself-splicing entity.We then examined whether deproteinized, matrix-

associated RNA can be spliced in the in vitro nuclearextract. For this purpose, we deproteinized the matrixpreparation (see Materials and Methods) and added thenaked matrix RNA to the in vitro splicing extract undersplicing conditions. The profile of the hybridizing RNAspecies did not change in 1 h of incubation, except that thelariat forms (lariat exon and free lariat) were debranched (34)(Fig. 4c and c'), in agreement with the results of controlexperiments (Fig. 4a). We believe that splicing events werenot detected because the RNA input far exceeded thecapacity of the in vitro splicing system for spliceosomeassembly and function (the precursor whose fate we aremonitoring by the Northern assay is only a minute fraction ofthe total amount of precursors in the matrix RNA popula-tion, derived from all the expressing genes). This interpre-tation is consistent with the following result (data notshown). When a labeled SP6 RNA precursor was added to

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116 ZEITLIN ET AL.

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y

d

FIG. 4. (a) Control experiments to monitor the migration of the various matrix RNA species after deadenylation and debranching. TheNorthern profile of 10 ,ug of matrix RNA from a mock reaction under deadenylation conditions (lane MC) is compared with the profile of 10,ug of the same RNA that was deadenylated with RNase H in the presence of oligo(dT) (lane H). A second sample of deadenylated material(10 ,ug) was incubated with nuclear extract under debranching conditions (lane DB). Two sets of samples from each reaction mixture wereelectrophoresed on the same gel, and after blotting, the two halves of the membrane were hybridized separately with IVS2 and exon 1 probes.Arrows indicate the products of the deadenylated or debranched species. Abbreviations are as described in the legend to Fig. 3. Panels b tod' show comparatively the results of the time courses of experiments in which the matrix preparation was incubated by itself under in vitrosplicing conditions (b), deproteinized matrix RNA was incubated in a HeLa cell splicing extract (c and c'), and the matrix preparation wascomplemented with the splicing extract (d and d'). Symbols and abbreviations are as in other figures. Species x is an artifactual band appearingoccasionally in such blots. The probes used in each case are indicated. Hybridization in panels c' and d' was done without washing off thesignal from the exon 1 probe. Lane MT in panels c and c' is the same as the 60-min lane in panels d and d', displayed for comparison. Thearrows in panel c' indicate the products of debranching. Some material was lost during manipulation from the samples of lanes 60 (b) and 30(d). RNA was deadenylated before electrophoresis for better display, especially of the lariat exon species. The size markers (nucleotides) inlanes M of panels b and c' are a mixture of HindIII fragments of bacteriophage and HpaII fragments of pBR322.

the splicing extract in the presence of deproteinized nuclearmatrix RNA, it was spliced with approximately 1/10th of theefficiency observed in the absence of matrix RNA and onlyafter a lag period between 30 and 60 min.We then asked whether we could complement the matrix

with soluble splicing factors. Indeed, when a sample of thematrix preparation was supplemented with nuclear extract

under splicing conditions, we observed that during thecourse of the reaction the amount of the precursor declined,whereas the amount of free lariat increased (Fig. 4d'). Mostimportantly, these events were evident as early as 5 min ofreaction time, suggesting that the characteristic lag period ofthe in vitro splicing reactions with SP6 precursors and HeLacell extracts was absent in this case. (The particular extract

60 120 mnir

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SPLICING AND THE NUCLEAR MATRIX 117

100-

50-

10-

La

0

P

5 10 20 30 60Time(mifn)

FIG. 5. Kinetics of the decrease of pre-mRNA (P) with corre-sponding increase of the free intron lariat (La) during complemen-tation of the nuclear matrix with splicing extract. The intensities ofthe precursor (P), the lariat, and the Y-form (nicked lariat) weredetermined by densitometry and normalized to the intensity of themature mRNA at each time point. The values for the lariat andY-form at each time point were added to yield the value for freelariat (La). The sum of the normalized intensities for P and La ateach point was constant (±410%). Ratios of [P/(P + La)] x 100(circles) or [La/(P + La)] x 100 (stars) are plotted. The closedsymbols are from the data of Fig. 4d'. The open symbols are from anindependent experiment (not shown).

we used in all these experiments exhibited a lag of approx-imately 30 min with SP6 substrates.) We note that a labeledSP6 precursor added to a reaction mixture containing nu-clear matrix and splicing extract was spliced very ineffi-ciently, presumably because of competition by the matrix forsoluble splicing factors (data not shown). We also note thatwe repeated the characterization of the free lariat speciesafter complementation (using a debranching assay as in Fig.4a) to confirm its identity (data not shown).Assuming that the increase of the free lariat with time in

the complementation reaction is a reflection of all the splic-ing events, we would conclude that the matrix-associatedprecursor is indeed spliced under these conditions. How-ever, the overall picture is complicated by the following

