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Experimental Cell Research 251, 135–146 (1999)Article ID excr.1999.4490, available online at http://www.idealibrary.com on

The Intranuclear Movement of Balbiani Ring PremessengerRibonucleoprotein Particles

Om Prakash Singh, Birgitta Bjorkroth, Sergej Masich, Lars Wieslander,* and Bertil Daneholt1

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, SE-17177 Stockholm, Sweden;and *Department of Molecular Genome Research, Stockholm University, SE-10699 Stockholm, Sweden

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Specific premessenger ribonucleoprotein (pre-mRNP)articles, the Balbiani ring (BR) granules in the sali-ary glands of the dipteran Chironomus tentans, cane visualized in the electron microscope when theyssemble on the genes, move through nucleoplasm,nd bind to and translocate through the nuclear pores.s shown by BrUTP labeling and immunoelectron mi-roscopy, newly synthesized BR RNP particles, re-eased from the BR genes, appear early in all nucleo-lasmic regions of the cell nucleus and they saturatehe nucleoplasmic pool of BR particles after 2 h ofabelling. It is concluded that within the nucleus theR particles move randomly. Furthermore, estimatesf minimum diffusion coefficients for the BR particlesre compatible with the view that the particles diffusereely in the interchromosomal space, although it isot excluded that the random movement could belightly retarded. Once the particles get bound to theuclear pore complexes, they seem committed to trans-

ocation through the nuclear pores. © 1999 Academic Press

Key Words: heterogeneous nuclear ribonucleopro-eins; premessenger ribonucleoproteins; intranuclearransport; nucleocytoplasmic transport; electronicroscopy.

INTRODUCTION

The structure and organization of the cell nucleusffect the flow of premessenger RNAs (pre-mRNAs)rom the genes on the chromosomes to the nuclearores in the nuclear envelope. In the diploid nucleus,he individual chromosomes appear as more or lessondensed chromosome territories, which change con-ormation and location within the nucleus [1, 2]. Be-ween the chromosome territories there is a complexystem of interconnected channels [2–4]. The activeenes are located in the periphery of the chromosomeerritories [5, 6], and the completed pre-mRNAs areelivered as ribonucleoprotein (RNP) complexes [7]

1 To whom correspondence and reprint requests should be ad-ressed. Fax: 146-8-313529. E-mail: bertil.daneholt@cmb.ki.se.

135

ove through the network of channels and reach otheregions of the nucleus and eventually the nuclear poresor exit to the cytoplasm.

It has been observed that specific pre-mRNAs appearn fiber-like tracks from the site of synthesis towardhe periphery of the cell nucleus [8, 9]. It has beenrgued that these observations of accumulated pre-RNA indicate that the RNA is actively transported

long specific paths, guiding the RNA toward the pe-iphery of the nucleus [9] and even all the way towardhe nuclear envelope [8]. The notion that pre-mRNAppears in nuclear matrix preparations has also beenaken as an argument for the hypothesis that RNAoves along nuclear fibers [10, 11]. On the other hand,

t has been reported for a specific pre-mRNA, as well asor bulk polyadenylated nuclear pre-mRNA, that it isvenly distributed in the interchromatin space, sug-esting that upon release from the chromosomes, pre-RNA moves in a spatially uniform fashion through

he interchromatin space, and the basis for the mobil-ty is diffusion [4]. Thus, it remains to be establishedhether the intranuclear movement of pre-mRNA isirectional or random [12–14].Most studies of the behavior of specific mRNA tran-

cripts have been carried out by confocal microscopy inombination with in situ hybridization. The site ofranscription can be approximately identified, al-hough it is not possible at this resolution level torecisely demarcate the transcription unit, to decidehether there is an adjacent maturation/storage site,nd to determine where the transport pathway actu-lly starts. Furthermore, it is difficult in diploid cells toollow the flow through the complex interchromatinhannel system. Finally, the steady-state distributionf mRNA is analyzed, and thus the newly synthesizedNA is not specifically followed.Here we present an ultrastructural approach to

tudy the flow of a specific type of pre-mRNP particles,he Balbiani ring granules in the salivary glands of theipteran Chironomus tentans. The BR pre-mRNAs are5–40 kb in size, are only marginally reduced in sizepon splicing (four small introns) and encode large-

0014-4827/99 $30.00Copyright © 1999 by Academic Press

All rights of reproduction in any form reserved.

sized salivary polypeptides [15]. The transcripts areapovstuBkHmpilm

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136 SINGH ET AL.

ssociated with proteins to form the BR pre-mRNParticles (50 nm in diameter) [16]. The exceptional sizef the BR particles makes it feasible to study the indi-idual particles in the electron microscope during bothynthesis and transport [16–19]. Furthermore, the in-erchromosomal space in the salivary glands is conspic-ous and is easily demarcated. Finally, the kinetics ofR RNA synthesis and nucleocytoplasmic transport isnown from early radioactive labelling studies [20, 21].ere we demonstrate that the newly synthesized pre-RNA can be labeled by BrUTP and that labeled BR

articles can be revealed in the electron microscope bymmunoelectron microscopy. By varying the period ofabeling, we were able to follow the intranuclear move-

ent of the newly made BR particles.

MATERIAL AND METHODS

Material. C. tentans was reared as described by Case and Dane-olt [22]. Salivary glands from fourth instar larvae were usedhroughout the present study.

Incubation of salivary glands. Intact salivary glands were iso-ated from fourth instar larvae in hemolymph at 18°C. The bodyavity of the larvae was gently opened by a fine pair of scissors, andhe ligaments attaching the glands to the body wall were carefullyut to avoid damage to the glands. Glands were incubated at 18°C for2.5, 45, 90, and 120 min in 20 ml of hemolymph containing 4 mMrUTP (Sigma). The hemolymph was replenished after 60 min in-ubation. Control experiments were performed in parallel with norUTP added.Electron microscopy. The glands were fixed in 2% glutaraldehyde

or 2 h at 4°C and in 1% osmium tetroxide for 30 min at 4°C,ehydrated, and embedded in Agar 100 resin. Thin sections (60–75m) were cut by a Reichert Ultracut ultramicrotome and stainedith lead citrate and uranyl acetate. The specimens were examined

n a JEOL 100CX electron microscope. For further details, see Bjork-oth et al. [23].

