11

REVIEW

The nuclear matrix: structure and composition

RON VERHEIJEN1'*, WALTHER VAN VENROOIJ2 and FRANS RAMAEKERS1

'Department of Pathology and department of Biochemistry, University of Sijmegen, The Xetherlands

•Author for correspondence at: Department of Pathology, University Hospital of Nijmcgcn, Geert Grootcplein Zuid 24, 6525 GANijmcgcn, The Netherlands

Summary

IntroductionThe pore complex-lamina

The nuclear pore complexesThe nuclear lamina

The nucleolar residueThe internal matrix

Morphological and biochemical aspectsHeterogeneous nuclear RNP particlesSmall nuclear RNP particlesNuclear actin

Enzymes involved in DNA and RNAmetabolism

Virus-specific proteinsAssociated transcripts

The nuclear matrix and RNA transportBehaviour of nuclear matrix components during

mitosis

Key words: nuclear matrix, pore complex-lamina,nucleolar matrix, internal matrix, chromosomal scaffold.

Introduction

The term nuclear matrix was first introduced byBerezney & Coffey (1974) to denote a highly structuredresidual framework obtained from rat liver nuclei bysequential salt extractions, detergent and nucleasetreatments. The isolated three-dimensional structureconsisted almost entirely of protein. Subsequentstudies showed that when protease inhibitors wereincluded in all isolation steps and ribonuclease (RNase)was omitted, the isolated nuclear matrix containedRNA as the second most abundant component (Her-man et al. 1978; Miller et. al. 1978a; Shaper <?<«/. 1979;Berezney, 1980; van Eekelen & van Venrooij, 1981;Mariman et al. 1982«; Fey et al. 1986a,b). In thisreview the term nuclear matrix is defined as thebiochemical entity that can be isolated after sequentialextraction of cells with non-ionic detergents, nucleasesand high-salt buffers (Shapere/ al. 1979). With respectto the nucleases, it should be stated here that severalauthors use only deoxyribonucleases (DNases), whileothers use DNases in combination with RNases. Nu-clear matrices have been isolated from a wide variety ofmammalian and non-mammalian cell types (reviewedby Shaper et al. 1979; Barrack & Coffey, 1982).

Journal of Cell Science 90, 11-36 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

However, it has been shown not to be an obligatorynuclear component (Lafond & Woodcock, 1983).

In general, nuclear matrix preparations from differ-ent cells and tissues possess some common structuralentities: (1) the residual elements of the nuclearenvelope, also designated the pore complex-lamina;(2) the residual nucleoli; (3) a granular and fibrousinternal matrix structure that extends throughout theinterior of the nucleus.

In recent years various studies have implicated thenuclear matrix as being involved in nuclear activitiessuch as DNA metabolism (reviewed by Berezney,1984; Jackson et al. 1984; Razin et al. 1985; Vogelsteinet al. 1985; Zehnbauer & Vogelstein, 1985; Nelson etal. 1986), DNA transcription, processing and transportof RNA (Zeitlin et al. 1987; reviewed by Berezney,1984; Jackson et al. 1984; Vogelstein et al. 1985;Zehnbauer & Vogelstein, 1985), steroid hormone ac-tion (reviewed by Barrack & Coffey, 1982; Diamond &Barrack, 1984; Kirsch et al. 1986; Alexander et al.1987) and viral replication (reviewed by Berezney,1984; Simard et al. 1986). The nuclear'matrix alsoseems to play a role in carcinogenesis (Berezney, 1984).It is important to note that the suggested role for thenuclear matrix in various nuclear functions is basedprimarily on the recovery of several relevant functional

11

Chromntin

Nucleolus

Innernuclear membrane

Nuclear lamina

Outernuclear membrane

Rough endoplasmicreticulum

Fig. 1. Schematic view of acell nucleus. The nuclearenvelope is composed ofinner and outer nuclearmembranes, which fuse at theregions where nuclear poresare situated. The nuclearlamina is localized on thenucleoplasmic site of the innermembrane.

molecules in nuclear matrix preparations, and thuscomprises only circumstantial evidence. This aspecthas to be borne in mind in discussing the relation ofparticular nuclear functions with the nuclear matrix.

Although in this review we want to focus mainly onrecently characterized nuclear matrix proteins, increas-ing evidence indicates that heterogeneous nuclear RNA(hnRNA or pre-mRNA) plays a structural role in theorganization of the internal nuclear matrix (Miller et al.1978*7; Berezney, 1980; Brasch, 1982; Gallinaro et al.1983; Long &"Schrier, 1983; Fey et al. 1986a,b).Therefore, part of this review will be dealing withRNA processing and transport in the nucleus as well.The main thesis proposed in this respect is that RNApolymerase II transcripts, soon after the initiation oftheir synthesis, bind to proteins to set up a structuralbackbone. Among these proteins are the so-called coreproteins. Subsequently, other functional moleculesthat are involved in RNA and DNA metabolism maybe tethered to this hnRNP structure. During process-ing and transport the RNA remains involved in themaintenance of the integrity of the internal matrix andis not released until after the last maturation step, thatis after changing its set of core proteins for proteinsknown to be associated with cytoplasmic mRNA.

The pore complex-lamina

The major structural components of the nuclear envel-ope are the inner and outer nuclear membranes enclos-ing a lumen or perinuclear space, as well as the nuclearlamina and pore complexes. The outer membrane onthe cytoplasmic side appears to be continuous with therough endoplasmic reticulum and is covered with

ribosomes on its outermost surface. The inner mem-brane is smooth. The nuclear pore complexes aresituated in those regions where the two membranesfuse (Franked al. 19816; Gerace, 1986).

The nuclear lamina is a fibrillar meshwork of pro-teinaceous material, which is intercalated between thechromatin and the inner membrane of the nuclearenvelope (see Fig. 1).

In preparing nuclear matrices, the nuclear envelopeis exposed to buffers containing non-ionic detergents,nucleases and high-molarity salt buffers. Morphologi-cally, only the pore complexes and the nuclear laminaseem to be resistant to such treatments. This residualframework is, therefore, mostly referred to as thenuclear pore complex-lamina, and is considered to be apart of the nuclear matrix. It should be noted, how-ever, that much of the data about the pore complex-lamina have not originated from studies of nuclearmatrix preparations but from investigations on isolatednuclear envelopes.

The nuclear pore complexes

The nuclear pore complexes are large organelles thatform channels for nucleocytoplasmic transport throughthe nuclear envelope (reviewed by Newport & Forbes,1987). They have also been postulated to serve as gene-gating organelles capable of interacting specificallywith expanded (transcribable) portions of the genome(Blobel, 1985).

The pore complexes are non-randomly distributedon the nuclear surface. Their three-dimensional struc-ture has been determined by electron microscopy usingnuclear envelopes from Xenopus oocytes (Unwin &Milligan, 1982). These authors found the pore complex

12 R. Verheijen et al.

to be a symmetrical structure (outer diameter ±120 nm) framed by two widely separated, coaxial rings.Each ring is composed of eight globular subunits, andattaches to the nuclear membranes. Connected to theserings and extending radially inwards from them along acentral plane are elongated structures called spokes.These spokes appear to contact a large central sphericalparticle, the plug.

Until 1982 none of the pore complex polypeptideconstituents had been defined and characterized. Inrecent years, however, the nuclear envelope of theXenopus laevis oocyte (see Fig. 2) has been shown tocontain one principal polypeptide of 68K (K = 10 Mr)that appeared to be a major component present in bothlamina and pore complex preparations (Stick &Krohne, 1982; Benavente et al. 1984a). Gerace et al.(1982) identified a prominent 190K nuclear pore com-plex glycoprotein (gpl90) in rat liver nuclear envel-opes. On the basis of its biochemical characteristics,these authors have suggested that the protein is in-volved in anchoring the pore complex to the nuclearenvelope membranes.

Davis & Blobel (1986) have identified and character-ized a 62K protein of the nuclear pore complex from ratliver. This protein was shown to be synthesized as asoluble cytoplasmic precursor of 61K. After incorpor-ation of the protein into the nuclear fraction the

protein seems to be modified by addition of A'-acetyl-glucosamine residues.

gpl90 as well as the 62K protein remain associatedwith the pore complex-lamina after Triton X-100extraction at low-ionic strength. However, the interac-tion of both proteins with the pore complex-laminafractions was found to be destabilized in the presence ofelevated salt concentrations.