VS2 probe Exon-2ATP AMP-PCPM30Mg Mg mg MT sup

-0E-La

_ La

observations. The relative amount of mature mRNA in thematrix at zero time is much higher (at least 20-fold) than thatof the precursor, when both are monitored and comparedone-to-one by using an exon 1 probe (Fig. 4c and d, lane 0).Thus, if the final ligation of the two exons does indeed takeplace it cannot be visualized in a reliable way, because theautoradiographic signal of the preexisting mature mRNA isso strong that a small increase cannot be monitored. Inaddition, the amounts of exon 1 and lariat exon do not seemto decline with time, as one would expect if these specieswere active. Also, because of the amount of preexistinglariat exon we cannot monitor in a reliable way the newlyformed lariat exon, which might be short-lived.We can explain the above observations by making the

reasonable assumption that one kind of particle (RNP assem-bly) contains exclusively precursor molecules, whereas theexon lariat and exon 1 are present in a different particle. Athird type of assembly should contain mature mRNA, aloneor together with the free intron lariat.

This view is consistent with the results we obtained whenwe examined by densitometric scanning the quantitativerelationship between the precursor and the free lariat in thekinetic study of the reaction. Since the hybridization signalof the mature mRNA remains practically unchanged duringthe course of the reaction, we used its intensity at each timepoint as an internal standard to normalize the intensities ofsignals derived from the precursor and the lariat for differ-ential losses during sample manipulation. The sum of pre-cursor and lariat at each time point was constant, as ex-pected from a precursor-product relationship through anintermediate (lariat exon). This relationship (decrease in theamount of precursor with corresponding increase of intronlariat) is presented in the graph of Fig. 5. The graph alsoindicates that under these conditions the appearance of thelariat is rapid with a half-time of approximately 10 min.

Since the free lariat can appear only by cutting at the 5'splice site and branching, followed by cutting at the 3' splicesite of the lariat exon, we are certain that at least three of thefour events taking place during the two-step splicing reactiondo occur. Thus, the fourth event (ligation), which cannot bemonitored, might also occur, unless the 3' endonucleolyticcut and the ligation event are uncoupled in this particulartype of in vitro reaction. Although we cannot formallyexclude this latter possibility, we note that several attempts

* - -probe -Exon-l1 IVS2

30t sp-60'pt sup3pt sup-60-pt

..- La

p p

y

y

Ela b b

FIG. 6. (a) The Northern profile of the nuclear matrix RNA species (control, lane MT) is compared with the profiles of complementationreactions performed in the presence of Mg2" and ATP, Mg2' alone, or P--y-methylene ATP (a nonhydrolyzable ATP analog). (b and b')Nuclear matrix was complemented with in vitro splicing extract, and at 30 or 60 min samples were briefly centrifuged to pellet the matrix.RNA in the supernatant and the pellet was analyzed by Northern blotting without prior deadenylation. The hybridization probes used for theanalysis are indicated. Hybridization in panel b' was done without washing off the signal from the exon 2 probe. The arrowhead indicates thelariat exon. Symbols and abbreviations are as in previous figures.

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118 ZEITLIN ET AL.

2 3 4 2

E-La _0 _ P_La "iLa

..p

Y

Li

a

El

La

b

FIG. 7. (a) Complementation of the matrix preparation withnuclease-treated splicing extract. Lane 1, Matrix RNA standard.Lane 2, Products of a splicing reaction containing matrix anduntreated extract (control). Lane 3, Products of a splicing reactioncontaining matrix and splicing extract that had been digested for 30min with micrococcal nuclease in the presence of Ca2+. The calciumions were chelated with EGTA before the addition of the treatedextract to the reaction mixture. Lane 4, Products of a splicingreaction performed after addition to the matrix of mock-treatedextract (simultaneous addition of micrococcal nuclease, Ca2+, andEGTA and incubation for 30 min at 30°C). All complementationreactions were for 60 min at 30°C. Samples were deadenylated anddeproteinized before electrophoresis. Abbreviations are as in otherfigures. In this particular experiment, the appearance of free linearintron (Li) is also detected, which is unusual for in vitro reactions.(b) Control reactions. Lane 1, Products of a splicing reactioncontaining an SP6 precursor RNA and a sample of the same

micrococcal nuclease-treated extract used for matrix complementa-tion (panel a, lane 3). Lane 2, Products of a splicing reactioncontaining the same SP6 precursor and a sample of untreatedsplicing extract. The SP6 precursor contained the same portion ofthe rabbit 1-globin gene as that present in ptkRl (Fig. 2). Precursor(10 ng) was incubated with 15 ,ul of treated or untreated extractunder splicing conditions for 2 h at 30°C. Reaction products were

deproteinized before electrophoresis. In this particular gel (5%urea-polyacrylamide), the spliced product (m) and the IVS1-exon 2lariat (E-LA) comigrate.

