Close coupling between transcription and exit of mRNP from the cell nucleus

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Close coupling between transcription and exit of mRNP from the cell nucleus Karin Kylberg, Birgitta Björkroth, Birgitta Ivarsson, Nathalie Fomproix, Bertil Daneholt⁎ Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, SE-171 77 Stockholm, Sweden

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Transcription is intimately coupled to co-transcriptional formation of mRNP particles and their

Received 3 September 2007

preparation for export. In the dipteran Chironomus tentans we have now investigated whether on-

Revised version received

going transcription is closely linked also to the ensuing transfer of the mRNPs from genes to

5 February 2008

cytoplasm. The assembly and nucleocytoplasmic transport of a specific mRNP particle, the

Accepted 8 February 2008

Balbiani ring (BR) RNP granule, were visualized in larval salivary glands by electron microscopy.

Available online 10 March 2008

When transcription was inhibited with DRB or actinomycin D (AMD), the growing BR mRNPs disappeared from the genes. The two inhibitors affected the distribution of BR mRNPs in the

Keywords:

nucleoplasm and in the nuclear pores in essentially the same way. At the nuclear pore

mRNP particles

complexes (NPCs) the basket-associated and translocating mRNPs were substantially reduced in

mRNA export

number, the translocating RNPs being essentially absent after 90 min treatment. Remarkably,

Nucleocytoplasmic transport

the amount of BR mRNPs in the nucleoplasm did not change. We conclude that on-going

Nuclear pore complex

transcription is required for the mRNPs to exit from the cell nucleus. Interruption of transcription

Nuclear basket

seems to primarily affect the intranuclear movement of BR mRNPs and/or prevent the binding of

Transcription

mRNPs to the NPCs rather than to directly interfere with translocation per se.

Electron microscopy

Introduction Messenger ribonucleoprotein (mRNP) complexes are formed on the chromosomes and transfer the genetic information from the genes in the cell nucleus to the polysomes in the cytoplasm [1–4]. The RNA component is synthesized as a precursor, pre-mRNA, by RNA polymerase II (RNA pol II) [5]. When the pre-mRNA emerges from the polymerase, it is immediately coupled to the proteins to form a ribonucleoprotein (RNP) fibril, 5 to 10 nm in diameter [6–8]. If the transcript is long, the fibril can be further packed into an RNP granule. The RNA-associated proteins mainly consist of members of two protein families: heterogeneous nuclear RNP (hnRNP) proteins [4] and serine-argininerich (SR) proteins [9]. The organization of the various proteins

© 2008 Elsevier Inc. All rights reserved.

along the elementary pre-mRNP fibril is non-random [4], but little is known about the more precise arrangement of the proteins. The pre-mRNP complex is extensively remodeled in conjunction with the capping, splicing and polyadenylation of the primary transcript, which takes place almost exclusively on or in the immediate vicinity of the gene [10–14]. The chemical reactions are carried out by assemblies of proteins, often called molecular machines, that vary considerably in complexity. Capping requires three enzymes, splicing more than 100 proteins (the spliceosome) and cleavage/polyadenylation 5–10 proteins. However, recent work has shown that within the cell the molecular machines mentioned do not work as independent entities but interact closely with the transcription machinery,

⁎ Corresponding author. Fax: +46 8 31 35 29. E-mail address: [email protected] (B. Daneholt). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.02.003