Immunoelectron microscopy. After incubation in the presence ofrUTP, the salivary glands were processed for immunoelectron mi-roscopy as earlier described [24, 25]. In short, the glands were fixedith 4% paraformaldehyde and 0.1% glutaraldehyde for 25 min at

oom temperature, cryoprotected with 2.3 M sucrose, and frozen bymmersion in liquid nitrogen. Ultrathin cryosections were picked upn nickel grids (coated with formvar and coal) and processed formmunocytochemistry using an anti-BrdU antibody (diluted 1:10)Boehringer, Mannheim). The labeling was visualized with goat anti-ouse IgG 1 IgM (H1L) antibodies conjugated to 6-nm gold parti-

les (diluted 1:50) (Jackson Laboratories, West Grove, PA). The sec-ions were stained with uranyl acetate, embedded in polyvinyllcohol (9–10 kDa; Aldrich, Steinheim, Germany), and analyzed in aEOL 100CX electron microscope. The control specimens, which hadeen incubated in the absence of BrUTP, were processed in parallel.For semiquantitative analysis of the labeling of Balbiani ring (BR)

articles, each individual BR particle in the section of the cell nu-leus studied was classified as labeled or unlabeled. The BR particlesere analyzed on electron micrographs with a total magnification of0,0003, and only readily identified BR particles were taken intoccount. In the BRs themselves, only the RNP particles with aiameter of 40–50 nm were examined, i.e., those being almost fin-shed. A BR RNP particle was designated labeled if one or more goldarticles were detected on top of the particle or within a distance of0 nm. To present the data in a manner that was easy to survey, a

ed was scanned by a Hewlett-Packard scanner using the Adobehotoshop software program. Then, the position and labelling ofach BR particle, established at high magnification, was indicated onhe low-magnification micrograph as a red dot if labeled and as alue dot if unlabeled. The sections in the control experiments (witho BrUTP added during incubation) showed very few gold particles,nd virtually no labeled BR particles were detected (less than one perucleus).The diffusion coefficient of the BR particles was calculated from

he experimental data using the formula D $ l2/6t, where D standsor the diffusion coefficient (expressed as mm2/s), l is the mean dis-ance travelled by the particle, and t is the time. To calculate theredicted value for the diffusion coefficient of a spherical particleith a 50-nm-diameter we used the formula D 5 kT/6phr, where D

s the diffusion coefficient (expressed as mm2/s), k is Bolzmann’sonstant (138,054 3 10216 erg/°K), T is the absolute temperature, h ishe viscosity of the solvent, and r is the radius of the particle. In thisatter calculation of D, it has been assumed that the temperature is0°C and the intranuclear viscosity is 8.1 cP according to Lang et al.26].

Splicing test. The degree of splicing of BR RNA in BRs anducleoplasm was determined according to Bauren and Wieslander27]. In short, salivary glands were fixed in ethanol:acetic acid (3:1)or 30 min on ice. The glands were washed in 70% ethanol for 30 minn ice and transferred to ice-cold ethanol:glycerol (1:1). Subse-uently, the glands were put in an oil chamber, and BR1s from 30uclei and nucleoplasmic samples from five nuclei were isolated by ae Fonbrune micromanipulator. The microdissection was monitorednder a phase-contrast microscope. The samples were transferred to00 ml of of an extraction solution (20 mM Tris–HCl, pH 7.5, 1 mMDTA, 0.5% SDS, 100 mg/ml proteinase K, and 50 mg/ml yeast RNA),nd the extraction was carried out for 30 min at room temperatureollowed by phenol:chloroform and chloroform extractions and etha-ol precipitation. The nucleic acid pellet was dissolved in 50 ml ofNase I buffer (10 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 10 mMithiothreitol) containing RNasin (800 U/ml), RNase-free DNase I60 U/ml) was added, and the sample was incubated at 37°C for 20in. After phenol:chloroform and chloroform extractions, the RNAas precipitated in ethanol.RNA was reverse transcribed in a buffer containing 25 mM Tris–Cl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5M of each of the four NTPs, 200 U of reverse transcriptase (Super-cript, GIBCO BRL), and 0.0002 pmol of an oligodeoxynucleotiderimer in a final volume of 20 ml. The primer was 19 bases long andositioned 392–411 bases downstream of intron 3 in the BR1 pre-RNA. The reaction was carried out for 1 h at 37°C. The sample was

hen heated in boiling water for 5 min and transferred to ice. TheDNA reaction mixture (10 ml) was directly introduced into 100 ml ofPCR buffer. The final PCR solution consisted of 10 mM Tris–HCl

pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 200 mM of each of the fourNTPs, 0.4 mM of each primer, 0.04 mM kinased 59 primer, and 2.5

of Taq polymerase (Perkin–Elmer). The 59 primer was 18 basesong and corresponded to a sequence located 48–30 bases upstreamf intron 3 in the BR1 pre-mRNA. This primer was 32P-labeled by T4olynucleotide kinase. The 39 primer was 19 bases long and corre-ponded to a sequence located 360–379 bases downstream of intron; this intron is 67 bases in length. The PCR solutions were heatedor 30 s at 94°C followed by 26 cycles of 30 s at 55°C, 1 min at 72°, and0 s at 94°C. The samples were phenol:chloroform extracted once,oncentrated by ethanol precipitation, and dissolved in 10 ml ofoading buffer. The samples were heated in boiling water for 3 minefore loading onto a 6% polyacrylamide sequencing gel. After elec-rophoresis, the gel was dried and the radioactivity localized anduantitated by phosphorimage analysis (Molecular Dynamics).