Recently, Gerace and co-workers reported on theidentification of eight structurally distinct pore com-plex proteins with common epitopes, isolated from ratliver cells (Snow et al. 1987). These polypeptides withapparent molecular weights of 210, 180, 145, 100, 63,58, 54 and 45 (Xl0J) copurified with the pore com-plexes under various conditions of ionic strength andnon-ionic detergent, and were characterized usingmonoclonal antibodies. All members of this group ofproteins contained multiple O-linked A:-acetylglucos-amine residues (see also Holt et al. 1987). Usingimmunoelectron microscopic techniques it was foundthat the proteins recognized by the monoclonal anti-bodies were situated on the cytoplasmic as well as onthe nucleoplasmic surfaces of the nuclear membranes,but were absent from the lumen. Because of somesimilarities between the biochemical properties of the63K protein and the 62K protein described by Davis &Blobel (1986), it was concluded by Snow et al. (1987)

Fig. 2. Electron micrograph showing the nuclear envelope manually isolated from oocytes of A', laevis (for details seeFranke & Scheer, 1970; Scheer, 1972). In the upper picture numerous pore complexes are denoted by vertical arrows,brackets denote some of intranuclear tangles of the fibrils associated with the pore complexes. In the lower picture thearrows denote the individual annular granules on either side of the pore margin, the arrowhead points to the centralgranule. N, nucleoplasmic side; C, cytoplasmic side; o, outer side; i, inner side of the nuclear envelope. Note that in thesecells the nuclear lamina at the inner aspect of the nuclear envelope is very thin and barely detectable. In whole-mountpreparations it appears as a single layer of a loose filamentous meshwork (Scheer et al. 1976; Aebi et al. 1986). Bars,0-1 /im. (Courtesy of Professor Dr W. W. Franke.)

The nuclear matrix 13

that these two pore complex constituents are probablyidentical.

Berrios et al. (1983) have identified a glycoproteinassociated with nuclear matrix pore complex-laminapreparations obtained from Drosophila melanogasterembryos. The molecular weight of this protein wasinitially estimated to be 17X103, but more recentstudies have shown that it was only 2x 10 smaller thangpl90 described by Gerace et al. (1982) in rat liver.This Drosophila glycoprotein is therefore designatedhere as gp 188. It seems to be the homologue of the ratliver gpl90, since the two polypeptides have somebiochemical properties in common (Filson et al. 1985).Antisera prepared against gpl90 were found not tocross-react with gpl88 (Filson et al. 1985). In contrast,one of the two antibodies raised against gpl88 cross-reacted weakly with glycoproteins of similar molecularweight in isolated nuclear fractions from Xenopusoocytes, as well as chicken, opossum and rat liver.There was no detectable release of gpl88 from thenuclear fraction after treatment with Triton X-100 orDNase I and RNase A. Extraction of the residualnuclear material with 1 M-NaCl resulted in the apparentsolubilization of approximately 10-20% of the glyco-protein, but the majority of this component resistedsalt extraction (and even 5 M-urea) and was foundassociated with the nuclear matrix pore complex-lamina.

Berrios et al. (1983) have also reported on theexistence of a 188K protein in the nuclear matrix porecomplex-lamina fraction of Drosophila embryos,which is distinct from gpl88. This protein was ident-ified as an ATPase/dATPase and appeared not to beglycosylated, as it was completely resistant to digestionby endoglycosidase H. It remains to be determined ifthe ATPase/dATPase is a real constituent of the porecomplex in vivo.

Nuclear matrix preparations described by manyauthors differ considerably with respect to the high-ionic strength conditions under which they are isolated.It is not known to what extent the pore complexproteins described above remain associated with suchdifferent preparations. When nuclear envelopes areexposed to rigorous extraction treatments involvingsolutions of high-ionic strength or non-denaturingdetergents, the basic structural elements of the porecomplexes are still identifiable (Franke et al. 19816).An interesting postulation in this respect is that onlythe components on the cytoplasmic surface would besensitive to salt extraction (Davis & Blobel, 1986),whereas the components on the nuclear surface wouldremain attached to the lamina by a link that is resistantto extraction with high-salt solution. Whether or not itslink to the lamina makes the nuclear part of the porecomplex resistant to high-salt extraction remains to beexamined.

The nuclear laminaThe nuclear lamina consists of a proteinaceous layersituated subjacent to the inner nuclear membrane(reviewed by Franke et al. 19816; Gerace & Blobel,1982; Gerace, 1986; Krohne & Benavente, 1986;Newport & Forbes, 1987). The lamina is usually co-purified together with the pore elements. The predomi-nant polypeptides in such preparations are the lamins,proteins in the molecular weight range of 60-80(XlO3), which are immunologically related. In mam-mals and avian species three main lamin proteins, i.e.lamins A, B and C, have been characterized, while atleast five different lamins have been described inamphibia and one or two in certain invertebrates(reviewed by Krohne & Benavente, 1986). Immuno-histochemical studies using specific antibodies to thelamin proteins have confirmed the localization of theseproteins at the rim of the nucleus (see, e.g., Fig. 3).

Several authors have detected lamin precursors(Gerace et. al. 1984; DagenaiseZ al. 1985; Lehnere/a/.1986), while additional minor components that displaythe biochemical properties characteristic of the laminshave been described for rat and chicken by Lehner etal. (1986). This indicates that the composition of thenuclear lamina in these species is probably morecomplex than previously assumed.

Recently, McKeon et al. (1986) and Fisher et al.(1986) have characterized the cDNA clones for humanlamins A and C. From the protein sequences deducedfrom these cDNAs it became apparent that lamin A hasan additional region of approximately 9K at its car-boxyl terminus as compared to lamin C (Fisher et al.1986). Both lamin sequences show a marked homologywith the intermediate filament proteins (reviewed byFranke, 1987). In vitro translation studies performedby Laliberte et al. (1984) showed that lamins A and Care encoded for by different mRNAs, with lamin Abeing a precursor approximately 2K larger than maturelamin A.

Using a mouse monoclonal antibody (IFA) raisedagainst a common domain of all intermediate filamentproteins, Lebel & Raymond (1987), as well as Osborn& Weber (1987), have shown that lamin B also sharessome sequence homology with the intermediate fila-ment proteins. This homology of the lamins to theintermediate filament proteins may account for thefibrillar nature of the lamina as seen, for example, inFig. 4.

Lamin B is thought to fulfil a role in anchoring thelamina to the inner membrane of the lipid bilayer, sincethis protein is more resistant to chemical extractionsfrom nuclear membranes compared with lamins A andC (Gerace & Blobel, 1982; Lebel & Raymond, 1984). Italso remains selectively associated with membranevesicles after nuclear envelope disassembly duringmitosis (Burke & Gerace, 1986).


Recent studies by Georgatos & Blobel (\987a,b)have demonstrated that lamin B also constitutes anintermediate filament attachment site at the nuclearenvelope. Their approach consisted of in vitro bindingstudies with isolated bovine lens vimentin and avianerythrocyte nuclear membranes. Removal of lamin Bfrom the nuclear envelope by urea extraction orblocking with anti-lamin B antibodies were found toreduce the binding of vimentin to these membranes.Other techniques, such as immunoprecipitation, ratezonal sedimentation and affinity chromatography,pointed to a specific vimentin—lamin B associationunder;/? vitro conditions. The 6-6K carboxyl terminusof vimentin was found to be involved in this inter-action, whereas the binding was positively influencedwhen lamin A was present. From these data Georgatos& Blobel (19876) concluded that intermediate fila-ments may be anchored directly to the nuclear lamina.These anchorage places are suggested to be restrictedto certain distinct foci along the lamina, coincidingwith nuclear pores, and not to be uniformly distributedover the nuclear surface (see also Goldman etal. 1985).

Fig. 3. Peripheral localizationof the nuclear lamina in mouseP19 cells as detected by anantiserum to lamin B. The setof pictures was obtained bythe use of a confocal scanninglaser microscope (Brakenhoffet at. 1985), which scannedthrough the nucleus frombottom (1) to top (9) at 2-j.imintervals. Bar, 10/tm.(Courtesy of Dr G. J,Brakenhoff & Dr R. vanDriel.)

A direct association between the cytoskeletal frame-work and the nuclear lamina had been described byCapco et al. (1982) by using whole-mount microscopyto visualize nuclear matrices prepared from mouse 3T3fibroblasts and HeLa cells. Their electron micrographsshowed that cytoskeletal filaments were attached to thenuclear lamina. Two-dimensional gel electrophoresisrevealed vimentin to be present in their nuclear matrixfractions. Several other investigators have demon-strated the presence of vimentin and cytokeratins innuclear matrix preparations from cells grown in sus-pension culture (see, for example, Verheijen et al.1986a).

