In the Nucleus and Cytoplasm of Chicken Erythroleukemic Cells, Prosomes Containing the p23K Subunit Are Found in Centers of Globin (Pre-)mRNA Processing and Accumulation

Experimental Cell Research 250, 569 –575 (1999) Article ID excr.1999.4556, available online at http://www.idealibrary.com on

RAPID COMMUNICATION In the Nucleus and Cytoplasm of Chicken Erythroleukemic Cells, Prosomes Containing the p23K Subunit Are Found in Centers of Globin (Pre-)mRNA Processing and Accumulation Flora De Conto,* ,† Sergey V. Razin,* ,‡ Ge´rard Geraud,* Cristina Arcangeletti,* ,† and Klaus Scherrer* ,1 *Institut Jacques Monod, Universite´ Paris 7, 2, Place Jussieu, Tour 43, 75251 Paris Cedex 05, France; †Istituto di Microbiologia, Universita` degli Studi di Parma, Via Gramsci 14, 43100 Parma, Italy; and ‡Institute of Gene Biology, RAS, Vavilov Ul. 34/5, 117334 Moscow, Russia

Prosomes were originally identified as 20S particles associated with untranslated mRNA; they also constitute the core of the 26S proteasomes. The cellular distribution of three types of prosomes characterized by the presence of subunits with molecular masses of 23, 27, and 30 kDa was analyzed using an immunocytochemical approach on cultured chicken erythroblasts. The prosomes containing the p27K and p30K subunits were found in diffuse distribution in both nuclei and cytoplasm. In contrast, the prosomes containing the p23K subunit, although relatively rare in the nuclear space, were found concentrated in one or two large spots. Using in situ hybridization with an a A-globin gene-specific riboprobe we found that the p23K-type prosomes colocalize in the nucleus with centers of globin (pre-)mRNA processing, and of mRNA accumulation in the cytoplasm. This result suggests there is local coincidence of specific-type prosome function with processing and, possibly, transport of a particular kind of (pre-)mRNA. © 1999 Academic Press Key Words: prosomes; proteasomes; MCP; globin genes; mRNA; pre-mRNA; processing centers; accumulation centers.

INTRODUCTION

Prosomes were originally discovered as a subfraction of cytoplasmic RNPs containing mRNA in its nontranslated state [1–3], but they constitute also the core of the 26S proteasomes (for review see [4]). Since the discovery of the multicatalytic proteinase (MCP) activity of these 20S particles (for review see [5]), interest in the participation of prosomes in proteolysis has superseded that in other aspects of their function. The recent 1 To whom correspondence and reprint requests should be addressed. Fax: (33-1) 44 27 76 47. E-mail: [email protected].

discovery of the RNase activity of some of the alpha subunits of the prosomes [6, 7] has, however, reemphasized our original demonstration of their interaction with mRNA [1–3]. Most interestingly, recent data show that the proteasome system is fully dispensable in adapted EL-4 lymphoma cells [8] and can be replaced, most likely, by the Tricorn proteinase observed by Baumeister and colleagues [9]. In these adapted cells, however, the 20S particles (prosomes) subsist. Although prosomes are most abundant in the cytoplasm, a variable number of prosomes was always observed in the nuclei of all cells investigated [10, 11] (discussion in [4]); their presence there, however, has not been analyzed any further and, hence, is at present not understood. It should be underlined that in cells of higher eukaryotes (in contrast to yeast cells) there are many different kinds of prosomes characterized by different subunit composition (discussed in [4]). Recently, we have investigated the nuclear prosomes in several types of cells and found specific patterns of nuclear localization characteristic of particles of specific subunit composition (Pilotti et al., submitted). Furthermore, specific types of prosomes show patterns of specific cytodistribution related to functional sectors of differentiated cells, e.g., along the bile canaliculi in hepatocytes [12] and in the sarcomeres of muscle fibers [11, 13], in a pattern identical to that of some mRNAs [14]. To understand further the possible function of nuclear prosomes (beyond their obvious role in protein degradation), we have studied here the distribution of three particular types of prosomes in chicken erythroid cells. In parallel experiments, the distribution of alpha globin gene transcripts was studied, using hybridization in situ with the corresponding probes. A striking colocalization of, exclusively, the p23K-type prosomes and globin gene transcripts was observed in the nu-