RESULTS

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137INTRANUCLEAR MOVEMENT OF PRE-mRNP

isualization of Balbiani Ring Pre-mRNP Particlesduring Assembly and Transport

The active BR genes have been extensively studied,nd the formation and transport of the transcriptionroduct has been described in detail (Fig. 1H) [16–19].n the proximal region of an active gene, the nascentNPs appear as thick RNP fibers growing longer and

onger downstream the gene, while in the middle andistal regions the 59 end region of the RNP fiber iseing packed into a dense globular structure, whichncreases in diameter along the gene (Fig. 1H). In Fig.B, a distal segment of an active BR gene is displayed:he approximate position of the chromatin axis haseen indicated by a dashed line, and the growing,talked RNP granules radiate perpendicularly fromhe axis. Close to the end of the gene, the globularortion of the RNP particle attains a diameter of 50m, which is the same as the diameter of the completedR RNP granules that have been released into theucleoplasm (Fig. 1C). After being bound to the nuclearore complex, the RNP particle changes conformationnd passes through the central channel of the complexs an extended rod (Figs. 1D–1F). Concomitant withhe translocation through the pore, the tightly packedlementary RNP fibril is unfolding and enters the cy-oplasm as an extended structure (e.g., Fig. 1F), whichs immediately engaged in protein synthesis, and theolysomes become associated with the tubular endo-lasmic reticulum (Fig. 1G; see also Fig. 1H). Thus, its feasible to directly visualize the assembly, the in-ranuclear flow, and the nucleocytoplasmic transportf the BR pre-mRNP particle on the ultrastructuralevel.

he Flow of Newly Synthesized BR RNP Particles

We adopted a strategy to BrUTP-label newly synthe-ized RNA that was introduced by Wansink et al. [28]nd Jackson et al. [29] for immunocytochemical pur-oses. Isolated salivary glands were incubated for var-ous times (22.5–120 min) in the presence of the mod-fied nucleotide BrUTP, which entered the glands andas incorporated into newly synthesized RNA (cf. thefficient uptake of exogeneous ATP according to Ref.30]). The glands were fixed in formaldehyde and glu-araldehyde, cryosectioned according to standard pro-edures, and the distribution of BrUTP-labeled RNAas visualized in immunoelectron microscopy experi-ents by an anti-BrdU antibody followed by a gold-

onjugated goat anti-mouse antibody [24, 25]. It shoulde emphasized that pre-mRNP complexes in generalill be labeled, but as we can identify the BR particles

n the sections, we can selectively analyze the labelingf this specific pre-mRNP particle. As shown in Fig. 1,

rowing BR RNP particles were labeled (gold particlesre denoted by arrows). BR particles in the nucleo-lasm (C) and during translocation (D, E) were alsoeen labeled. Finally, gold particles were recorded inhe cytoplasm (G), although in this compartment, theabel attributed to BR RNA cannot be specifically de-

arcated. It should be pointed out that there weressentially no gold particles in the specimens whenrUTP was omitted from the incubation medium (inverage, far less than one BR particle per nucleus andection was labelled). The labeling was sensitive toctinomycin D, an inhibitor of RNA synthesis (data nothown). We conclude that the BrUTP has been readilyncorporated into BR pre-mRNA, and the BR RNA isvidently transported into the nucleoplasm andhrough the nuclear pores into the cytoplasm during a0-min incubation period. The level of incorporationeems appropriate to study the flow in a semiquanti-ative manner.

Although BrUTP-labeled RNA can be transported tohe cytoplasm in mammalian cells [28], it has beenrgued that splicing and transport is likely to be de-ective as bromoruridine-containing transcripts are notroperly spliced in vitro [2, 31]. It was, therefore, mostmportant to evaluate possible deleterious effects onhese parameters in the insect system studied here.uring the course of the incubation, the BRs main-

ained their size, which has proven a good criterion forhe level of transcription in the BRs. Within the BRshe number and distribution of the active genes as wells the structure and density of the growing RNP par-icles along the individual genes were approximatelyhe same throughout the experiment (cf. Fig. 1A). Fi-ally, the amount of BR1 RNA was measured after 90in incubation in the presence and absence of BrUTP,

nd no major differences were noted (see below). Thus,ll our data support the view that transcription con-inues essentially unchanged in the presence of therUTP.To evaluate whether the intranuclear flow and the

ucleocytoplasmic transport go on undisturbed in theresence of BrUTP, we determined whether the num-er of BR particles remained the same in the nucleo-lasm and in the stage of translocation after 90 minncubation in the presence of BrUTP (the control wasncubated in parallel but with no BrUTP added). Thelands were analyzed with conventional electron mi-roscopy providing optimal morphology, which was es-ecially important to clearly visualize and count theranslocating particles (Fig. 1F). The relative density ofhe nucleoplasmic particles remained essentially un-hanged (data not shown). The nuclear pore results areresented in Table 1. Both the BR particles docked tohe nuclear pore complex (D) and those translocatingT) were counted, and again no major differences were

138 SINGH ET AL.

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139INTRANUCLEAR MOVEMENT OF PRE-mRNP

oted between the glands incubated in the presencend in the absence of BrUTP. Finally, the D/T quo-ient, which is a sensitive parameter for disturbancesn the translocation process, was calculated and it wasbout the same in the BrUTP-labeled glands and in theontrol (4.5 vs 4.2). We conclude that the morphologicalata obtained, in combination with the kinetics ofrUTP-labelling described above (see also further be-

ow), suggest that the transport of BR particles is es-entially unaffected by the presence of BrUTP duringhe incubation of salivary glands in the time spantudied (0–120 min).Finally, the efficiency of splicing was determined

sing the approach devised by Bauren and Wieslander27]. One of the BRs, BR1, and a sample of nucleoplasmere dissected by micromanipulation from fixed sali-ary glands, the RNA was extracted, and the relativemounts of unspliced and spliced RNA was measuredy an RT-PCR approach. The primers were chosen tovaluate the removal of intron 3 in the BR1 transcript.he PCR products were separated in a polyacrylamideequencing gel and were detected and quantitated by ahosphorimager (Fig. 2). The position in the gel of annspliced product was determined by analysis of aCR product from DNA (lane 1), and the position of apliced product by analysis of a RT-PCR product fromytoplasmic RNA (lane 2). The BR samples fromrUTP-labeled and control glands are shown in lanes 3nd 4, respectively. Most of the BR1 RNA in the BRsas already spliced within the BRs and to about the