Another indication of the attachment of intermediatefilaments to the nuclear envelope has been reported byStaufenbiel & Deppert (1982), who showed that afterisolation of nuclei from cells grown in suspensionculture the majority of the cytokeratin and vimentinfilaments had collapsed onto the nuclear surface butstill constituted a filamentous system. These collapsedfilaments could be partially unfolded again by centrifu-gation through an isotonic buffer.


Fig. 4. Rotary-shadowed platinum/carbon replica of an isolated and cntical-point-dned BHK—nuclear matrix preparationshowing the lamina as a meshwork of anastomosing 8-10nm filaments. Cell extraction was essentially performed asdescribed by Simard el al. (1986) (0-5% Triton X-100, 5 fig ml"1 DNase I, 2lM-NaCl). Bar, 1-OjUm. (Courtesy of Dr V.Bibor-I lardy.)

16 R. Yerlieijen el al.

A remarkable observation in the field of intermediatefilament-nuclear matrix interactions has recently beenmade by Carmo-Fonseca et al. (1987). These investi-gators isolated nuclear matrix-intermediate filamentscaffolds from cultured rat ventral prostate cells andisolated rat uterine epithelial cells. Subsequently, thescaffolds were critical-point dried, platinum-carbonreplicated and examined by electron microscopy. Insuch preparations the intermediate filaments were notseen to abut on the nuclear lamina, but rather to belooped and to follow the nuclear surface. Short, directconnections of a cross-bridge type (5-7 nm in diam-eter) extended laterally from the intermediate filamentsand fused with the nuclear pore complexes (see Fig. 5).These cross-bridges appeared to be about 75-100 nmlong in uterine epithelial cells and were shown to beassociated with cytokeratin filaments, while in fibro-blasts they were considerably shorter (approximately14 nm long) and probably associated with vimentinfilaments. The cross-bridges were not recognized byanti-cytokeratin antibodies. Considering the very shortlength of these linking structures, the authors con-cluded that their finding does not contradict theobservations of Georgatos 8c Blobel (1987a,b). AlsoFey et al. (1984a) have clearly shown interactions ofintermediate filament structures with the nuclear per-iphery (see Fig. 6).

Apart from investigations on cytoskeleton-nuclearlamina interactions, other studies have established thatduring interphase the lamina is in intimate contact withthe peripheral chromatin (Boulikas, 1986; reviewed byHancock & Boulikas, 1982; Hubert & Bourgeois, 1986;Hancock & Dessev, 1987). Such interactions are prob-ably important for stabilizing or maintaining certainaspects of higher-order chromosome architecture (Leb-kowski & Laemmli, 19826).

In conclusion, the lamina not only determines thenuclear shape and the spatial organization of the porecomplexes, but seems also to be directly involved inanchoring both the intermediate filaments and thechromosomes at the nuclear periphery during inter-phase.

The nucleolar residue

The nucleolus is the site of synthesis and processing ofpre-ribosomal RNA, and of assembly of the ribosomalproteins and ribosomal RNA into pre-ribosomal par-ticles. Only a relatively small number of the manynuclear proteins are confined exclusively to thenucleolus and presumably play specific roles in itsstructure and function. One such major nucleolar-specific protein is nucleolin or C23. This phosphoryl-ated protein (HOK/pI 5-5) is probably involved in pre-rRNA transcription and ribosome assembly (Bugler et

al. 1982). Small nuclear RNPs (U3 and U8 in particu-lar), which may function in rRNA processing, are alsoaccumulated in the nucleolus (Epstein et al. 1984;Reddy et al. 1985). Many authors have dealt with themorphological and biochemical aspects of the nucleolus(for reviews see Jordan & Cullis, 1982; Goessens, 1984;Hadjiolov, 1985; Sommerville, 1986).

At the Eighth European Nucleolar Workshop in 1983an attempt was made to standardize nucleolar no-menclature. Thus, the nucleolar matrix was defined asthe residual structure left after extraction procedures toreveal the nuclear matrix (Jordan, 1984). Such nu-cleolar matrices retain the size and shape of the originalnucleoli. However, as nucleolar residues present innuclear matrix preparations may have been subjectedto treatment with DNase I only or with a combinationof DNase I and RNase A, it is necessary to extend thedefinition given by Jordan (1984). The term nucleolarmatrix is used here to indicate the residual nucleolus innuclear matrix preparations in which only DNase I hasbeen used in the nuclease step.

Little is known about the composition of the nu-cleolar remainders in nuclear matrix preparations. It isdifficult to detect any morphological resemblance be-tween the nucleolar residues found in nuclear matricesand structural components of the intact nucleolus.Aggregation and condensation of nucleolar structures,probably due to the presence of divalent cations, makeinterpretation of electron-microscopic images very dif-ficult. Procedures using EDTA, however, can yieldfairly decondensed nucleolar matrices. This is illus-trated by the studies of Long & Ochs (1983), whoprepared chromatin-depleted nuclei from Frienderythroleukaemia cells under conditions that avoid theuse of high salt concentrations. These authors partiallydigested the DNA, and then washed the cells twice in2mM-EDTA. Compared with the amounts in wholenuclei, the amounts retained in the resulting structureswere approximately 1 % of the DNA, 65 % of the totalRNA, 70% of the hnRNA, 74% of the snRNA, 29%of all protein and 2% of the histories. Although inelectron micrographs of nuclei treated in this way nomorphological evidence was found for residual nu-cleoli, immunofluorescence studies showed that pro-tein C23 was located in distinct centrally localizedregions. Exposing these EDTA-prepared chromatin-depleted nuclei to 2mM-MgCl2 resulted both in thereformation of morphologically distinct residual nu-cleoli and in aggregation of matrix fibrils. Similareffects have been observed in rat liver nuclei byGalcheva-Gargova et al. (1982) and also by Hubert etal. (1981), Bouvier et al. (1980) and Aaronson & Woo(1981).

For some time it was not clear whether the nucleolarmatrix structures and the intranuclear matrix networkwere composed of distinct or identical constituents.


Fig. 5. A. Rotary-shadowed platinum/carbon replica of an isolated, extracted and critical-point-dried rat uterine epithelialcell. Cell extraction was performed according to Fey et al. (1984a) in order to obtain nuclear matrix-intermediate filamentscaffolds. Nuclear pore complexes are seen attached to intermediate filaments through short filamentous cross-bridgesapproximately 5 nm in diameter (arrows). B. Lateral view of the thin filaments, extending from the intermediate filamentsto the nuclear matrix in a similar preparation as shown in A. The thin filaments are seen to abut on the nuclear lamina(arrows) and pore complexes (open arrow). Filaments with identical diameters link adjacent intermediate (cytokeratin)filaments (arrowheads). Bars, 0'2,i(m. (Courtesy of Dr M. Carmo-Fonseca & Dr A. Cidadao; see also reference Carmo-Fonseca*?/ al. (1988).)


The first experiments that provided evidence for differ-ent protein compositions of these two structures wereperformed in rat liver by Berezney & Coffey (1977).Later, Todorov & Hadjiolov (1979) found five distinctprotein bands with apparent molecular weights of 30,40, 56, 70 and 82 (XlO3) enriched in the nueleolarmatrix fraction. Distinct nueleolar matrix proteinsfrom rat liver were found also upon two-dimensionalgel electrophoresis by Comings & Peters (1981). Theyfound many basic proteins specific for the nucleolus,the most prominent one being a 33K protein.

The elegant studies of Franke et al. (1981a) led tothe isolation of a nueleolar matrix from Xenopus oocytenuclei comprising filaments of about 4nm diameter,which were densely coiled into higher-order fibrils of30-40 nm diameter. This matrix was resistant to treat-ments with low-salt and high-salt buffers, DNases andRNases, sulphydryl agents and non-ionic detergents,and contained a single 145K/pI 6-2 protein (see alsoBenavente et al. 1984a,b). Furthermore, Olson &Thompson (1983) showed that a 160K polypeptide waspredominantly found in the nueleolar matrix fractionfrom Novikoff hepatoma ascites cells prepared bydigestion with DNase I and extraction with high-salt.