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0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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cleus and cytoplasm of AEV cells, which transcribe the globin domain but do not synthesize hemoglobin. MATERIALS AND METHODS Cell culture. AEV cells of the line HD3 (clone A6 of line LSCC [15]) were grown in suspension in Dulbecco’s modified Eagle’s medium supplemented with 8% fetal bovine serum and 2% chicken serum. Prosome-specific monoclonal antibodies. Monoclonal antibodies against the prosomal proteins (p-mAbs) used in this study have been described previously [16, 17]. These mAbs are available from ICN Biomedicals (Orsay Cedex, France). The prosomal subunits tested were p23K (hybridoma clone 35A), p27K (hybridoma clone IB5), and p30K (clone 62A32). The prosome subunits named according to our terminology [18] correspond to the human subunit or gene names published by Hendil and colleagues [19] and ourselves [20] in the following manner: p23K, subunit HC7-I; p27K, Pros-27 (or iota); and p30K, Pros-30 (or HC2). Visualization of prosomal antigens in cells. The cells were collected on microscope slides using a “Cytospin” centrifuge. Prior to immunostaining, all samples were fixed with cold methanol as described previously [11]. The fixed cells were washed three times (5 min each) in PBS [7 mM Na 2HPO 4, 1.5 mM KH 2PO 4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl]. After washing, the cells were preincubated for 15 min with 1% BSA (bovine serum albumin) in PBS and then incubated with prosomal monoclonal antibodies (p-mAbs) in PBS buffer supplemented with 0.2% BSA (incubation buffer) for 30 min at 37°C in a humid chamber. After incubation, the cells were washed three times (5 min each) with PBS. Then the prosomal antibodies bound to antigens were revealed by FITC- or Cy3-conjugated antimouse IgG (Sigma Immunochemicals), as described in the manufacturer’s manual. Negative controls were carried out by an identical procedure, except that the first antibody was replaced by the incubation buffer alone. In situ hybridization. Hybridization in situ was carried out as described in the Boehringer (Mannheim) manual. Briefly, HD3 cells were fixed in 1% paraformaldehyde in PBS for 20 min at room temperature before treatment with a solution of 70% ethanol and 3% H 2O 2, to avoid endogenous peroxidases. Cells were then permeabilized with 0.2% Triton X-100 in PBS for 10 min, washed carefully in PBS, and immersed in 0.1 M glycine in PBS for 5 min. Cells were then washed in PBS and treated with 0.25% acetic anhydride, 0.1 M triethanolamine buffer for 10 min prior to incubation at 42°C for 16 h with riboprobes (0.5 ng/ml) in hybridization buffer (50% deionized formamide, 53 SSC, 10% dextran sulfate, 2.53 Denhardt’s solution, 10 mM dithiothreitol, 20 mM vanadyl ribonucleotide complex). To prepare strand-specific probes, the 1.0-kb HindIII–HindIII chicken genomic DNA fragment containing the p gene or the 1.8-kb chicken genomic DNA fragment containing the a A gene (for the map see [21]) was cloned into the pSP73 vector (Promega). The fragments were then transcribed in the direction opposite to that of globin gene transcription with the T7 RNA polymerase, using the Boehringer (Mannheim) kit for preparation of digoxigenin-labeled RNA. After hybridization, the digoxigenin-labeled probe was detected by incubation with anti-digoxigenin-AP, FAB fragments [Boehringer (Mannheim)] followed by incubation with tyramide, as described in the manual for the TSA-DIRECT (tyramide signal amplification) kit (DuPont, NEN). Confocal laser-scanning microscopy and image analysis. Analysis of patterns of specific prosome proteins or of p23K-type prosomes– globin RNA colocalization in HD3 cells was performed using the TCS (Leica Germany) confocal imaging system, equipped with a 633 objective (plan apo; NA 1.4). For FITC, Cy3, and TRITC excitation, an argon– krypton ion laser adjusted at 488 and 560 nm was used. For each optical section, double-fluorescence images were acquired in

sequential mode. The signal was treated using line averaging, to integrate the signal collected over eight lines in order to reduce noise. For high resolution, we defined a set of acquisition parameters, which took into account Nyquist’s principle. The confocal pinhole was closed to yield a minimum field depth (about 0.6 mm), and a focal series was collected for each specimen. The focus step between these sections was generally 0.3 mm and the XY pixelization was set to 100 nm. Each selected section level was then processed to produce a single high-spatial-resolution red/green composite image. Negative controls were examined in parallel to assess the specificity of FITC, Cy3, and TRITC signals and the absence of any cross-talk. Photographs were printed on a sublimation laser printer (Colorease Kodak) with Photoshop software. No mutual cross-contamination of the green (FITC) and red (Cy3, TRITC) signals was detected (data not shown). Double-staining microfluorometry analysis was performed using Multicolor analysis software (Leica France) running on the TCS (Leica) confocal microscope. In the cytofluorogram, the pixel to pixel correlation between two channels (red and green) could be outlined; colocalized or closely related areas could be selected (yellow cloud) and quantified. The selected cytofluorogram was superimposed in white color onto the merged images of this same area.