FIG. 1. Immunoelectron microscopy of BR particles. (A) Segmentarticles attached to the chromatin axis. The position of the chromaarticles have been indicated by open arrows. (D–E) BR particleslastic-embedded material (added for comparison). (G) Cytoplasm.cription, during transport through the nucleoplasm, and during tRNA active in protein synthesis on the endoplasmic reticulum.

hromosome; N, nucleus; NE, nuclear envelope; C, cytoplasm; ER, endicated by small arrows. Bar: 1 mm in A and 100 nm in B–G.

elative Number of Docked and Translocating BR Particlesin BrUTP and Control Experiments

Dockedparticles (D)

Translocatingparticles (T) D/T

rUTP 46 10 4.6ontrol 38 9 4.2

Note. Sections through nine nuclei from three animals were ana-yzed. For each nucleus, the number of docked (D) and translocatingT) particles was determined as well as the length of the nuclearnvelope analyzed. The particle frequencies were expressed as theumber of particles per 100 mm of nuclear envelope. The averagealues for the nine nuclei were calculated as well as the D/T quo-ients.

ame extent in BrUTP-labeled glands (77%) and inontrol glands (88%). Thus, the splicing of intron 3ccurs mainly cotranscriptionally in both cases, whichs in agreement with the data of Bauren and Wies-ander [27]. When the reverse transcriptase was ex-luded from the reaction mixture, no RT-PCR productsere obtained (lanes 5 and 6). Finally, when the nu-

leoplasmic sample from BrUTP-labeled glands (lane) was compared with that from control glands (lane 8),e noted that almost all BR1 RNA molecules were

pliced in both the BrUTP-labeled glands (96%) andhe control glands (98%), again in agreement with theesults of Bauren and Wieslander [27]. Thus, splicingf BR RNA proceeds with the same kinetics and at theame location in BrUTP-labeled glands as in controllands.We conclude that transcription, splicing, and trans-

ort of BR pre-mRNP particles continue essentiallynchanged in BrUTP-labeled salivary glands. An anal-sis of the distribution of BR UTP-labeled BR RNParticles should make it possible to follow the move-ent of the BR particles from their site of synthesis,

ia nucleoplasm, through the nuclear pores, and intoytoplasm.

inetic Analysis of the Movements of the BR Particles

To follow the flow of BrUTP-labeled BR RNP parti-les from the site of synthesis, the BRs, through the

the cell nucleus. (B) Portion of an active BR gene with growing RNPaxis is indicated by a dashed line. (C) Nucleoplasm. Unlabeled BR

transit through nuclear pores. (F) Translocating BR particle fromSchematic drawing showing BR pre-mRNP particles during tran-

slocation through the nuclear pore and finally the cytoplasmic BRfollowing designations are used: BR, Balbiani ring; PC, polyteneplasmic reticulum. Gold particles bound to BR particles have been

FIG. 2. Splicing of BrUTP-labeled pre-mRNA from BR1. Lanes 1nd 2 show size markers: lane 1, the unspliced product (PCR ofNA); lane 2, the spliced product (RT-PCR of cytoplasmic RNA). TheT-PCR analyses of BR1 pre-mRNA isolated from 30 microdissectedR1s are presented in lanes 3–6: lane 3, BrUTP; lane 4, no BrUTP;

anes 5 and 6, as lanes 3 and 4, respectively, but no reverse tran-criptase added. The RT-PCR analyses of BR1 pre-mRNA from mi-rodissected nucleoplasm from five nuclei are presented in lanes 7nd 8: lane 7, BrUTP; lane 8, no BrUTP.

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140 SINGH ET AL.

ucleoplasm and to and through the nuclear pores, thealivary glands were incubated in the presence ofrUTP for 22.5, 45, 90, and 120 min. For each time

FIG. 3. Nuclear distribution of labeled (red) and unlabeled (blue2.5 min incorporation. A few labeled BR particles distant from the Bin incoporation. (D) 120 min incorporation. The following designucleolus; N, nucleus; C, cytoplasm. The BRs are encircled to better slectron micrograph has been brightened to better bring out the pos

oint two experiments were carried out, and one cellucleus in each experiment was analyzed in detail; i.e.,ach BR RNP particle in a central section through the

R RNP particles after different periods of BrUTP incorporation. (A)have been denoted by small arrows. (B) 45 min incorporation. (C) 90ons are used: BR, Balbiani ring; PC, polytene chromosomes; Nu,

their demarcation toward the nucleoplasm. The nucleoplasm of thens of the colored BR particles. Bars, 5 mm.