Recent studies of Olson et al. (1986), also performedon Novikoff hepatoma cells, revealed a nueleolarmatrix fraction that was enriched in polypeptides withmolecular weights of 28, 37-5, 40, 70, 72, 110 and160X103. The 110K protein was recognized by anantibody directed against protein C23 (Olson et al.1981). About 25 % of the protein, 50 % of the RNA andless than 4% of the DNA of untreated nucleoli wererecovered in such nueleolar matrix preparations. Olsonet al. (1986) also showed that pre-ribosomal RNPparticles are major constituents of nueleolar matrixpreparations extracted with DNase I and high-saltsolutions. RNase A treatment during the DNase Idigestion stage, together with the inclusion of 1 %/3-mercaptoethanol in the high-molarity salt washes,reduced the protein content to 15 % and the RNAcontent to about 2-5% of that of untreated nuclei.Under such conditions protein C23 and the 160Kprotein were also removed. The remaining polypep-tides were largely represented in the 30-50K range,and electron microscopy revealed only amorphousmaterial instead of the granular elements usually foundin nueleolar matrix preparations (Olson et al. 1986).

In preparing nueleolar matrices from mouse L-cells,Shiomi et al. (1986) used 50/xgmr1 DNase I, aconcentration five times higher than that used by Olsonet al. (1986), and reproducibly obtained core nucleoli(the nueleolar fraction that remains after extensiveDNase I action, without a high-salt extraction) with aminimum protein content. The nueleolar matrix frac-tions prepared by Shiomi et al. (1986) contained about5 % of the amount of DNA present in isolated nucleoli,

16% of the RNA and less than 4% of its originalprotein content, being enriched for proteins of 34K,36K, 43K, 57K, lamins A and C (70 and 62K). Alsohigher molecular weight proteins, including a lOOK/pI6-8 and a phosphorylated 160K/pI5-5 protein werefound in these preparations. A portion of ribosomalspacer DNA remained tightly bound after treatmentwith2M-NaCl (see also Bollaef al. 1985). Shiomi et al.(1986) found the C23 protein to be quantitativelyreleased from the nueleolar matrix by 2M-NaCl, whichis in contrast to the results of Olson et al. (1986). Thiswas verified with a specific antibody test indicating thatthe 110K protein was distinct from protein C23. Theobservations of several investigators that the nucleolusis often in close contact with the nuclear envelope (Rae& Franke, 1972; Goessens, 1979; Bouteillee/ al. 1982;Bourgeois et al. 1981, 1984) may explain the presenceof lamins A and C in the nueleolar matrix preparationsof Shiomi et al. (1986). Evidence for a nucleolus-nuclear envelope junction in the form of a 'pedicle' or'stalk' has been presented by Rae & Franke (1972) inmouse hepatocyte nuclei and more recently by Hubertet al. (1984), who isolated nucleoli containing nuclearshells from membrane-depleted rat liver nuclei (seealso Bouviere/ al. 19856). Hubert e? al. (1984) showedthe nucleoli to be anchored to the peripheral lamina bya pedicle that was continuous with an intranucleolarnetwork. The pedicle and the network that supportsthe nueleolar DNA were composed mainly of non-histone proteins insoluble in 2M-NaCl.

In conclusion, the nueleolar residues in nuclearmatrix preparations appear to be complex structurescomprising several different elements, including manyproteins not yet adequately characterized.

The internal matrix

Morphological and biochemical aspectsSeveral authors have provided evidence of the protcin-aceous nature of the internal matrix, based on the factthat a fibrillar structure is observed in the nucleus whenboth DNA and RNA are removed by nucleases (Capcoet al. 1982). Other authors (Galcheva-Gargova et al.1982; Kaufmann & Shaper, 1984; Kaufmann et al.1986) have observed a complete reorganization of theinternal matrix structure, depending on the isolationconditions used. In this regard there is concern aboutaggregation or precipitation of otherwise soluble nu-clear components, due to the high-salt concentrationsrequired to extract chromatin during matrix prep-aration, or about cross-links formed by oxidation ofsulphhydryl groups.

Other studies indicated that the structural integrityof the internal network depends on the maintenance of


. • • • /

20 /?. Verheijen et al.

certain metalloprotein interactions during matrix iso-lation, e.g. with Ca2+ and Cu2+ (Lebkowski &Laemmli, 1982a,b) or with Mg2+ (Bouvier et al.1985a). These latter authors found the intranuclearstructures in HeLa nuclear matrix preparations to becomposed of residual (DNase- and salt-resistant) RNPcomplexes of both nucleolar and non-nucleolar origin.These intranuclear structures comprised two distinctbut superimposed networks, which appeared as thinfibrillar elements (2-3 nm) and as thick fibrogranularelements of varying size. Both networks disappeared asa result of RNase digestion in the presence of low-ionicstrength EDTA before extraction of nuclei in 2M-NaCl.However, the thick fibrillar elements were preservedfrom being eluted in 2 M-NaCl when RNase was used inbuffers containing Mg2+. This remaining network wasenriched in two proteins of 49K and 70K. From theseresults Bouvier et al. (1985c/) concluded that in thepresence of Mg + interactions between certain RNPcomplexes are established, which then become able toform a salt-resistant intranuclear network.

Herman et al. (1978) showed that after removal of99 % of the chromatin in a two-step extraction pro-cedure, both steady-state and newly synthesizedhnRNA were associated with the remaining nuclearstructure. Their suggestion that the integrity of thenuclear matrix is dependent on the RNA was incontrast with the conclusion of Miller et al. (1978a)that RNase treatment of the nuclear matrix does notalter the morphology of this network.

Considering the results obtained by the manyworkers on nuclear matrix structure and composition,it will be obvious that the presence or absence of theinternal network in nuclear matrix preparationsdepends on the experimental protocol used. The effectsof divalent cations, the molarity of extraction buffers,the effect of high-salt treatment, the extent of disul-phide cross-linking during preparation of the matrices,the order in which the various preparation steps areapplied, the use of ( N H ^ S C ^ instead of NaCl for theextraction itself, the presence of endolytic enzymesother than proteases inhibited by phenylmethylsul-phonyl fluoride (PMSF) or phenylmethylsulphonylchloride (PMSC), and many other factors that are usedfor the preparation of the matrix structure have not yet

Fig. 6. A. Whole-mount transmission electronmicrographs of the nuclear matrix-intermediate filement(NM-IF) scaffold from a breast carcinoma cell line. Thechromatin-depleted nuclear matrix (NM) is apparently inassociation with intermediate filaments (IF) largelyconsisting of cytokeratins. Note the nuclear pores (NP)present in the nuclear lamina. Bar, 0-5jum. B. Immunogoldstaining of intermediate filaments (IF) as described by Feyet al. (1984«) in a similar preparation as in A, using anti-cytokeratin antibodies. Bar, 01 /*m. (Courtesy of Dr E. G.Fey.)

been studied sufficiently. Some of these technicalproblems have been tackled in recent papers butanswers are still incomplete (Long & Ochs, 1983;Kaufmann & Shaper, 1984; Staufenbiel & Deppert,1984; Fey et al. I984a,b, I986a,b; Mirkovitch et al.1984; Verheijen et al. 1986a).

Electron-microscopic studies performed by Pouche-let et al. (1986) have shown the existence of a well-defined network in nuclei of resting mouse lympho-cytes in situ. These authors prepared nuclear matricesfrom formaldehyde-fixed cells. The nuclease step insuch isolations was performed either with DNase onlyor with DNase in combination with RNase. In bothtypes of preparations three well-defined networks wereobserved: the lamina, an intra-chromatin network andan inter-chromatin network. This latter structure couldbe superimposed on the internal network of isolatednuclear matrices. Its sensitivity to pepsin digestion wasincreased tenfold when the digestion was preceded bytreatment with RNase. This latter finding indicatesthat RNA appears to be essential for the maintenanceof this structure. Also Fey et al. (1986a) have shownthat the morphology of the internal matrix changeddrastically when RNases were used in the extractionbuffers (see Fig. 7).

In conclusion, the relevance of an internal matrixstructure in vivo is still a matter of controversy and atpresent time many questions are still unanswered. It isbeyond the scope of this review to compare all thedifferent isolation procedures for the preparation ofnuclear matrices or to discuss all the indications thatsupport or deny the existence of an extranucleolarnetwork in vivo. We merely conclude that considerableevidence indicates that when an internal matrix struc-ture is obtained without prior treatment with RNase,hnRNA is found to be an integral constituent of thisstructure (Miller et al. 1978«; Berezney, 1980; Brasch,1982; Gallinaroe/ al. 1983; Long & Schrier, 1983; Feyet al. 1986a,b).