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and also to some extent with each other [10,11,15,16]. This interplay permits a sophisticated regulation of the formation of the mature mRNP complex on the chromosomal level. RNA pol II influences the pre-mRNA processing steps by various mechanisms. The C-terminal domain (CTD) of the RNA polymerase II plays a particularly important role [5,10,13,17]. In the preinitiation complex, when bound to promoter-associated proteins, the CTD is hypophosphorylated while in the active elongating state it becomes hyperphosphorylated. In the latter state the CTD adopts a more extended shape, which facilitates the interaction of the CTD with the growing transcript, and delivery of processing factors. In fact, capping, splicing as well as polyadenylation depend on the inflow of proteins to mRNA via the CTD-tail. In addition, the transcription machinery can recruit processing factors to the pre-mRNA via interactions with transcription initiation factors and transcription elongation factors (for discussion, see Refs. [10,15]). The mRNPs are also prepared for export concomitant with transcription. The key export factor is the export receptor, Mex67: Mtr2 in yeast and NXF1(TAP):p15 in metazoans, which enables the mRNP particles to be transported to and through the nuclear pores [18,19]. The receptor is coupled co-transcriptionally to the mRNP by an evolutionarily conserved transport complex, called TREX, which is composed of a multisubunit THO complex, the RNA helicase UAP56 and the adaptor protein Aly/REF (in yeast the latter two are designated Sub2 and Yra1, respectively) [20,21]. In yeast, the THO complex is bound to the RNA polymerase and recruits Sub2 and later on Yra1 to the transcription machinery. Sub2 and Yra1 are transferred to the nascent mRNP, and the export receptor Mex67:Mtr2 binds to the mRNP via the adaptor Yra1. In metazoan, the receptor-loading process is analogous but on intron-containing genes the THO complex is not associated with the polymerase but exerts its function as part of spliceosomes. UAP56 and Aly/REF are delivered to the spliced mRNP and Aly/REF recruits the export receptor NXF1:p15. Intronless genes in metazoans use the same receptors and adaptors, but it remains to be shown whether the loading is done via the polymerase as in yeast or whether the receptor can be added directly onto the growing mRNP also in this case. There are likely to be additional export pathways. For example, additional adaptors, SR proteins in metazoans and the SR-like Npl3 in yeast [22–24], have been identified and extensively studied. The SR proteins provide an alternative to the Aly/ REF (Yra1 in yeast) pathway and are regulated by phosphorylation; these proteins bind to the pre-mRNA in a hyperphosphorylated state, but later on become hypophosphorylated and then gain affinity for the export receptor complex [25,26]. It has been revealed that the nascent RNP complexes and their maturation on the genes are monitored by a surveillance machinery, the RNA exosome [27–29]. This body contains a complex of 3′ to 5′ exonucleases, retains aberrant mRNAs, and ultimately degrades them to ensure that only properly processed transcripts are exported. When mRNP complexes have been released from the genes, they move freely by Brownian diffusion in the interchromatin space [30–37]. However, the mRNP particles can also interact transiently with a fibrous network in the nucleoplasm [38–40]. In chromatin-containing regions, the movement of the mRNPs may be restricted, and mRNPs can be trapped and even immobilized in small cavities [36,37]. The movement is essentially

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without direct ATP dependence, but energy deprivation might trap more mRNP particles due to alterations in chromatin structure [36,37]. The mRNPs travel randomly in the nucleoplasm and appear throughout the nucleus [33,37]. Eventually, they will bind to nuclear pore complexes (NPCs) anywhere along the nuclear envelope, translocate through the nuclear pores, and enter cytoplasm. A nuclear pore complex (NPC) has a cylindrical core with a central, 25-nm wide, channel filled with a meshwork of very thin filaments [41–44]. On the nuclear side, there are eight fibrils anchored to the core and extending into the nucleoplasm. At their distal ends, the fibrils bifurcate and can laterally interdigitate with their neighboring fibrils to form a ring-like structure, the terminal ring. The nuclear fibrils and the terminal ring constitute the nuclear basket. On the cytoplasmic side, there are eight shorter fibrils extending from the core into the cytoplasm. In studies of exceptionally large mRNPs, it has been possible to visualize the passage of an mRNP through the complex NPC [45]. The mRNP particle binds to the tips of the fibrils of the nuclear basket, enters the basket, and docks at the entrance to the central channel of the core. The tightly packed RNP fibril unfolds and passes through the central channel with the 5′ end in the lead. When the fibril exits into the cytoplasm, the mRNP is immediately engaged in protein synthesis. The passage of the mRNPs through the NPC is not yet well understood in molecular terms. A basket fibril is formed from coiled-coil homodimers of the protein Tpr [46], and the mRNPs are likely to bind to the tip of the fibril [45,47], which contains the C-terminus of the protein [48,49]. During the passage through the central channel the RNP particle has to traverse the fibrous network. The fine filaments are likely to consist of extended, FGrich nucleoporins [50]. As the export receptors have special affinity for these structures, they are well suited to facilitate the passage of the export complex through the central channel

Fig. 1 – An overview of a cell nucleus in a salivary gland cell. Balbiani rings (BR) and polytene chromosomes (Pc) are indicated. The stain represents the distribution of the RNA-binding protein hrp45. The scale bar corresponds to 5 µm.

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[51,52]. As recently shown, the export receptor is located at the 5′ end of the RNP complex [53], which is in good agreement with the observation that the 5′ end of the mRNP is in the lead through the translocation [54]. The core structure of the RNP fibril is also reorganized to a major extent during the passage through the NPC, as the hnRNP and SR proteins confined to the cell nucleus leave the mRNP [3,4]. At the exit from the channel, the 5′ end of the mRNP complex is restructured in a process governed by the RNA helicase Dbp5 [55,56]. This process is especially important as it makes the translocation irreversible (the ratchet principle) [57]. The export receptor is removed from the mRNP and returns into the nucleus. Furthermore, the capbinding complex is displaced from the 5′ end, which paves the way for the initiation of protein synthesis [58]. More and more information suggest that the many steps in gene expression are more intimately linked and coordinated than earlier believed [15]. As mentioned above, RNA pol II transcription is closely coupled to the events taking place in the immediate vicinity of the gene, i.e. processing of the premRNP as well as the formation of the mRNP export complex. It is, however, an open possibility that on-going transcription