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141INTRANUCLEAR MOVEMENT OF PRE-mRNP

FIG. 3—Continued

nucleus was scrutinized and classified as labeled orutpclo3ttgRaTa

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142 SINGH ET AL.

nlabeled. Within the BRs, only BR RNP particles inhe distal region of the BR genes (almost completedarticles) were investigated, as their labeling intensityorresponded most closely to the finished product de-ivered to the nucleoplasm. The analysis was carriedut at a total magnification of about 30,0003, and0–40 partly overlapping micrographs were used forhe analysis of each single nucleus. The results werehen transferred to a low-magnification electron micro-raph showing the whole nucleus, each labeled BRNP particle being pointed out at its proper position asred dot and each nonlabeled one as a blue dot (Fig. 3).he two experiments for each time point were in goodgreement with each other.In the low-magnification electron micrographs pre-

ented in Fig. 3, the cell nucleus (N) is clearly demar-ated from the surrounding cytoplasm (C) in each of theour cells. Furthermore, in each nucleus, one prominentarge Balbiani ring (BR), portions of one or more of theour polytene chromosomes (PC), and large areas of sur-ounding nucleoplasm can be discerned. In three of thelectron micrographs presented (Figs. 3B–3D), there islso a densely stained nucleolus, which is always closelyonnected to a large chromosome (clearly revealed inig. 3C). It should be emphasized in this context that

n salivary gland nuclei the chromatin material, i.e.,he polytene chromosomes, is clearly separated fromurrounding nucleoplasm, and the nucleoplasm com-rises large regions within the nucleus, permittingnalysis of the flow of the BR RNP particles.After 22.5 min incubation, the labeled BR RNP par-

icles were essentially confined to the BRs but someppeared in the nucleoplasm (Fig. 3A). Labeled parti-les were seen distant from the BR (arrows in Fig. 3A),ome of them even present in the opposite pole of theell nucleus. As the labeling in the control was veryow, each dot is significant in the BrUTP experiments.fter 45 min of incubation, the labeling in the BR had

ncreased slightly, but the most drastic change washat the number of labeled BR particles had now in-reased in the nucleoplasm and they were spread allver the nucleus (Fig. 3B). There was no distinct label-ng gradient from the BR and outward into the nucle-plasm. As the increase in nucleoplasmic labeling wasbout fourfold, by far most of the labeled BR particlesecorded after 45 min represent those that have left theRs during the last 22.5 min of the experiment. Thus,

he even distribution observed after 45 min stronglyupport the conclusion, based on the 22.5-min experi-ent, that BR particles reach all nucleoplasmic re-

ions within 22.5 min. When the incubation was ex-ended to 90 min, the result was essentially the sames that for 45 min: There was no further increase inabeling within the BR, but the relative labeling wastill increasing in the nucleoplasm (Fig. 3C). Finally,

her change in labeling in either the BR or the nucle-plasm (Fig. 3D).To further describe the distribution of the labeled

nd unlabeled BR particles, at least in a semiquanti-ative manner, the labeled and unlabeled particlesere counted in the BR and in six spherical zones

urrounding the BR (Fig. 4). The increment in radiusetween adjacent zones was 5–6 mm. It should beoted that the large BR RNP particles are generatednly in BR1 and BR2, which are located close togethern chromosome IV. All BR particles are, therefore,ynthesized from essentially the same region of theucleus. The results are presented in Table 2 and ex-ressed as percentages of labeled BR particles out of allR particles recorded in the BR or the zone studied. BRarticles translocating through the pores were few andften difficult to identify in the cryosections (less clearorphology relative to plastic sections; Figs. 3D and

E vs 3F) and were, therefore, not suitable for quanti-ative analysis.

The labeling of the BR particles was high alreadyfter 22.5 min incubation (47%) and reached a plateauefore 45 min (55–60%), probably as early as after 30in incubation. As it is known that the transcription

ime for a BR transcript is 20 min [32], these resultsuggested that the time needed to saturate the BrUTPool was less than 10 min. Furthermore, the data sup-orted the notion that the finished BR products are nottored in the BRs but are released into the nucleoplasmmmediately upon synthesis [20]. The high degree ofabeling (55–60%) suggested that essentially all BRarticles contained BrUTP-labeled RNA, as many par-icles are not likely to be recorded as labeled due to theimitations of the immunoelectron microscopy tech-ique (e.g., all particles are not superficial in the sec-ions, and all BrUTP residues in the particles are notccessible to the primary antibody). We conclude thatfter 30 min incubation, the BRs deliver BR particleso the nucleoplasm, and these newly synthesized nu-leoplasmic particles are likely to exhibit a labelingrequency on the order of 55%.

Labeled BR particles were recorded in the nucleo-lasm already after the shortest incubation period22.5 min). As there is no storage of newly synthe-ized BR particles within the BRs [20], it is to bexpected that BrUTP-labeled particles appear in theucleoplasm soon after the start of the experiment.fter 22.5 min incubation, we found that 2–3% of theR particles were labeled in the nucleoplasm, after5 min about 10%, after 90 min 35– 40%, and after20 min about 50% (Table 1). Thus, the labeling after20 min was close to the 55% expected when all theR particles are fully labeled and have been synthe-ized during the experiment. Evidently, the labelingas increasing up to about 90 min and was then

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143INTRANUCLEAR MOVEMENT OF PRE-mRNP

apidly leveling off. It should be noted that the pro-ortion of labeled BR particles is underestimatedfter 22.5 min, and to a lesser extent after 45 min,ue to the fact that the released particles were notully labeled until after 30 min incubation (saturatedrUTP pool and the entire transcripts labeled). Weonclude that after 120 min incubation essentiallyll BR particles present in the nucleoplasm have

FIG. 4. Nucleoplasmic zones used in the quantitative analysis ofucleolus. The numbers indicate the different zones studied. The re

TABLE 2

Labeled BR Particles as Percentages of Total BR Particlesn BRs and in Six Nucleoplasmic Zones after Different Timesf BrUTP Incorporation

Zone

Incubation time (min)

22.5 45 90 120

BR 47 58 60 541 4 10 39 462 5 9 36 563 1 13 37 504 0 8 39 515 3 6 32 506 0 7 36 NDa

Note. Four nuclei have been analyzed, one for each time point. Theuclei were chosen for comparison on the basis of a similar periph-ral location of the BR (cf. Fig. 4).

a Not determined due to small size of the nucleus.

een synthesized during the incubation, and the BRarticles present at the start of the experiment hadeen transported to cytoplasm or have been de-raded.In conclusion, already during the shortest time of

ncubation (22.5 min), the few labeled BR particlesecorded appeared in many regions of the cell and hadeached also distant parts of the cell in relation to theosition of the BR (Fig. 3A). After 45 min and longer,hen many more labeled BR particles are present, it isvident that they were distributed all over the cell andn all zones studied (Fig. 4). Thus, the most strikingesult is that BR particles released from the BRs moveapidly within the cell nucleus and appear early in allegions of the nucleus. It is obvious that newly madearticles rapidly intermingle with older ones in a nu-leoplasmic pool of BR particles. After 120 min incuba-ion, essentially all the nucleoplasmic particles presentt the outset of the experiment had turned over, andost of them, perhaps all, had been exported to the

ytoplasm (cf. Ref. [21]).