A relevant question is whether the presence of aninternal matrix is a general feature of cells. Usingprocedures similar to those published by Merman et al.(1978) and Miller et al. (1978a) a relatively stableRNA—matrix association can be found in various typesof cells (Berezney, 1980; van Eekelen & van Venrooij,1981; Brasch, 1982; Gallinaro et al. 1983; Long et al.1979; Fey et al. \986a,b). The adult chicken erythro-cyte nucleus, however, in which virtually no DNA orRNA synthesis takes place, was found to lack aninternal nuclear matrix. Also, mild extraction pro-cedures resulted in empty shells of pore complex-lamina together with loose aggregates of core histones(Lafond & Woodcock, 1983). In contrast, rat livernuclei showed a typical intranuclear salt-resistant skel-eton after the same treatments. These results indicatethat an internal matrix is not an obligatory nuclear


Fig. 7. Transmission electron micrographs of HeLa nuclear matrix preparations in unembedded resin-free sections (0-2fimthick) as described by Fey et al. (1986c;). A shows an RNP-containing nuclear matrix preparation, and B an RNP-depletednuclear matrix. The RNP-containing nuclear matrix reveals fibres (F) that extends throughout the nucleus, formingcontinuous associations between nucleoli (Nu) and the nuclear lamina (L). Cytoplasmic filaments (Cy) are observed inassociation with the lamina. The RNP-depleted nuclear matrix displays a distortion of nuclear shape. The interior of thenuclear matrix is composed of condensed and fragmented filament aggregates (FA). The distortion of the interior bydigestion with RNase suggests that RNA is an important structural component of the nucleus. Bars, l-Of/m. (Courtesy ofDr E. G. Fey.)

component and that in erythrocytes it is apparently notrequired for the spatial organization of chromatin. Incontrast, much more active 5-day-old embryonic eryth-rocytes did contain an interchromatinous nuclearmatrix (Lafond & Woodcock, 1983). Subsequent ob-servations from the same group showed that an internalnuclear matrix is generated during the reactivation ofchick crythrocyte nuclei in mouse L-cell cytoplasts.The nuclei enlarge and chromatin decondenses, ac-companied by an influx of proteins from the hostcytoplasm and the onset of RNA synthesis (Lafond &Woodcock, 1983). Recently, Woodcock & Woodcock(1986) have used the same experimental system toidentify 15 major polypeptides that, after a 16-hreactivation period, had migrated into the nucleus.Five of the identified proteins in the 30-70K molecularweight range appeared to be nuclear matrix proteins;two of these had their counterparts in L-cell nuclei.During the concanavalin-A-induced stimulation oflymphocytes Setterfield et al. (1983) observed that the

nuclear volume increased up to sixfold, together withan extensive synthesis of stable interchromatinousmatrix proteins. All these results suggest a correlationbetween the presence of nuclear matrix structures andnuclear 'activity'.

In summary, the composition and organization ofhnRNA as part of the granular and fibrous internalnuclear matrix structure still require more precisecharacterization. The results obtained to date permitthe conclusion that when nuclear matrices are isolatedin the absence of RNase, hnRNA can be isolatedalmost quantitatively as an intimate part of it. Con-sidering the complicated composition of the nuclearmatrix it will be obvious that it is not a static structure,but must display considerable dynamic activity.

Heterogeneous nuclear RNP (hnRNP) particlesShortly after hnRNA has been synthesized it associateswith proteins to form fibrillar ribonucleoprotein(RNP) particles and granules resembling 'beads on a


string' that extend away from the DNA-protein axis.These fibres (7 nm thick) and granules (20-25 nmdiameter) are mostly referred to as the nuclear RNPnetwork. hnRNP structures can be isolated from cellnuclei in a wide range of sedimentation values(30-250 S), depending on the isolation procedure ap-plied (reviewed by LeStourgeon et al. 1981; Holoubek,1984). The monomeric forms of these structures arehnRNP particles of 30-40 S. Next to an RNA fragmentin the range of 125-800 nucleotides, these 40 S hnRNPparticles contain a set of proteins that comprise75-90% of its mass (Holoubek, 1984; Dreyfuss, 1986).Although non-specific binding of proteins to the RNAduring the isolation of the particles has never beenexcluded, it is generally accepted that a distinct set ofso-called core proteins is present in 40 S hnRNPparticles. According to the nomenclature of Beyer et al.(1977), HeLa cells contain the following core proteins:A1(34K), A2(36K), B1(37K), B2(38K), C1(41K) andC2(43K) (reviewed by LeStourgeon et al. 1981;Holoubek, 1984; Dreyfuss, 1986). Proteins Cl and C2appear to play a role in hnRNA processing, as amonoclonal antibody to these proteins inhibits in vitrosplicing of an mRNA precursor, while depletion ofthese proteins from the splicing extract abolishes itscapacity to splice pre-mRNA (Choi et al. 1986). Two-dimensional gel separations of the core proteins havemade it necessary to extend the nomenclature of Beyeret al. (1977), as has been done by Wilk<?/ al. (1985) andCeWs etal. (1986).

Recently, Dreyfuss' group analysed the compositionof hnRNP complexes obtained from HeLa cells byimmunopurification with monoclonal antibodies. Bytwo-dimensional gel electrophoresis they identified aset of over 24 proteins in the molecular weight range of34-120K as consistent hnRNP components, of whichthe A, B and C proteins appeared to be just onesubgroup. Chromatography on single-strandedDNA-agarose indicated that almost all these proteinsare single-stranded nucleic acid binding proteins (G.Dreyfuss, personal communication). The A, B and Cproteins are also bound to the matrix-associatedhnRNA (van Eekelen & van Venrooij, 1981; Marimanet al. 1982a; Dreyfuss et al. 1984).

Nuclear hnRNP particles can be isolated either by alengthy low-salt extraction procedure or by sonicationof isolated nuclei (reviewed by Holoubek, 1984). Theextraction of hnRNP particles from intact nuclei isdependent on the action of nuclear RNases and on theslightly alkaline pH that is required for the release ofthe particles from nuclei. Since the nuclear matrix hasbeen shown to be extremely susceptible to proteolyticactivities (Miller et al. 1978a), it is likely that thisprocedure may also release RNP particles from thenucleus by proteolytic degradation of the nuclearstructure. The sonication procedure, on the other

hand, releases RNP particles by the shearing forcesemployed. Faiferman & Pogo (1975) have shown thatthe yields of particles from disrupted nuclei are pro-portional to the shearing forces applied. It is evidentthat this procedure also destroys the delicate nuclearinfrastructure. In studying the release of RNA fromthe nuclear matrix several authors have found thathnRNA is bound tenaciously to other matrix com-ponents and that it can be separated from the attachingstructure only after disruption of the nuclear integrity(Longed al. 1979; van Eekelen & van Venrooij, 1981).

In summary, it can be concluded that isolation ofhnRNP particles necessarily implies the fragmentationof the internal nuclear matrix. As a consequence,depending on the procedure used, varying quantities oflarge hnRNP complexes and RNP core particles musthave been present in nuclear matrix preparationsisolated by various workers during the last decade. Thecomplexity of the nuclear matrix and salt-resistanthnRNP structures has been compared by Gallinaro etal. (1983). Apart from small nuclear RNAs about 40proteins in the 25-120K molecular weight range werecharacterized as common constituents of the nuclearmatrix and the hnRNP particles. In addition, the pre-mRNAs and maturation products present in bothstructures were compared. The results confirmed thesimilarity of the structures, strongly suggesting thatpre-mRNA in the nuclear matrix and in the salt-resistant complexes derived from hnRNP share acommon constitutive unit (Gallinaro et al. 1983).

Small nuclear RNP (snRNP) particlesEukaryotic cells contain a group of metabolicallystable, capped RNAs known as U RNAs (for reviews,see Holoubek, 1984; Brunei et al. 1985). The majorrepresentatives are designated U1-U6 RNAs, while inthe higher eukaryotes four minor U RNA species(U7-U10) have been described as well (Reddy et al.1985). The U RNAs are found in discrete ribonucleo-protein particles, which all appear to play a role in theprocessing of pre-mRNA (reviewed by Padgett et al.1986; Maniatis & Reed, 1987). In interphase cells thelocalization of U3 RNP (and probably U8 RNP) isrestricted to the nucleolus, whereas the other U snRNPparticles are mainly located in the nucleoplasm.

The association of U RNAs with the nuclear matrixwas first reported by Zieve & Penman (1976) and Milleret al. (19786), who demonstrated that, like hnRNA,small nuclear RNAs remained in the nuclear matrixafter removal of nuclear membranes and chromatin.Similar observations were made by Herlan et al.(1979), Maundrell et al. (1981) and Ross et al. (1982)for Tetrahymena, duck erythroblast and chicken eryth-roblast nuclear matrices, respectively.