could also be closely coupled to subsequent stages in the information transfer. The transport system would then sense the transcriptional state and could rapidly respond to a change in production of and demand for mRNPs. To explore the possibility of a signaling system between ongoing transcription and transport to and through the nuclear pore, we have now studied the behavior of a specific mRNP particle in a model system for gene expression, the salivary glands in the dipteran Chironomus tentans. In this system it is feasible to visualize in the electron microscope how a specific mRNP particle, the Balbiani ring (BR) RNP granule, is assembled on the gene and moves via the nucleoplasm to and through the nuclear pore complexes [45]. We have now studied how this flow is affected by interruption of transcription by DRB and actinomycin D. A striking effect was seen at the NPCs: the number of basket-associated and translocating BR mRNPs rapidly declined after initiation of the drug treatments. Thus, the exit of mRNPs from the cell nucleus was reduced or even abolished upon cessation of transcription. However, the amount of nucleoplasmic mRNP particles remained essentially the same. We conclude that upon block of transcription the mRNP

Fig. 2 – Immunofluorescence microscopy of Balbiani rings after transcriptional inhibition. Salivary glands were isolated from Chironomus tentans fourth instar larvae and incubated for 0, 10, 30 and 90 min with DRB (A–D), actinomycin D (E–H) or without inhibitors (I–L). The Balbiani rings (arrowheads) were challenged with a primary antibody against the RNA-binding protein hrp45 and a Texas Red-conjugated secondary antibody, and examined in a laser scanning confocal microscope. The scale bar equals 5 μm.

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particles are prevented either from moving to the nuclear envelope or from being bound to NPCs. The mechanisms involved and the nature of the signal from the transcription machinery are being discussed.

Materials and methods Experimental material C. tentans midges were raised under laboratory conditions according to Case and Daneholt [59]. Salivary glands were isolated from fourth instar larvae.

Incubation of salivary glands Sister salivary glands were gently dissected out at 18 °C and tested for damage with Trypan Blue in ZO medium [60]. In each experiment, an intact gland was transferred to hemolymph and either DRB (5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole) (Sigma) or actinomycin D (Calbiochem) was added to a final concentration of 30 μg/ml and 10 μg/ml, respectively. As control, the corresponding sister gland was incubated in normal hemolymph with no drug added. The glands were incubated in an airtight container at 18 °C for 5, 10, 30 or 90 min before fixation. Glands that were destined for 5 or 10 min incubations were preincubated in pure hemolymph for 5 min to recover after dissection. For each

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drug treatment and incubation period, three separate incubations with single glands were performed.

Electron microscopy After incubation the glands were fixed for 2 h at 4 °C with 2% glutaraldehyde in 0.05 M sucrose and 0.1 M Na-cacodylate buffer (pH 7.2). Subsequently, the glands were washed overnight in the Na-cacodylate buffer, postfixed in 1% OsO4 for 1 h at 4 °C, rinsed in the buffer and transferred to 70% ethanol. The glands were brought through a graded series of ethanol (70 to 99%) at room temperature. Agar 100 was then introduced stepwise: the glands were kept in ethanol/Agar in a 3/1 ratio for 30 min, in a 1 + 1 ratio over night, and in pure Agar for 1–6 h. Finally, the glands were left to polymerize at 60 °C for at least 2 days. Ultrathin sections (about 70 nm) were cut by a Reichert Ultracut S ultramicrotome and stained with uranyl acetate and lead citrate before being photographed in a Philips CM 120 electron microscope at 60 kV.

Analysis of electron micrographs In each salivary gland three nuclei, cut roughly in the midplane, were selected for quantitative measurements. The circumference of each nucleus was determined, with a map measure tool, in arbitrary length units that was converted into μm. All spherical BR particles, partly or entirely located within a distance of 50 nm

Fig. 3 – An active Balbiani ring gene visualized by electron microscopy. A portion of a salivary gland cell is shown in panel A with nucleoplasm (Npl), cytoplasm (Cpl) and a Balbiani ring (BR) indicated. As shown in the schematic drawing in panel E, a transcriptional loop can be subdivided into three regions: proximal (p), middle (m) and distal (d). In the proximal region (p) the growing RNP is seen as a thick fiber, whereas in the middle region (m) the peripheral part of the RNP is starting to fold and form a globular portion. In the distal region (d) the globular portion is further expanded. The released RNP particle is a ring-like spherical particle. Examples of electron micrographs of the proximal, middle and distal regions are exhibited in panels B, C and D, respectively. Broken white lines in B through D depict the path of the chromatin axis. A few representative growing RNP particles have been indicated with arrows in B–D. The scale bars correspond to 1 μm (A) and 100 nm (B–D).