DISCUSSION

he Intranuclear Movement of BR Pre-mRNPParticles Is Random

In the present study we have analyzed the intranu-lear flow of newly synthesized BR pre-mRNP particles

distribution of BrUTP-labeled BR particles. BR, Balbiani ring; Nu,ts are presented in Table 2.

thesul

generated in the BRs. The BR particles proved to beermqsotpdApcrrapn

phpsal(ttpeptiitfTomet

sfmrisnsetor

Is the Random Movement of BR Pre-mRNP Particles

nisittwttbsdm5ltatsop5uptbcsicimcl5atpfwiitmfAtces

t

144 SINGH ET AL.

fficiently labeled with BrUTP and could be readilyecorded in various regions of the nucleoplasm. Theovements of the BR particles could be at least semi-

uantitatively studied by a zonal analysis. The mosttriking result is that the BR particles appeared notnly in proximal nucleoplasmic regions but also in dis-al nucleoplasmic regions even after the shortest incor-oration time (22.5 min); no distinct concentration gra-ient of the newly made particles could be detected.long with additional production of BrUTP-labeled BRarticles, the proportion of labeled BR particles in-reased in parallel in all regions of the nucleoplasm toeach a plateau after 120 min incorporation. Thus, theesults strongly support the view that the completednd released BR particles join a large nucleoplasmicool of BR particles, and they move in a random man-er within the cell nucleus.Our analysis of the intranuclear movement of BR

articles is in good agreement with a study by Bing-am and co-workers [4], who investigated steady-statere-mRNA in polytene nuclei in Drosophila using initu hybridization and confocal microscopy. Zachar etl. [4] demonstrated that a specific pre-mRNA wasocated at a high concentration at the transcription siteprimary zone) and at a lower concentration evenlyhroughout the interchromatin space (designated ex-rachromosomal channel network). Furthermore, bulkolyadenylated nuclear pre-mRNA showed the sameven extrachromosomal distribution, indicating thatre-mRNA in general is likely to be located throughouthe interchromatin space. Finally, they noted that thenterchromatin space had a channel-like organizationn diploid imaginal disc nuclei similar to that in poly-ene salivary gland nuclei, suggesting that the space isunctionally equivalent in the two classes of nuclei.hus, the confocal study of steady-state pre-mRNA andur own ultrastructural and kinetic analysis of theovement of newly synthesized BR particles are in

xcellent agreement, and they suggest that the in-ranuclear mobility of pre-mRNP is random.

It should be emphasized that the large interchromo-omal space in the C. tentans salivary gland cells of-ered exceptional possibilities to identify a directed

ovement from the site of synthesis, the Balbianiings, toward the periphery of the cell nucleus, shouldt be present. However, we did not see newly synthe-ized BR particles following one or a few specific tracks,or did we see a front of newly made particles movinglowly toward the nuclear envelope. Thus, we found novidence for a directed movement of BR particles fromhe BR genes to the nuclear pores. A complex, but stillrdered movement is not formally excluded, but weegard this alternative as less likely.

Free or Restricted?

Our study does not provide direct information on theature of the random movement. However, it seemed

mportant to us to try to evaluate whether free diffu-ion alone could account for the results obtained and,n addition, to consider whether intranuclear struc-ures could influence the movement. First, supposinghat the BR particles diffuse freely in the nucleoplasm,e made two calculations of the diffusion rate based on

he experimental data available. If we assume that itakes 22.5 min for newly synthesized BR particles toecome evenly distributed within the nucleus by diffu-ion (Fig. 3A; see also the interpretation above of theata from the 45-min experiment), the mean displace-ent would be $25 mm (the diameter of the nucleus is

0 mm; it should be noted that the BRs are usually notocated in the center of the nucleus), which correspondso a diffusion coefficient of $0.08 mm2/s (see Materialsnd Methods). A more realistic minimum estimate ofhe diffusion coefficient would be obtained if it is as-umed that the BR particles are distributed through-ut the nucleus in 15 min, as the BR particles arerobably not detectable in the nucleoplasm until after–10 min incubation due to the time required to buildp the BrUTP pool and to transcribe a substantialortion of the gene. Under the assumption mentioned,he diffusion coefficient for the BR particle would thene $0.12 mm2/s. The two minimum estimates should beompared with the diffusion coefficient predicted for apherical particle, 50 nm in diameter, diffusing freelyn solution. Assuming an intranuclear viscosity of 8.1p [26], the predicted coefficient for the 50 nm particles 1.1 um2/s (see Materials and Methods). Thus, the

inimum diffusion coefficients for the BR particlesalculated on the basis of the experimental data areower than the predicted diffusion coefficient for a0-nm particle diffusing freely in solution, but only byfactor of about 10. We conclude that our estimates of

he diffusion coefficient for the BR particles are com-atible with the view that the BR particles diffusereely in the interchromatin space. Such a conclusionould be in good agreement with a recent study of the

ntranuclear movement of fluorescein-labeled oligo(dT)ntroduced into rat myoblasts [33]. A large fraction ofhe oligo(dT), probably bound to hnRNP particles,oved at diffusion rates comparable to that predicted