Miller et al. (19786) and Gallinaro et al. (1983)found a quantitative association of all snRN As with the


nuclear matrix. Zieve & Penman (1976), however,characterized U2, U3, U4 and U6 RNA as beingnuclear matrix-associated, whereas Ul RNA wasmainly lost upon chromatin extraction. In contrast,Ciejek el al. (1982) have shown that only some of the UsnRNAs were tightly associated with chicken oviductmatrices. They did not find a quantitative associationnor a specific enrichment of one or more of thesnRNAs.

snRNPs can be released efficiently from isolatednuclei or from nuclear matrices by disintegration of thenuclear structure, for example by sonication. To agreat extent the snRNPs are released in the form of 10 SsnRNP particles; however, they are partly found inassociation with larger particles (30-60 S) containinghnRNA. Part of the snRNA is believed to be base-paired to these particles (reviewed by Padgett et al.1986). In cross-linking experiments using 4'-amino-methyl-4,5',8-trimethylpsoralen (Pogo et al. 1982) itwas shown that in chromatin-depleted nuclei snRNAs,mainly Ul and U6, can be cross-linked to hnRNA.Similar experiments on whole cells in vivo indicatedthat both Ul and U2 RNAs can be found base-pairedto hnRNA (reviewed by Padgett et al. 1986). Althoughthe latter experiments and the association of snRNAwith hnRNP complexes suggest a close correlationbetween the structural organization of hnRNA andsnRNA, Pogo and co-workers were able to removesnRNA from nuclear matrices by treatment with 1 %sodium deoxycholate, leaving the hnRNA in the struc-ture (Pogo et al. 1982). This suggests that at least someof the snRNA and hnRNA are in different nuclearorganizations.

Sera from patients with connective-tissue diseasesoften contain antibodies directed against snRNP par-ticles. Anti-Sm sera recognize the complete set of Ul,U2, U4, U5 and U6 (U1-U6) RNP particles, whereasanti-Ul RNP sera exclusively react with Ul RNP-specific proteins (Pettersson et al. 1984; Habets et al.1985fl). Up to 12 polypeptides have now been ident-ified as constituents of the snRNPs U1-U6, i.e. 70K,A(32K), A'(31K), B'(27K), B"(26K), B(25K),C(22K), D(16K), D'(15K), E(12K), F(11K) andG(9K) (Bringmann & Luhrmann, 1986). Proteins70K, A and C are unique to the Ul RNP particle,whereas A' and B" are specific constituents of the U2RNP particle.

Vogclstein & Hunt (1982) showed that upon prep-aration of nuclear matrices of 3T3 cells a subset of theantigens recognized by anti-Sm sera was retained in thematrices. Judging from the intensities of immunofluor-csccnce, 65 % of the Sm antigens remained in theresidual matrix after extraction with 2M-NaCl. Treat-ment of the matrices with RNase removed the Smantigens, which implies that they are associated with

the matrix structure via RNA. When a human autoim-mune serum containing anti-La antibodies was used asa control, immunodecoration of the matrices did notoccur. This means that the La RNP particles (contain-ing polymerase III transcripts such as pre-tRNAs andprecursor forms of 7 S RNA and 5 S rRNA; Rinke &Steitz, 1982) were lost during matrix preparation.Similar experiments to immunodecorate isolatedmatrix structures with antibodies directed against URNP antigens have been performed by van Eekelen elal. (1982), Spector <?/ al. (1983) and, more recently, byReuter et al. (1984). They showed that several antigensrecognized by these sera are almost quantitativelyretained in nuclear matrices prepared from culturedcells. There is, however, some uncertainty as towhether these antigens can be removed by RNasetreatment or not. An explanation for such discrepanciescan probably be found in the fact that these human serarecognize different antigenic polypeptides in an snRNPparticle.

Cell fractionation studies performed by Habets et al.(19856) have shown that the Ul RNP-specific 70Kantigen and the B'/B (U1-U6) RNA-associated anti-gens are more tightly complexed with the nuclearmatrix compared with the other U RNP proteins, sincesubstantial amounts of both the 70K and B'/B wereresistant to subsequent treatments with detergents,DNase, RNase and high-salt solution (see also Verhei-jen et al. 19866). The other U snRNP-associatedproteins, for example the A and D antigens, werereadily extracted under these circumstances. There-fore, using a serum that contains a strong anti-70Kactivity one would expect an RNase-resistant immuno-fluorescence, as indeed has been found (Verheijen el al.1986a). In contrast, a human serum that recognizespredominantly the Sm antigens will not decoratenuclear matrices as clearly after RNase treatment.

The fact that some antigens, for example, the UlRNA-specific 70K protein, are more tightly associatedwith the nuclear matrix compared with other U RNPproteins, suggests that such proteins can be involved inbinding U RNAs to the hnRNA—matrix complex.

Using immunoelectron microscopy with Sm anti-bodies on Novikoff hepatoma cells, Spector et al.(1983) found the snRNPs to be localized in a reticularnuclear network. This localization of snRNPs wasaltered when PtK2 cells had been treated with 5,6-dichloro-1-jS-D-ribofuranosylbenzimidazole (DRB),which inhibits 60-75 % of the hnRNA synthesis.Drug-treated cells showed an accumulation or clump-ing of antigens recognized by anti-Sm antibodies in thecentral region of the nucleus. This effect on thedistribution of snRNPs by inhibition of the hnRNAsynthesis supports the assumption of a functionalassociation between these two nuclear components(Spector etal. 1983).

24 R. \ erheijen et al.

Fakan et al. (1986) have applied immunoelectronmicroscopic techniques to mouse and Drosophila tis-sue-culture cells, using monoclonal antibodies directedagainst hnRNP core proteins or against U RNP pro-teins. Their studies provided direct evidence for anassociation of Ul RNP and possibly also of other URNP species with extranucleolar RNA during earlytranscription elongation. In addition the results ofthese authors confirmed the presence of hnRNP pro-teins within the growing RNP chains in the transcrip-tion complexes.

In summary, the limited number of studies per-formed on the interaction between snRNA or snRNAand nuclear matrix structures indicates a specificinteraction between these two complexes. It will be ofgreat interest to elucidate the nature of such aninteraction and to establish its functional significance.

Nuclear actinConsiderable evidence has accumulated over the pastdecade to show that actin is a constituent not only of thecytoplasm but also of interphase nuclei in a widevariety of cells (for a review, see Scheer et al. 1984).Actin has also been demonstrated as a major protein inmanually isolated and cleaned nuclei of Amoeba andamphibian oocytes, in which concentrations of3-4mgml~' can be found (Krohne & Franke, 1980;Gounon & Karsenti, 1981).

Experiments by Manley et al. (1980) have indicatedthat at least part of the nuclear actin may be involved inRNA transcription. The transcription of protein-coding genes in eukaryotic systems is performed byRNA polymerase II, which in vitro requires sup-plementation with crude cellular extracts to initiateaccurate transcription. Such cellular extracts containmultiple factors, some of them recognizing specificpromotor elements such as the TATA box (Breathnach& Chambon, 1981). One of these factors has beenpurified and characterized as a protein that is strikinglysimilar to actin (Egly et al. 1984).

In a completely different approach, Scheer et al.(1984) observed that injection of antibodies againstactin into oocytes resulted in the cessation of transcrip-tion by RNA polymerase II, loop retraction andchromosome condensation. Moreover, even strongerinhibition was observed after injection of actin-bindingproteins from different sources, such as fragmin fromPhysarum polycephalum and an actin modulator pro-tein from mammalian smooth muscle. The idea thatactin is involved in some way in RNA transcription isalso supported by the finding that this protein is tightlyassociated with purified RNA polymerase II (Smith etal. 1979) and possibly involved in the regulation ofpoly(A) metabolism mediated by poly(A) polymerase(Schroder et al. 1982).

Additional evidence for a possible role of nuclearactin in RNA metabolism has been documented inseveral studies by Nakayasu & Ueda (1984, 1985,1986). These authors have shown an interaction be-tween pre-mRNAs and actin filaments in the nuclearmatrix of mouse L-cells (Nakayasu & Ueda, 1985). In aprevious study it had been shown that actin filamentsare closely associated with small nuclear RNPs(Nakayasu & Ueda, 1984). Recently, the existence oftwo additional acidic species of actin in the nuclei ofmouse L-cells were reported next to the two common/3-actins and y-actins (Nakayasu & Ueda, 1986). Themost acidic actin (pi 5-1) was localized predominantlyin the nuclear matrix. Other authors have also foundactin as a major protein in nuclear matrix preparations(Capco et al. 1982; Staufenbiel & Deppert, 1984;Verheijen et al. 1986a; Nakayasu & Ueda, 1986).Although the possibility of contamination with cyto-plasmic actin cannot be totally excluded, the obser-vations described above justify the conclusion thatactin may be a major and well-defined nuclear matrixprotein that might have a defined function in RNAsynthesis in vivo.