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(one particle diameter) from the inner surface of the nuclear envelope, were classified as bound to the nuclear pore complex, whereas pear- or rod-shaped RNPs within the nuclear pore complex were regarded as translocating. The density of bound and translocating particles in each nucleus was obtained from the numbers recorded divided by the length of the nuclear envelope examined. The mean values of the densities of bound and translocating particles were calculated and plotted against time, i.e. each time point was represented by three experiments and nine nuclei. The shared zero point was based on seven experiments and 22 nuclei. The BR particle concentration in the nucleoplasm was determined from the micrographs (total magnification 18,900×) using a transparent lattice with squares 2.5 × 2.5 cm in size (corresponding to 1.32 ×1.32 μm in the specimen). The lattice was randomly placed onto the electron micrographs and the BR RNPs in ten randomly chosen squares were counted in each experiment.

Immunofluorescence Incubated salivary glands were fixed and permeabilized with 4% paraformaldehyde and 5% Triton X-100 in PBS for 20 min at room temperature and further treated with 5% Triton X-100 in PBS for 15 min. After washing in PBS, the glands were blocked in 1% milk in PBS and then incubated overnight at 4 °C with a primary polyclonal antibody against hrp45 (diluted 1:250) [61]. The specimens were rinsed three times in PBS and then incubated for 1 h with a Texas Red-conjugated donkey anti-

rabbit antibody (diluted 1:50) (Invitrogen). After washing in PBS the glands were mounted in Mowiol and examined in a laser scanning confocal microscope (LSM510 Meta, Zeiss) using 1 μm thick optical sections.

Results Regression of Balbiani rings upon treatment with DRB and actinomycin D DRB is a nucleoside analogue that inhibits the kinase activity of the elongation factor P-TEFb [62]. As this factor phosphorylates the CTD of the RNA pol II and enables the polymerase to escape from the promoter, DRB treatment will halt the polymerase at the promoter. However, the elongation per se is not dependent on the kinase and polymerases already on the template will traverse the entire gene. On the other hand, actinomycin D (AMD) inhibits all transcribing RNA polymerases by a physical interaction with the DNA molecule itself and disables the polymerases to proceed reading the genes. Because the transcriptional activity of BRs is correlated to the size of the BRs [63], the effect of DRB and AMD could be monitored by immunofluorescence microscopy. Salivary glands were isolated from fourth instar C. tentans larvae and treated with DRB or AMD for 0, 10, 30 or 90 min. Sister glands were incubated in parallel with no drug added. After incubation, the glands were fixed, permeabilized and challenged with a polyclonal antibody

Fig. 4 – Electron microscopic analysis of the effect of DRB on active BR genes. Salivary glands were dissected out and incubated in hemolymph supplemented with DRB for 0, 30 and 90 min. The upper panels display sections through BRs, while the lower panels show at higher magnification areas selected from the corresponding upper panels (boxed). Proximal (p), middle (m) and distal (d) segments have been indicated with letter and short arrow. Long thick chromatin fibers have been marked with long arrow. The scale bars represent 500 nm (upper) and 200 nm (lower).

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recognizing the SR protein hrp45, an abundant RNA-binding protein [64]. The immunolabeling was visualized with a Texas Red-conjugated anti-rabbit antibody. The three BRs and polytene chromosomes are shown in an overview of a cell nucleus in Fig. 1. The results of the transcription inhibition experiments are presented in Fig. 2. In control glands, the size of the BRs did not change suggesting that transcription was maintained during incubation in non-treated glands (Figs. 2I–L; BRs indicated by arrow heads). When the glands were treated with DRB, the BRs were reduced in size after 30 min incubation and had almost vanished after 90 min, indicating that transcription had been blocked (Figs. 2A–D). The BRs in AMD-treated glands gradually diminished during the course of the incubation (Figs. 2E–H), although they did not regress as much as during the DRB treatment. In conclusion, the transcription inhibitors DRB and actinomycin D caused shrinkage of the BRs, which indicated that the inhibitors had exerted their effect. Ultrastructural changes recorded in the glands during the treatment also strongly

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indicated that transcription had been blocked (see below). The incubation per se did not seem to affect transcription as the BRs in the control glands maintained their size.

Electron microscopy of BRs To examine transcription in the BRs after drug treatment we used electron microscopy. Salivary glands were isolated and incubated individually in hemolymph supplemented with DRB or actinomycin D for 0, 5, 10, 30 or 90 min. Sister glands were incubated in parallel without the drug. The glands were fixed, plastic embedded, sectioned, stained and examined in the electron microscope.

The structure of active BR genes A BR contains numerous active genes, which have earlier been extensively studied in the electron microscope [45,65]. An active gene is schematically presented in Fig. 3E. The active gene forms

Fig. 5 – Electron microscopic analysis of the effect of DRB on transport of BR RNPs across the nuclear envelope. The effect of DRB treatment on the passage of BR RNPs through the NPCs was examined after 0 (A), 30 (B) and 90 min (C). In A, a BR particle bound to the NPC is indicated by an arrow and a translocating particle by an arrowhead. A nucleoplasmic BR RNP is shown in D at higher magnification together with examples of BR particles still translocating through the pore after short DRB treatment (E–H). Spherical particles associated with the NPCs (E, F) are designated bound and the pear- or rod-shaped ones as translocating. N, nucleus; C, cytoplasm. The scale bars represent 500 nm (A–C) and 100 nm (D–H).