to detect free exon 2 in our complementation experiments(using an exon 2 probe) produced negative results (whereasthe probe detected precursor, exon lariat, and maturemRNA, as expected). Thus, uncoupling is consistent onlywith the hypothesis that the uncapped exon 2 is instantlydegraded upon production.The complementation reaction we described is ATP de-

pendent. The increase in free lariat was observed only whenmatrix and splicing extract were incubated in the presence ofATP and Mg2+ (Fig. 6). In the presence of Mg2' alone orMg2+ and a nonhydrolyzable ATP analog there was nodetectable increase of the free lariat species. However, inthe reactions in the absence of ATP we observed a decline inthe amount of precursor analogous to that seen in Fig. 4b.Additional experiments (data not shown) suggest that this isa nonspecific Mg2+ dependent event; there was no decreasein the amount of precursor when the matrix was incubated(in the presence or absence of extract) for 30 min undersplicing conditions without Mg2+ and in the presence ofEDTA. These preincubated preparations could then becomplemented by the addition of excess Mg2+ and extract.

In contrast to the putative precursor particle, which hasthe potential to become active by complementation withsoluble factors present in the splicing extract, the particle

containing exon 1 and lariat exon has presumably lost thispotential (the level of these species remains practicallyconstant throughout the reaction [Fig. 4d and d']).The hypothesis that mature mRNA is present in a third

type of particle, with different components or configurationthan the particle of the precursor, is consistent with thefollowing observation. Matrix supplemented with splicingextract was incubated under splicing conditions, and atvarious time points samples were briefly centrifuged to pelletthe matrix. The RNA species in the supernatant and pelletfractions were then examined by Northern analysis (Fig. 6band b'). We observed that the leftover of the precursorremains always in the pellet, whereas most of the maturemRNA is found in the supernatant. Thus, it seems that thepostulated mRNA particle is quite unstable in the splicingbuffer at 30°C.What is the nature of the soluble factors present in the

splicing extract that participate in the complementationreaction we described? We began investigating this questionby attempting to determine whether some of these factorscontain an RNA component. It has been established from invitro experiments that at least three or four and possiblymore small nuclear RNPs (snRNPs) are required for splicingby participating in the formation of the spliceosome (1, 4, 13,22; reviewed in reference 31). Thus, treatment of the splicingextract or some of its subfractions with micrococcal nucle-ase abolishes splicing of SP6 transcripts (13, 22). We usedthe same approach to examine our complementation reac-tion. Nuclear splicing extract was incubated withmicrococcal nuclease for 30 min, and then the nuclease wasinactivated by chelating the Ca2+ cofactor with EGTA. Asample of this treated extract was incubated with a rabbit,-globin SP6 precursor under in vitro splicing conditions, inparallel with a control reaction with the same SP6 substrateand untreated extract. In the presence of treated extract, theSP6 pre-mRNA remained intact (Fig. 7b, lane 1) (whereas itwas spliced with untreated extract, as expected [Fig. 7b,lane 2]). In parallel, another sample of the treated extractwas added to a splicing reaction containing nuclear matrix.The matrix-associated precursor of ptkR2 declined (Fig. 7a,lane 3), with corresponding increase of the free intron lariat,as in the control reactions containing untreated extract ormock-treated extract (Fig. 7a, lanes 2 and 4, respectively).This result indicates that with the exception of themicrococcal nuclease-resistant U5 snRNP (4), at least theother U snRNPs that are known to participate in splicing(see reference 4) are not necessary for complementation. Wenote that, as previously documented (see reference 14, andother references therein), all the U snRNAs (Ul and U6) areassociated with the matrix. When we electrophoresed inparallel, on a high-percentage (10%) urea-polyacrylamidegel, RNA extracted either from the matrix or from the invitro splicing extract, the ethidium bromide-stained profilesof all these snRNA species (that were identified by size andrelative mobilities) were practically identical in the twopreparations (data not shown).The complementation reaction we described (matrix plus

extract), in which the splicing profile changes in a time-dependent fashion, contrasts with the control reactions(deproteinized matrix RNA plus extract or incubation of thematrix alone), in which splicing events are not detected. Italso contrasts (because of the absence of time lag) with theusual in vitro splicing reactions with SP6 precursors, inwhich a lag period is observed before the appearance ofsplicing products. These comparisons indicate that theevents we monitor by complementation depend on the

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SPLICING AND THE NUCLEAR MATRIX 119

presence of preassembled RNP structures in the matrixpreparation. This interpretation is strengthened by the resultof the complementation experiment with nuclease-treatedextract, which indicates that these complexes contain mostof the known snRNPs required for splicing already associ-ated with the putative precursor particle.To our knowledge, this is the first time that a potential for

function can be assigned to an element associated with a

nuclear matrix preparation by a direct functional assay.

Although these results do not prove that the nuclear matrixitself is an in vivo structural prerequisite for splicing, our

system will be useful for further experiments to investigatethis issue. In addition, the assay we developed can now beused to easily identify complementing splicing factors byfractionation of the nuclear splicing extract.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants fromthe National Institutes of Health to A.E. and S.S. and by a gift to thelaboratory of A.E. from the Bristol-Myers company.

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