Enzymes involved in DNA and RNA metabolismEnzymes involved in DNA and RNA metabolism thathave been found in nuclear matrix fractions are numer-ous. These include DNA alpha and beta polymerases(Nishizawa et al. 1984; Smith et al. 1984; Foster &Collins, 1985), topoisomerase I (Nishizawa et al.1984), topoisomerase II (Halligan et al. 1984; Berrioset al. 1985), RNA polymerase II (Lewis et al. 1984),poly(A) polymerase (Schroder et al. 1984), DNAmethylase (Burdon et al. 1985) and DNA primase(Wood & Collins, 1986; Tubo & Berezney, 1987a,b).

Vims-specific proteinsIt has been shown that the nuclear matrix is animportant site of viral interaction (reviewed by Berez-ney, 1984; Simard et al. 1986). Viral DNA (see, e.g.,Smith et al. 1985) and virus-specific proteins (see, e.g.,Jones &Su, 1982; Bibor-Hardy et al. 1985; Khittoo etal. 1986) have been found enriched in nuclear matrixpreparations.

The bulk of large tumour antigen (large T) in simianvirus 40 (SV40)-infected cells is present in threesubnuclear locations: in the nucleoplasm, associatedwith the cellular chromatin and tightly bound to thenuclear matrix. Schirmbeck & Deppert (1987) haveanalysed the distribution of large T in lytically infectedmonkey cells and found that the amounts of large Tassociated with both chromatin and nuclear matrixincreased markedly after transition from early to latephase of viral infection. The amount of nuclcoplasmiclarge T increased only slightly. During the course ofinfection large T accumulated in the chromatin and in


the nuclear matrix fraction, in parallel with the increaseof viral DNA synthesis. Recent studies by the samegroup have indicated that the association of SV40 largeT with the chromatin and the nuclear matrix ismediated by protein-protein interactions, rather thanby sequence-specific DNA binding (Hinzpeter & Dep-pert, 1987).

Associated transcripts

Newly synthesized SV40 RNA appears to be quantitat-ively associated with the nuclear matrix, while itsprocessing and transport also appear to take place onthis structure (Ben Ze'ev & Aloni, 1983; Abulafia et al.1984). Similarly, influenza viral RNA sequences (Jack-son et al. 1982) and the primary transcripts as well asthe spliced RNA intermediates of adenovirus-specificgenes (Mariman et al. 1982a; van Venrooij et al. 19826,1985) have been shown to be tightly bound to thematrix structure. Rearrangements in the nuclearmatrix morphology after infection with adenovirustype 2 have been demonstrated by the electron-micro-scopic studies of Zhonghe et al. (1987).

Other studies concerning nuclear matrix-associatedtranscripts have been presented by Ciejek e£ al. (1982).RNA was isolated from oviduct nuclear matrices andanalysed by hybridization to cloned probes for ovalbu-min and ovomucoid mRNA. More than 95% of all ofthe precursors of these mRNAs, including varioussplicing intermediates, were associated with thematrix. Less than 50 % of the mature mRNA present inintact nuclei was recovered in the nuclear matrix.Schroder et al. (1987a) have also studied the release oftotal mRNA, as well as of specific high-abundanceovalbumin mRNA, from hen oviduct nuclear matrices.Their results confirmed the earlier findings of Ciejek etal. (1982), by demonstrating that ovalbumin pre-mRNA was almost quantitatively associated with theoviduct nuclear matrix, whereas only one-third of themature ovalbumin mRNA of whole nuclei was re-covered in the nuclear matrix fraction. In addition,they showed that the binding of both pre-mRNA andmatrix-bound mature mRNA displayed no differencein strength when the matrices were subjected totreatments with high-salt (3M-NaCl), urea (4M) , deter-gent (2% Triton X-100) or EDTA (SOmM). Themature mRNA, however, was released selectively fromthe nuclear matrix by either ATP, AMP plus pyrophos-phate, ADP or ATP analogues containing non-hydro-lysable a,/3 or j6,y bonds. Whereas mRNA translocationthrough the nuclear pores is dependent on hydrolysis ofATP or GTP, mRNA release from the nuclear matrixapparently does not require hydrolysis of the ft,yphosphodiester bond. From these results Schroder etal. (1987a) suggested that the release of RNA might becaused by a conformational change of a nuclear matrix

(or mRNP) component induced by ATP or its deriva-tives without cleavage of any high-energy bond. ThehnRNA remained completely bound to the matrix inthe presence of ATP. Furthermore, the release ofmature mRNA by ATP could be strongly inhibited byvarious inhibitors of topoisomerase II, by a mechanismnot yet understood. Other remarkable results were thatboth mature and pre-mRNA were released from thematrix structure in the presence of poly(A), ethidiumbromide or the copper chelator 1,10-phenanthroline(Schroder et al. 1987a). The general conclusionreached by these authors was that nucleoplasmic RNAtransport is apparently regulated not only duringpassage through the pore complex but also at the levelof RNA release from the nuclear matrix.

Although the possibility that the non-matrix-boundmRNAs in the studies of Ciejek et al. (1982) andSchroder et al. (1987a) are contaminations from cyto-plasmic mRNAs cannot be completely ruled out, thedata indicate that a substantial part of the processedmRNA in the nucleus is bound differently and not astightly to the nuclear matrix as are the mRNA precur-sors. Since nuclear mRNAs are associated with adifferent set of proteins, as compared to cytoplasmicRNA molecules (van Eekelen et al. 19186; van Ven-rooij et al. 1982a), one would expect the selection ofmature mRNA for nucleocytoplasmic transport tooccur by means of exchange of hnRNP proteins(Dreyfuss, 1986) with a set of mRNA-binding pro-teins. Possible candidates for such proteins that couldexchange with hnRNP proteins are the transportproteins described by Moffet & Webb (1983) or cyto-plasmic mRNP proteins (Wagenmakers et al. 1980;Setyono & Greenberg, 1981; van Eekelen et al. 1981a;van Venrooij et al. 1982a).

All these results support the concept that the nuclearmatrix may be the structural site for RNA processingwithin the nucleus of eukaryotic cells.

The nuclear matrix and RNA transport

Recently, Schroder et al. (19876) have reviewed nu-cleoplasmic mRNA transport and discussed require-ments for mRNA release. For this reason we will focusonly on the fact that different RNA species havedifferent rates of transportation.

A general finding is that smaller RNAs are trans-ported faster to the cytoplasm than the larger mRNAs.Newly synthesized globin mRNA (9 S), for example, isreleased into the cytoplasm about 5 min after initiationof its synthesis (Bastos & Aviv, 1977; Kinniburgh &Ross, 1979). This RNA is polyadenylated and spliced.Histone mRNA, which is non-polyadenylated,unspliced and similar in size to the globin mRNA, isreleased into the cytoplasm within 10 min (Adesnik &Darnell, 1972). Adenovirus pIX mRNA (about 9S ,


polyadenylated and unspliced) reaches the cytoplasmwithin 4 min after the start of synthesis (Mariman et al.19826). In contrast, the bulk of the late adenovirusmRNAs reach the cytoplasm only after 16 min (Mari-man et al. 19826), a phenomenon that has been foundfor most cellular mRNAs as well (see, e.g., vanVenrooij et al. 1975). The reason for these divergentrates of transportation between matrix-bound mRNAsis not understood. Probably most of the larger pre-mRNAs require more complicated processing patterns,which means that the rate of transportation of mRNAdepends mainly on the rate of maturation. Anotherpossibility is that mRNAs emerging rapidly into thecytoplasm are not assembled into the usual hnRNPstructures, as has been suggested by Pederson fortranscripts lacking introns (Pederson, 1983). In thisrespect it should be mentioned that the final maturationstep of mRNA, that is the binding of the typicalcytoplasmic mRNA-associated proteins (Wagenmakerset al. 1980), is accompanied by the release of hnRNA-associated proteins (van Eekelen et al. 19816; vanVenrooij et al. 1982a). It has also been established thatmature mRNAs in the nucleus cannot be considered asintegral components of the nuclear matrix, as areprecursor RNAs (Ciejek et al. 1982; Schroder et al.1987a).