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a transcription loop with a chromatin axis and growing RNP complexes perpendicularly attached to the axis via RNA polymerases. The BR gene is 35–40 kb in size [66], and an exceptionally large transcription product, a ring-like, spherical RNP particle with a diameter of 50 nm, is generated. The RNP particle is formed co-transcriptionally and the packing of the thin elementary RNP fibril into higher order structures can be followed along the gene. In the proximal (p) part of the gene the RNP fibril is coiled into a thick RNP fiber, which in the middle segment (m) is bent and forms a peripheral globular structure. Further along the gene, in the distal portion (d), the bent fiber grows and the globular portion enlarges. Upon completion of transcription, the stalk portion of the growing RNP is retracted into the globular portion and the ring-shaped spherical particle is formed. In electron micrographs from ultrathin sections through a BR, it is only possible to discern segments of individual active genes (Fig. 3A). Examples of proximal (p), middle (m) and distal (d) portions have been marked in the figure. A proximal, a middle and a distal segment are also displayed at higher magnification in Figs. 3B, C and D, respectively. Thick RNP fibers are seen in the proximal segment (B), the initiation of the formation of a peripheral globular portion in the middle segment (C) and stalked RNP particles with a globular portion in the distal region (D).

particles (E and F), while the pear- or rod-shaped particles are called translocating particles (G and H). We next examined the effect of DRB on the appearance of BR RNP particles at the nuclear envelope and describe here the

The effect of DRB on active BR genes At the start of the experiment the three gene segments (p, m and d) could be detected in the BRs (Fig. 4, left panel). After 30 min DRB treatment, distal segments with large stalked RNP particles (d) could be seen but not proximal or middle segments (Fig. 4, middle panel). Furthermore, long thick fibers (arrows) had appeared, each presumably representing the chromatin axis folded into a 30-nm chromatin fiber [67]. The thick fibers often appear as loops. After 90 min there were essentially only some thick fibers left and a few large BR RNP particles (Fig. 4, right panel). These results confirm earlier studies of the action of DRB [67,68], indicating that DRB blocks transcription at or close to the promoter. Upon addition of DRB, no new growing RNPs are added while the ones that are on the template can be completed. When the chromatin template is devoid of RNPs it is packed into a thick chromatin fiber and finally into higher order compact chromatin. We conclude that as expected DRB had blocked transcription early, most likely already at the promoter.

DRB reduced the transport of BR RNPs across the nuclear envelope Due to their giant size and abundance, the BR particles can be readily monitored not only during transcription, but also during transport from the genes, via nucleoplasm, to and through the nuclear pores [45]. The BR particles in nucleoplasm are easily recorded as large, densely stained granules (Figs. 5A and D). During translocation across the nuclear envelope the BR particles first bind to the basket of the nuclear pore complex (NPC) (arrow in Fig. 5A), unfold through the central channel of the NPC (arrowhead in Fig. 5A). This process can be stepwise followed at higher magnification in Figs. 5E–H (cf. Ref. [54]). Spherical particles associated with the NPC are designated bound

Fig. 6 – Quantitative analysis of the electron microscopic analysis of the effect of DRB on the passage of BR mRNPs through the NPC. DRB-treated salivary glands (solid line) were compared to control glands incubated in pure hemolymph (dotted line). For each time point nine nuclei from three glands were analyzed. Bound and translocating were classified as explained in the legend of Fig. 5. (A) The average number of bound BR RNPs per μm nuclear envelope (NE) plotted against time (0, 5, 10, 30 and 90 min). (B) The average number translocating BR RNPs per μm NE plotted against time. (C) BR particle concentration in the nucleoplasm (BR RNPs/μm2) plotted against time.

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effects seen after DRB treatment for 30 and 90 min (Figs. 5A–C). For our analysis we selected nuclei sectioned in the nuclear midplane to obtain maximal length of nuclear envelope and NPCs cut parallel to the central channel. After 30 min DRB treatment fewer particles were bound to the NPC (Fig. 5B), and there seemed more on top of the nuclear basket than within the basket (more particles like E than F). Furthermore, only a low number of translocating RNPs were found (G and H). After 90 min incubation with DRB the number of BR RNP particles had further decreased, and no translocating particles seemed to be present (Fig. 5C). It should be noted that the few pore passages that were found during the course of the DRB treatment appeared structurally normal, and so did the free nucleoplasmic particles (Figs. 5D–H). The number of BR particles in nucleoplasm did not seem reduced during the treatment (Figs. 5B–C). In the control glands the translocation and binding frequencies did not change indicating that the export was maintained with no drug added. Thus, there was a conspicuous reduction in the number of both bound and translocating BR RNPs at the nuclear envelope during DRB treatment. To get a quantitative measure of the changes observed, we determined the density of bound and translocating BR particles along the nuclear envelope. BR particles, partly or entirely within a distance of 50 nm from the inner surface of the nuclear envelope were counted as bound particles. The density values of bound and translocating particles were obtained by dividing the number of bound or translocating particles with the length of the nuclear envelope in μm. The