or average-sized hnRNP particles in aqueous solution.minor fraction moved 10-fold more slowly, indicating

hat it was associated with very large macromolecularomplexes. In the case of the BR particles, it is notxcluded that the random movement could also be re-tricted to some extent.If steric hindrance does influence the movement of

he BR particles, a few specific options have to be

considered. Due to the vast interchromatin space in thesmtcsrfpdatHsutpmbcol[afhrt

cnutccstdwpw

Bto[tnttrt[

e

Mehlin for professional assistance with Fig. 3. The work was sup-padrt

1

1

1

1

1

1

1

1

1

145INTRANUCLEAR MOVEMENT OF PRE-mRNP

alivary gland nuclei, it seems likely that the move-ent of the BR particles is only marginally affected by

he four large polytene chromosomes and the two nu-leoli. It is, however, possible that the interchromatinpace contains a delicate fibrous network, which couldestrict the movement of the BR particles. It is knownrom early micromanipulation and microinjection ex-eriments that the polytene chromosomes are embed-ed in a gel [34]. For example, when a small volume ofsuspension of carbon particles is injected into the gel,

he particles remain confined to the site of injection.owever, the particles disperse quickly when the gel is

olubilized, which can be accomplished by agitationsing a microneedle. As actin is an abundant protein inhe nucleoplasm of salivary gland cells (our own un-ublished data), it is possible that the gel consists of aeshwork of short actin filaments. Such an actin-

ased gel has, in fact, been identified and carefullyharacterized in the large-sized nuclei of amphibianocytes [35], and presumably it exists in somatic dip-oid cells as well (for discussion and references, see Ref.36]). It is not excluded that the interchromatin spacelso contains a more stable fibrous network, often re-erred to as nuclear matrix [10]. Future experimentsave to be specifically designed to decide whether theandom movement is restricted by a fibrous network inhe interchromatin space.

The BR particles are distributed in all regions of theell nucleus, and they seem to translocate throughuclear pores in all areas of the nuclear envelope [19;npublished data]. There is, therefore, no evidence forhe gene gating model, i.e., that a specific active gene isonnected to one or a limited number of adjacent nu-lear pores and that the gene transcripts exit exclu-ively through these nearby pores [37]. The observa-ion that the BR particles pass through poresistributed all along the envelope is in good agreementith the general notion that all nuclear pores are ca-able of both import and export of macromoleculesith no obvious specialization [38].It is still an open question how the randomly movingR particles in the nucleoplasm are selected for export

o the cytoplasm. Most or all BR particles in the nucle-plasm will eventually be transferred to the cytoplasm21]. Presumably, the BR particles are picked out forransport in a stochastic fashion and, once bound touclear pore complexes, they are committed for exit tohe cytoplasm. The molecular mechanisms involved inhe recruitment of the BR particles for transport, theecognition of the particles at the pore complex, andhe translocation proper are still essentially unknown16; see also Ref. 39].

We thank Professor Reiner Peters for valuable advice regardingstimation of diffusion coefficients. We are grateful to Dr. Hans

orted by the Swedish Natural Science Research Council, the Knutnd Alice Wallenberg Foundation, the Kjell and Marta Beijer Foun-ation, and the Gunvor and Josef Aner Foundation. O.P.S. was aecipient of a Guest Research Fellowship from the Karolinska Insti-ute and S.M. of a Research Fellowship from the Swedish Institute.

REFERENCES

1. Cremer, T., Kurz, A., Zirbel, R., Dietzel, S., Rinke, B., Schrock,E., Speicher, M. R., Mathieu, U., Jauch, A., Emmerich, P.,Scherthan, H., Ried, T., Cremer, C., and Lichter, P. (1973). Roleof chromosome territories in the functional compartmentaliza-tion of the cell nucleus. Cold Spring Harbor. Symp. Quant. Biol.58, 777–792.

2. van Driel, R., Wansink, D. G., van Steensel, B., Grande, M. A.,Schul, W., and de Jong, L. (1995). Nuclear domains and thenuclear matrix. Int. Rev. Cytol. 162A, 151–188.

3. Spector, D. L. (1990). Higher order nuclear organization: Three-dimensional distribution of small nuclear ribonucleoproteinparticles. Proc. Natl. Acad. Sci. USA 87, 147–151.

4. Zachar, Z., Kramer, J., Mims, I. P., and Bingham, P. M. (1993).Evidence for channeled diffusion of pre-mRNAs during nuclearRNA transport in metazoans. J. Cell Biol. 121, 729–742.

5. Fakan, S., Puvion, E., and Spohr, G. (1976). Localization andcharacterization of newly synthesized nuclear RNA in isolatedrat hepatocytes. Exp. Cell Res. 99, 155–164.

6. Kurz, A., Lampel, S., Nickolenko, J. E., Bradl, J., Benner, A.,Zirbel, R. M., Cremer, T., and Lichter, P. (1996). Active andinactive genes localize preferentially in the periphery of chro-mosome territories. J. Cell Biol. 135, 1195–1205.

7. Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G.(1993). hnRNP proteins and the biogenesis of mRNA. Annu.Rev. Biochem. 62, 289–321.

8. Huang, S., and Spector, D. L. (1991). Nascent pre-mRNA tran-scripts are associated with nuclear regions enriched in splicingfactors. Genes Dev. 5, 2288–2302.

9. Xing, Y., Johnson, C. V., Dobner, P. R., and Lawrence, J. B.(1993). Higher level organization of individual gene transcrip-tion and RNA splicing. Science 259, 1326–1330.

0. Berezney, R., Mortillaro, M. J., Ma, H., Wei, X., and Samara-bandu, J. (1995). The nuclear matrix: A structural milieu forgenomic function. Int. Rev. Cytol. 162A, 1–65.