Behaviour of nuclear matrix componentsduring mitosis

The onset of mitosis is accompanied by an extensiverearrangement of nuclear components (see Fig. 8). Asthe cell approaches mitosis, the nucleolus first de-creases in size and then disappears as the chromosomescondense and all RNA synthesis ceases. At prophasethe lamins become highly phosphorylated (Ottaviano &Gerace, 1985), followed by dissassembling of thenuclear envelope. In immunofluorescence localizationstudies it has been shown that during prophase manynuclear (matrix) proteins shift from their distinctnuclear locations to a diffusely cytoplasmic distri-bution, excluding the condensed chromosomes (Chalyet al. 1984). At telophase this process is reversed. Suchbehaviour has been documented for several pore com-plex proteins (see, e.g., Davis & Blobel, 1986; Snow e(al. 1987), the lamins (see, e.g., Gerace, 1986; Ver-heijen et al. 19866), snRNP proteins (see, e.g., Reuteret al. 1985; Spector & Smith, 1986; Verheijen et al.19866) and hnRNP proteins (see, e.g., Martin &Okamura, 1981). For some of these proteins themolecular state of association in mitotic cells has beeninvestigated. Lamin B in Chinese hamster ovary(CHO) cells, for example, has been found to remainassociated with phospholipid vesicles during mitosis,while lamins A and C are converted into a soluble form(Burke & Gerace, 1986). In a previous study, Lahiri &

Thomas (1985) found that the hnRNP core proteins inmitotic HeLa cells were not free but stably associatedwith high molecular weight RNA in the form ofhnRNP particles that sedimented between 80 and200 S. Recent studies by these authors (Lahiri &Thomas, 1987) showed that during mitosis at least95 % of the total cellular snRNPs was also present insuch hnRNP complexes, sedimenting at about 100 S.

Other nuclear proteins, when investigated with im-munocytochemical techniques, appear to be associatedwith the condensed chromosomes in mitosis (Chaly etal. 1984). Such behaviour has been specifically de-scribed for some nucleolar proteins (see, e.g., Pfeifle etal. 1986), topoisomerase I (see, e.g., Verheijen et al.19866) and topoisomerase II (Earnshaw et al. 1985).The question arises as to how this group of nuclear(matrix) proteins is associated with the chromatin. Themodels for chromosome architecture that are in voguehave been reviewed by Earnshaw (1986). In one ofthese models a non-histone scaffold supposedly main-tains the higher-order topological organization of DNAin mitotic chromosomes. Such scaffolds have beenvisualized in the electron microscope by Paulson &Laemmli (1977) after treating isolated mitotic HeLachromosomes with dextran sulphate and heparin inorder to remove the histones. Their preparationsconsisted of a subset of non-histone proteins attachedto intact chromosomal DNA. The proteinaceous com-ponent or chromosome scaffold has been isolated fromsuch structures after nuclease digestion and extractionof chromosomal proteins (Adolph et al. 1977; Lewis &Laemmli, 1982) and was initially proposed to be a rigidlinear axial backbone in each chromatid, responsiblefor the morphology of metaphase chromosomes (Paul-son & Laemmli, 1977). Among the scaffold proteins,Lewis & Laemmli (1982) found two prominent high-molecular weight polypeptides, Sc-1 and Sc-2, of about170 and 135K, respectively. Using a polyclonal anti-body that recognized chicken scaffold protein Sc-1,Earnshaw et al. (1985) have shown this polypeptide tobe topoisomerase II (see also Gassere/ al. 1986). Othercomponents that have been found in isolated chromo-some scaffolds are centromeric proteins (Earnshaw etal. 1984).

Chromosome scaffolds respond dramatically tochanges in the ionic environment. Scaffolds isolated inthe presence of 2 M-NaCl differ completely in appear-ance from those isolated at low ionic strength in thepresence of dextran sulphate and heparin (Earnshaw &Laemmli, 1983), even though the protein compositionof the two forms appears to be identical (Lewis &Laemmli, 1982). Exposure of isolated scaffolds tomillimolar concentrations of Mg2+ also causes a similardramatic alteration in scaffold appearance (see Fig. 9;and references, Earnshaw & Laemmli, 1983).


Fig. 8. Immunofluorescent localization of various nuclear antigens in MR65 cells (human lung carcinoma) in interphase(Al-Dl) and metaphase (A2-D2) with monoclonal antibodies directed against: A, the lamins (antibody 41CC4); B, theUl RNP-specific 70K protein (antibody 2.73); C, the hnRNP-associated C proteins (antibody 4F4); D, a nucleolus-associated cell proliferation marker (antibody Ki-67). X1150.

' ' • • « • - •

Fig. 9. Electron micrographs ofchromosome scaffolds preparedfrom highly purified HeLa mitoticchromosomes. A. Scaffoldprepared at low ionic strengthusing a dextran sulphate/heparinlysis mix. B. Scaffold prepared atlow ionic strength using a dextransulphate/heparin lysis mix,followed by exposure to S mM-MgC^. Centromere region,indicated by arrows. Bar, 1-0 jUm.(Courtesy of Dr W. C. Earnshaw;see also Earnshaw & Laemmh(1983).)

28 R. Verlieijen et al.

Hancock & Dessev (1987) have isolated chromo-somes by lysis with Nonidet P40 in low ionic strengthbuffer without divalent cations or EDTA. Thesechromosomes slowly stretched to up to several timestheir original length while conserving an identifiablechromosome morphology (see Fig. 10). This finding isincompatible with a rigid nature of a supposed shape-determining skeletal element. These authors furtherobserved that each chromatid was not linear, butconsisted of a spiraled fibre, many times the chroma-tid's length, which was unwound upon incubation ofpolyamine-stabilized chromosomes with deoxvcholateor diiodosalicylate (see Fig. 11). The winding of thisfibre was suggested by Hancock & Dessev (1987) asbeing determined by protein-protein interactions be-tween neighbouring gyres. These authors also con-cluded that if a skeletal element does exist in mitoticchromosomes it does not itself dictate the dimensionsof the chromosome, but rather are the form and

dimensions of the skeletal element determined by theDNA associated with it.

Using the same polyclonal antibody to chickentopoisomerase II as described above, Earnshaw & Heck(1985) have examined the distribution of this enzymein intact, swollen but unextracted, chromosomes fromMSB-1 chicken lymphoblastoid cells. Under the con-ditions used in their experiments, topoisomerase 11 waslocalized in numerous separate spots that appeared tobe 120-200 nm across when covered with bound anti-body. It was therefore concluded that these data didnot provide evidence for the existence of a rigid core-like scaffold structure.

In summary, despite the controversial experimentaldata, we think it is justifiable to conclude that, ifpresent /// vivo, the chromosomal scaffold is not a rigidstructure, but rather a component of the mitoticchromosome with dvnamic features.

Fig. 10. Mitotic chromosomes fromCHO cells, prepared by lysis with0-25% Nonidet P40 in 0-15 M-sucrose/0-2M-phosphate, pH7-5. Thelysate was stained with Hoechst 33258and examined in suspension byfluorescence microscopy. Thechromosomes showed a dramaticallyextended appearance (B,C) ascompared to their initial shape (A).Magnifications in A, B and C are thesame. Bar, 10;/m. (Courtesy of Dr R.Hancock; see also Hancock & Dessev(1988).)


Fig. 11. Mitotic chromosomes from CIIO cells prepared in a polyamine-containing medium by lysis with Nonidet P40.Lithium diioclosalicylate was added to a final concentration of 25 mM and the chromosomes were immediately examined byfluorescent staining with Hoechst 33258, showing that chromatids are wound in helical gyres. Bar, lOjUm. (Courtesy of DrR. Hancock; see also Hancock & Dessev (1988).)

This study was supported by the Netherlands CancerFoundation, grant no. NUKC 1984-11. The authors thankProfessor Dr W. W. Franke (Heidelberg, FRG) for provid-ing Fig. 2, Drs G. Brakenhoff and Dr R. van Driel(Amsterdam, The Netherlands) for Fig. 3, Dr V. Bibor-Hardy (Sherbrooke/Montreal, Canada) for Fig. 4, Drs A.Cidadao and M. Carmo-Fonseca (Oeiras, Portugal) for Fig.5, Drs E. G. Fey and S. Penman (Cambridge, USA) for Figs6 and 7, Dr W. C. Earnshaw (Baltimore, MD, USA) for Fig.9, and Dr R. Hancock (Quebec, Canada) for Figs 10 and 11.

We also acknowledge the kind gifts of the monoclonalantibodies 41CC4 (from Dr G. Warren; Heidelberg, FRG),2.73 (from Dr S. I loch; La Jolla, USA) and 4F4 (from Dr G.Dreyfuss; Evanston, USA).

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(Received 2 October 1987 - Accepted 25 January I9SS)

36 R. Verheijen el a\.

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