RESULTS

Distribution of Prosomes in AEV Cells To understand the experiments shown, it should be recalled that the HD3 cells investigated are AEV transformed cells expressing the v-erb oncogenes [15]. They correspond to chicken hemopoietic cells of the red lineage [22] arrested in early stages of differentiation [23, 24]. These cells abortively transcribe the globin genes [25, 26], but can be induced to differentiate and may attain up to 70% of hemoglobin (Hb)-producing cells [24]. The cells used here grew exponentially, and less than 5% were Hb positive; however, abortive differentiation occurred which led to some productive processing of globin pre-mRNA and transfer of mRNA to the cytoplasm. In the cultures used here, up to 50% of such “pseudoinduced” cells had some a A globin mRNA in the cytoplasm (data not shown). As in all other cells tested, in avian erythroblasts prosomal proteins were not found as free antigens outside the 20S particles; sucrose gradient analysis after dissociation of the mRNPs does not show immunoreaction outside the 19S–20S prosomal peak [18] (discussion in [4]). The IIF pictures therefore indicate the presence of 20S particles which show up in variable patterns due to their variant “mosaics” of subunit composition, and not free prosomal proteins. The results of immunostaining of AEV cells with the p23K-, p27K-, and p30K-specific p-mAbs are shown in the confocal series of Figs. 1a–1c. The cells shown are in fairly advanced stages of pseudoinduction. It is evident that three quite distinct patterns of immunofluorescence can be observed. All three p-mAbs labeled the cytoplasm quite strongly and the nuclei to different extent. The p23K p-mAb regularly stained one or two larger spots in the nuclei (arrows) and some faint cir-

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FIG. 1. Analysis of distribution of different kinds of prosomes in cultured chicken AEV-transformed erythroblasts. The following pmAbs were used for IIF: a–a0, p-mAb against p23K; b– b0, p-mAb against p27K; and c– c0, p-mAb against p30K. The pictures represent single confocal sections (0.3 mm) of Z series. [Arrows point to the Processing Centers (PCs) and the arrowhead points to the periphery of faint circular area(s) regularly observed.]

cular areas (arrowhead) which, given their size, might correspond to nucleoli (cf. Pilotti et al., submitted). In contrast, the p27K and p30K p-mAbs gave a more diffuse pattern in the nucleus which, as the confocal patterns show, seem to spare out some spherical areas (which might also correspond to nucleoli). Each type of mosaic prosome earmarked by a specific subunit seems, thus, to distribute in its own particular pattern. Negative controls without p-mAbs carried out as detailed under Materials and Methods showed the complete absence of any fluorescent signal. In the Nuclei of AEV Cells, the Pattern of p23K-Type Prosome Distribution Overlaps with That of Productively or Abortively Processed Globin Transcripts In a parallel set of experiments we studied the distribution of the embryonic alpha-type p gene transcripts using in situ hybridization with the corresponding strand-specific riboprobes. Negative controls in the absence of the riboprobe did not show any significant signal (data not shown). In confocal series, surprisingly, patterns were observed (Figs. 2a–2c) that were strikingly similar to the distribution of the p23K-con-

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taining prosomes shown above. The most obvious similarity in distribution of the p gene transcripts and the p23K-containing prosomes was that, in addition to cytoplasmic staining, one or two large spots of transcript accumulation can be observed in the nuclei. These might be interpreted as representing pre-mRNA processing centers (PCs). Indeed, pulse-chase experiments and steady-state labeling of RNA in erythroblasts have shown in the past that the majority of globin transcripts in the nuclei are already partially processed [27]. Similar hybridization patterns were obtained with a probe recognizing transcripts of the a A globin gene (Figs. 2d and 2g). There is, however, much less globin mRNA in the cytoplasm of these cells than in those shown in Figs. 2a–2c: such cells may, hence, correspond to earlier stages of abortive terminal differentiation. In view of the above-mentioned similarity of the patterns of globin RNA and of the p23K-containing prosomes, which accumulate in one or two dots within the nuclei, we undertook double-labeling experiments, combining in situ hybridization with the globin probe and immunofluorescence staining with prosomal antibodies. As can be seen in Figs. 2d–2f and 2g–2i, within the nuclear spots observed by IIF and confocal microscopy, the p23K p-mAb (green) colocalize (yellow) with the centers of accumulation of the a A gene transcripts (red). To confirm this observation and to test for the reality of (i) apparent partial asymmetry of the globin and prosome stain and (ii) the apparent but spurious coincidence in the cytoplasm, we undertook high-resolution coincidence analysis at the pixel level (Fig. 3; white dots indicate coincident pixels). Figure 3 shows that prosomes are present not only at the level of the processing pathway of globin transcripts, but also at cytoplasmic centers of accumulation of globin mRNA, most likely in an untranslated state (cf. Discussion). In answer to the first question, (i) the nuclear p23K prosomes seem to occupy only a particular sector of the PCs, possibly in a more peripheral area (arrowheads) sometimes protruding into the nucleoplasm in direction of the cytoplasm (fat arrows); some areas of the PCs seem to be devoid of 23K prosomes (slim arrows). The question about the cytoplasmic coincidence (ii) also seems to be answered in a positive manner: there are distinct accumulation centers (ACs) of cytoplasmic globin mRNA, where colocalization of the globin RNA with the p23K prosomes is revealed by pixel analysis (Fig. 3, white spots in the cytoplasm). Since prosomes are found exclusively on untranslated mRNA (cf. [4]), these pictures may indicate that cytoplasmic globin mRNA associated with the p23K prosomes localizes in specific areas, prior to or after translation.