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mean values were plotted against time in Fig. 6. DRB-treated glands and control sister glands were studied in parallel. As best seen in tangential sections of the nuclear envelope, the nuclear pore density did not change neither in DRB-treated nor in control cells (data not shown). Due to variability between glands and the limited number of observations, only major differences were considered significant. During DRB treatment the density of bound BR particles decreased and was about 25% of the start level after 90 min incubation (Fig. 6A). The effect on translocating particles was even more striking: already after 5 min DRB treatment the particle density had diminished to about 25% of the control level, and after 90 min no translocating particles could be recorded (Fig. 6B). The BR RNP particle concentration in the nucleoplasm remained essentially constant except for low 5 and 10 min values (Fig. 6C). As this “drop” was also seen in the non-treated sister gland controls, it does not seem to be due to the drug treatment but reflects a variation between animals as to particle concentration in nucleoplasm. Thus, DRB caused a rapid decrease in BR RNP export across the nuclear envelope, the decline of translocating BR particles being initiated within the first 5 min of incubation. As both bound and translocating particles decreased in number or vanished, there is no evidence that the block appears during the passage of BR RNP through the nuclear pore; the interruption of the RNP export is likely to occur prior to or at the binding of the BR RNPs to the NPC. BR particles remained seemingly intact in the nucleoplasm and spread throughout nucleoplasm as in control cells.

Fig. 7 – Electron microscopic analysis of the effect of actinomycin D on active BR genes. Salivary glands were incubated in actinomycin D for 0, 30 and 90 min, respectively. The appearance of the transcriptional BR loops after treatment is shown in the upper panel. The lower panel corresponds to higher magnification of the areas selected from the corresponding upper panels (boxed). Proximal (p), middle (m), and distal (d) segments have been indicated. The scale bars equal 500 nm (upper) and 200 nm (lower).

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The effect of actinomycin D on active BR genes The effect of actinomycin D on transcription of BR genes was also investigated by electron microscopy. In the non-treated glands (0 min), the proximal, middle and distal segments of the active genes can be discerned (Fig. 7, left panel). In parallel with the regression of the BRs during the AMD treatment (Figs. 2E–H), the amount of growing particles decreased but proximal as well as middle and distal regions could be seen. However, the BR RNP particles seemed more dispersed, presumably indicating that the density along the chromatin axis had decreased. We conclude that during AMD treatment many growing RNP particles had disappeared from the genes and been lost from all three segments of the genes. This result is in agreement with the known action of actinomycin D as an inhibitor of transcription elongation. The disappearance of halted BR mRNP particles from the template are likely to be due to degradation by nuclear exosomes (see Introduction). Actinomycin D not only affects RNA polymerase II, but also the transcription of RNA polymerases I and III. The inhibition

of RNA polymerase I activity was clearly indicated in the electron micrographs by segregation of the nucleolar structure. Nucleolar fragments were also seen in the nucleoplasm (data not shown). We conclude that AMD had the predicted inhibitory action on both mRNA and rRNA transcription.

Actinomycin D blocked exit of BR RNPs from the nucleus To look for effects on nucleocytoplasmic transport BR RNP particles associated with the nuclear envelope, actinomycin D-treated glands were examined in the electron microscope. Treatment with AMD for 30 or 90 min resulted in fewer bound and translocating particles (Figs. 8B–C) compared with the control (Fig. 8A). Those that were still associated with the nuclear envelope exhibited normal morphology (Figs. 8E–H). The effect of AMD was also quantified in the same way as earlier done in the DRB experiment. The density of bound BR RNPs decreased during the treatment and seemed to level off at 40% of the start value after 90 min incubation (Fig. 9A). More striking was the decrease in the density of translocating RNPs:

Fig. 8 – Electron microscopic analysis of the effect of actinomycin D on the transport of BR RNPs across the nuclear envelope. After different actinomycin D incubation times, 0 min (A), 30 min (B) and 90 min (C), the transport of BR particles across the nuclear membrane was monitored. Bound BR RNPs are indicated with arrowheads and a translocating BR RNP with arrow. A nucleoplasmic particle (D) and particles still engaged in NPC-binding (E, F) and translocation (G, H) after actinomycin D treatment. N, nucleus; C, cytoplasm. The scale bars correspond to 500 nm (A–C) and 100 nm (D–H).