1. Agutter, P. S. (1995). Intracellular structure and nucleocyto-plasmic transport. Int. Rev. Cytol. 162B, 183–223.

2. Rosbash, M., and Singer, R. H. (1993). RNA travel: Tracks fromDNA to cytoplasm. Cell 75, 399–401.

3. Xing, Y., Johnson, C. V., Moen, P. T., Jr., McNeil, J. A., andLawrence, J. B. (1995). Nonrandom gene organization: Struc-tural arrangements of specific pre-mRNA transcription andsplicing with SC-35 domains. J. Cell Biol. 131, 1635–1647.

4. Strouboulis, J., and Wolffe, A. P. (1996). Functional compart-mentalization of the nucleus. J. Cell Sci. 109, 1991–2000.

5. Wieslander, L. (1994). The Balbiani ring multigene family:Coding repetitive sequences and evolution of a tissue specificcell function. Progr. Nucleic Acids Res. Mol. Biol. 48, 275–313.

6. Daneholt, B. (1997). A look at messenger RNP moving throughthe nuclear pore. Cell 88, 585–588.

7. Skoglund, U., Andersson, K., Bjorkroth, B., Lamb, M. M., andDaneholt, B. (1983). Visualization of the formation and trans-port of a specific hnRNP particle. Cell 34, 847–855.

8. Skoglund, U., Andersson, K., Strandberg, B., and Daneholt, B.(1986). Three-dimensional structure of a specific pre-messenger

RNP particle established by electron microscope tomography.

1

2

2

2

2

2

2

2

2

2

29. Jackson, D. A., Hassan, A. B., Errington, R. J., and Cook, P. R.

3

3

3

3

3

3

3

3

3

3

R

146 SINGH ET AL.

Nature 319, 560–564.9. Mehlin, H., Daneholt, B., and Skoglund, U. (1992). Transloca-

tion of a specific premessenger ribonucleoprotein particlethrough the nuclear pore studied with electron microscope to-mography. Cell 69, 605–613.

0. Daneholt, B. (1975). Transcription in polytene chromosomes.Cell 4, 1–9.

1. Edstrom, J.-E., Ericson, E., Lindgren, S., Lonn, U., and Ryd-lander, L. (1977). Fate of Balbiani-ring RNA in vivo. ColdSpring Harbor Symp. Quant. Biol. 42, 877–884.

2. Case, S. T., and Daneholt, B. (1978). The size of the transcrip-tion unit in Balbiani ring 2 of Chironomus tentans as derivedfrom analysis of the primary transcript and 75S RNA. J. Mol.Biol. 124, 223–241.

3. Bjorkroth, B., Ericsson, C., Lamb, M. M., and Daneholt, B.(1988). Structure of the chromatin axis during transcription.Chromosoma 96, 333–340.

4. Tokuyasu, K. (1980). Immunocytochemistry on ultrathin frozensections. J. Histochem. 12, 381–403.

5. Visa, N., Alzhanova-Ericsson, A., Sun, X., Kiseleva, E., Bjork-roth, B., Wurtz, T., and Daneholt, B. (1996). A pre-mRNA-binding protein accompanies the RNA from the gene throughthe nuclear pores and into polysomes. Cell 84, 253–264.

6. Lang, I., Scholz, M., and Peters, R. (1986). Molecular mobilityand nucleocytoplasmic flux in hepatoma cells. J. Cell Biol. 102,1183–1190.

7. Bauren, G., and Wieslander, L. (1994). Splicing of Balbiani ring1 gene pre-mRNA occurs simultaneously with transcription.Cell 76, 183–192.

8. Wansink, D. G., Schul, W., van der Kraan, I., van Steensel, B.,van Driel, R., and de Jong, L. (1993). Fluorescent labeling ofnascent RNA reveals transcription by RNA polymerase II indomains scattered throughout the nucleus. J. Cell Biol. 122,283–293.

eceived March 16, 1999

(1993). Visualization of focal sites of transcription within hu-man nuclei. EMBO J. 12, 1059–1065.

0. Egyhazi, E., Pigon, A., and Rydlander, L. (1982). 5,6-Dichlorori-bofuranosyl-benzimidazole inhibits the rate of transcription ini-tiation in intact Chironomus cells. Eur. J. Biochem. 122, 445–451.

1. Fay, F. S., Taneja, K. L., Shenoy, S., Lifshitz, L., and Singer,R. H. (1997). Quantitative digital analysis of diffuse and con-centrated nuclear distributions of nascent transcripts, SC35and poly(A). Exp. Cell Res. 231, 27–37.

2. Egyhazi, E. (1976). Quantitation of turnover and export to thecytoplasm of hnRNA transcribed in the Balbiani rings. Cell 7,507–515.

3. Politz, J. C., Browne, E. S., Wolf, D. E., and Pederson, T. (1998).Intranuclear diffusion and hybridization state of oligonucleo-tides measured by fluorescence correlation spectroscopy in liv-ing cells. Proc. Natl. Acad. Sci. USA 95, 6043–6048.

4. D’Angelo, E. G. (1946). Micrurgical studies on Chironomus sal-ivary gland chromosomes. Biol. Bull. 90, 71–87.

5. Clark, T. G., and Merriam, R. W. (1977). Diffusible and boundactin in nuclei of Xenopus laevis oocytes. Cell 12, 883–891.

6. Le Stourgeon, W. M. (1978). The occurrence of contractile pro-teins in nuclei and their possible functions. In “The Cell Nu-cleus” (H. Busch, Ed.), Vol. VI, pp. 305–326. Academic Press,New York.

7. Blobel, G. (1985). Gene gating: A hypothesis. Proc. Natl. Acad.Sci. USA 82, 8527–8529.

8. Dworetzky, S. I., and Feldherr, C. M. (1988). Translocation ofRNA-coated gold particles through the nuclear pores of oocytes.J. Cell Biol. 106, 575–584.

9. Ohno, M., Fornerod, M., and Mattaj, I. W. (1998). Nucleocyto-plasmic transport: The last 200 nanometers. Cell 92, 327–336.