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after 30 min 25% was left and after 90 min only 10% (Fig. 9B). The concentration of BR particles in the nucleoplasm remained stable throughout the experiment, perhaps with a slight transient decrease early in the experiment (Fig. 9C). In

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conclusion, like DRB, actinomycin D reduced the density of bound and especially translocating BR RNPs at the nuclear envelope, indicating that the export of BR RNPs from nucleus to cytoplasm drastically decreased. At the same time, nucleoplasmic BR RNPs remained essentially unchanged as to number and distribution. We conclude that the export block is likely to occur prior to or at the binding of the BR RNPs to the NPC.

Discussion Block of transcription prevents BR mRNPs from leaving the nucleus

Fig. 9 – Quantitative analysis of the electron microscopic analysis of the effect of actinomycin D on the exit of BR RNPs from the nucleus. For each time point (0, 5, 10, 30 and 90 min) nine nuclei from three glands were scrutinized, and bound, translocating and nucleoplasmic particles were quantified as described in the legend of Fig. 6. The AMD-treated glands (solid line) were compared to control glands (dotted line). (A) Average values of bound BR particles per μm nuclear envelope (NE) plotted against time. (B) The average number of translocating BR particles per μm NE plotted against time. (C) BR particle concentration in the nucleoplasm (BR RNPs/μm2).

In this study we have investigated a possible link between transcription and export of a specific mRNP species in the salivary glands of C. tentans, the Balbiani ring mRNP particles. Two transcriptional inhibitors with different mode of action, DRB and actinomycin D, were used to block transcription, and the effect on BR mRNP export was studied with electron microscopy. The two drugs gave essentially the same effects on mRNA export: the number of particles associated with the basket of the NPCs was reduced, while particles translocating through the nuclear pores almost disappeared. Thus, the export of mRNPs diminished drastically during the drug treatments. Although DRB can interfere with other processes than transcription, e.g. co-transcriptional splicing and 3′ end formation [69], we believe that in the present study the crucial effect was inhibition of transcription as actinomycin D elicited a similar response as DRB. We conclude that the exit of mRNPs from the cell nucleus is closely coupled to on-going transcription. Such a coupling is not true for RNA in general, as e.g. transfer RNA is transported to the cytoplasm in the salivary gland cells during actinomycin D treatment [70]. The most remarkable observation in our study was that the number and distribution of mRNP particles in the nucleoplasm remained essentially unchanged during the transcription block. As it is known that essentially all BR particles in the nucleoplasm will ultimately be exported to the cytoplasm [71], we had expected to see a gradual reduction of the amount of nucleoplasmic BR mRNPs and an ensuing decrease in the number of bound and translocating RNPs at the NPCs. However, in spite of a rapid loss of both bound and translocating BR mRNPs, the RNP particles in the nucleoplasm (by far most of them synthesized before the block of transcription) remained essentially the same. Evidently, the passage of mRNPs through the NPCs had continued, while the provision of the NPCs with new mRNPs had failed. The inhibited production and the blocked exit from the nucleus of BR mRNPs had resulted in a roughly unchanged nucleoplasmic pool of BR particles. The existence of such a pool suggests that the reduced exit of mRNPs cannot simply be a consequence of lack of BR mRNPs to be transported to the NPCs; the information transfer has to be inhibited. The primary block is unlikely to be within the NPC itself as no accumulation of BR mRNPs occurred within the basket or nuclear pore. Instead, we have to consider two other options, viz, that the mRNP particles cannot be transported to the nuclear periphery or that they are prevented from binding to the NPC.

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Block of intranuclear movement or binding to the NPC? Let us first examine the possibility that the BR mRNPs are prevented from moving to the NPCs. Normally, the BR RNP particles are released from the genes immediately upon completion of transcription and move randomly in nucleoplasm [33]. The BR RNP particles are not stored neither in the vicinity of the genes, nor at special loci in nucleoplasm. Moreover, the mobility of the nucleoplasmic BR particles is not constrained by chromatin [36,37], as the vast nucleoplasmic regions between the polytene chromosomes lack chromatin. The apparent diffusion rate of BR particles in nucleoplasm is in agreement with the view that the mobility is by Brownian diffusion; no transport machinery has to be invoked. However, a certain portion of the BR particles are attached to a fibrous network in the nucleoplasm, which suggests that the particles can be transiently associated with fibers [39]. One possible mechanism for the retarded or inhibited movement during the transcription block could then be that the particles attach but do not leave the fibers; they become immobilized for a shorter or longer time. Although photobleaching experiments in mammalian cells suggest that neither DRB [35] nor actinomycin D [34] affects the mobility of mRNPs in nucleoplasm, it will be necessary to track individual BR particles in the salivary gland cells to firmly establish whether or not the observed interaction between the particles and the nucleoplasmic fibers is changed upon block of transcription. Two recent observations support the possibility that ongoing transcription could be closely coupled to the movement of mRNP particles to the nuclear pores. The first set of data concerns the mobile nucleoporin Nup98 [72,73]. This protein shuttles between nucleus and cytoplasm and is essential for RNA export [74]. In nucleoplasm it seems to interact with other, less mobile components. It is supposed to direct the export of RNPs to and/or through the NPCs, presumably as a component of the RNP particles. Remarkably, upon block of transcription Nup98 loses its mobility both within nucleoplasm and NPC. The explanation close at hand is that as no more RNPs are formed, no RNPs are available for export. Considering our own data, an alternative explanation could be that the mobility is lost because the RNP particles (with the associated Nup98) have been “frozen” on the fibrous network. The second observation supporting an attachment to a nuclear structure was reported by Tokunaga et al. [75]. They studied export of microinjected pre-mRNAs after treatment of mammalian cells with transcriptional inhibitors. The injected, and subsequently spliced fluorescent pre-mRNPs accumulated in a novel type of nuclear foci close to the SC35 speckles, designated TIDRs (transcriptional inactivation dependent RNA domains). This finding favours the view that the mRNPs are arrested in the nucleoplasm upon transcriptional inhibition. Let us then consider the alternative explanation for the low number of mRNPs at the pore upon cease of transcription: the particles do move to the nuclear envelope but they cannot bind to the NPC. Normally, the particles bind to the tip of the basket fibrils, enter the basket, unfold and pass through the central channel with the 5′ end in the lead [45,54]. That the binding of the BR particle to the basket fibrils represents a discrete step during translocation has been demonstrated in experiments with antibodies to the nucleoporin Nup153 [76]. Injection of these antibodies into salivary gland nuclei resulted in an accumulation

of BR particles on top of the baskets; the particles could bind but did not enter the baskets. Thus, if the particles do reach the NPC, it is the initial binding of the particle to the basket that fails as no particles are seen on top of the basket. We conclude that an inhibition of the binding of BR particles to the basket could explain the observed block of exit of mRNPs from the nucleus, but there is still no specific experimental support for this alternative.

The nature of a signal between transcription and mRNPs in transit The nucleoplasmic BR RNP particles are evenly distributed throughout nucleoplasm [33], while the BR genes are located in two giant chromosomal puffs, the BRs, on polytene chromosome IV. It seems, therefore, unlikely that the nucleoplasmic particles interact directly with the transcription machinery at active genes. Furthermore, this conclusion is further supported by the fact that independent of the level of transcription (with or without inhibitors), there is only a very low number of completed particles in the immediate vicinity of the BR genes. Thus, it seems closer at hand to suggest that there is a signal system mediating the communication between the active genes and the large number of widely distributed mRNPs in the nucleoplasm. A signal could be directed towards the mRNP particles themselves to make or maintain their transport competence. Alternatively, it could be targeted towards the transport machinery (adapters, receptors, and other export factors) and/or the transport pathway (nucleoplasmic fibers and nuclear basket). Thus, presently many different targets and mechanisms have to be kept in mind. Two examples of possible targets deserve to be specifically commented upon. Some of the hnRNP proteins in the nuclear mRNP particles leave the particles prior to the exit of mRNPs from the nucleus [3,4]. One of them, hnRNP C, harbours a retention signal that can override the nuclear export signal, perhaps with potential of keeping the mRNPs confined to the nucleus if not being removed from the complex [77]. Thus, the regulation of the displacement of hnRNP C (or similar proteins) could govern the exit of mRNPs from the nucleus. Another set of nuclear mRNP proteins, the SR proteins, could play a crucial role but by another mechanism. Some of the SR proteins are known as adaptors for RNA export receptors [22,24]. The BR mRNP particles contain an SR protein, hrp45, that is added co-transcriptionally to the BR transcripts and accompanies the mRNA to the NPC [64]. Recently, additional SR proteins have been recorded in the BR mRNPs, and all of them shuttle [78]. As it is known that SR proteins only bind the export receptor in a hypophosphorylated state [25], the structure of the mRNP export complex could be modified by regulation of the level of phosphorylation of an SR protein, which could govern whether the mRNPs are released from nucleoplasmic fibers or perhaps whether they bind to the NPC. Our results provide evidence for direct communication either between the transcription machinery and the nucleoplasmic mRNP particles ready for export or between the machinery and the export factors and the transport pathway, the latter perhaps involving both nucleoplasmic fibers and NPCs. As shown for other interactions between molecular machines in the nucleus, such a communication could act in both directions to optimize a

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rapid and efficient use of the resources of the cell [16]. If this signaling system is only global or whether it can also operate at the level of individual genes remains to be established.

Acknowledgments The study was supported by the Swedish Research Council, European Community (3D-EM Network of Excellence), Human Frontier Science Program Organization, and Knut and Alice Wallenberg Foundation.

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