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FIG. 2. Colocalization of areas of globin gene transcript accumulation with the p23K prosomal nuclear spots. (a– c) Localization of the p gene transcript in HD3 AEV cells by in situ hybridization (three consecutive confocal sections). (d–f and g–i) Localization in the same cell of the a A gene transcript (d, g; red), p23K-containing prosomes (e, h; green), and superimposition of the two patterns (f, i; yellow indicates colocalization). FIG. 3. Colocalization at pixel level of the p23K-type prosomes (green) with the globin (pre-)mRNA (red) in nucleus and cytoplasm. The results of double-staining microfluorometry analysis within the selected area of the original pictures are shown in Figs. 2g–2i. (a) The 1:1 pixel to pixel correlation between the signals in the two channels (red and green) within the colocalized or closely related areas (yellow clouds in Fig. 2i) is shown by white spots. The arrowheads and slim arrows show respectively the sectors of the nuclear globin RNA processing centers infiltrated by prosomes and relatively free of prosomes. The fat arrows point to the areas where the globin RNA coincident with p23K-type prosomes seems to protrude into the nucleoplasm, possibly in direction of the cytoplasm. Note the large cytoplasmic spots where partial dissociation of globin mRNA (red) and coincident prosomes (white dots) can be suspected. (b) is a magnification of (a).

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DISCUSSION

To allow interpretation of the IIF (indirect immunofluorescence) data shown, it must be recalled that in no cells tested so far were free prosomal proteins found, except under heat-shock conditions [28] (discussion in [4]). The IIF patterns shown relate, hence, systematically to prosome particles and not to free protein antigens. The main result of the present investigation is the observation of a striking colocalization of the p23Ktype prosomes present in erythroid cell nuclei, with the centers of processing (PCs) of the a-type globin gene transcripts. Another observation of interest is that of p23K prosome-associated globin mRNA in restricted areas of the cytoplasm (ACs). These findings hint to the possibility that there are unknown mechanisms involving prosome function which may be at the basis of the colocalization observed. It should also be pointed out that the observation of different staining patterns, with antibodies to three different subunit proteins of the 20S prosome/proteasome particles, is in itself important: it demonstrates once again that prosomes of higher eukaryotes have a variable mosaic composition. Indeed, it is clear that the p23K-containing prosomes cannot possibly contain either the p27K or the p30K subunits. Otherwise, all three antibodies would give similar patterns of staining in the cytoplasm and the nuclei. In recent years, the main emphasis in research on the system of prosomes/proteasomes has been largely confined to studies of MCP activity, often believed to be the main if not exclusive functionally significant feature of this system [5]. The new evidence mentioned in the Introduction about the RNase activity of prosomes [6, 7] and the absence of proteasome function in adapted human lymphocytes [8] has questioned this view. It seems to confirm our old [1] as well as more recent data (discussion in [4]) which suggest that prosomes have other functions beyond proteolysis. The results of the present paper imply that prosomes may be present and, hence, possibly associated in some way with the pathway of nuclear RNA metabolism and transport. From our earlier studies on nuclear and cytoplasmic (globin) mRNA and RNPs, several notions have emerged which relate to the observations reported here. By the criterion of two-dimensional gel electrophoresis, nuclear and cytoplasmic mRNAs were found to be associated with different sets of proteins [29]. However, the fact that prosomes are present in both the nucleus and the cytoplasm might constitute a notable exception to this rule. This is of particular interest in view of their presence in (globin) mRNP of the cytoplasm [3] as well as in the nuclear proteinaceous matrix and matrix-type RNPs (Pilotti et al., submit-

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ted). Although not formally proven (actually in investigation), a possible participation of the prosome system throughout the (pre-)mRNA processing and transport pathways can, hence, be suspected. It is known that prosomes interact directly with actin [10], which is a component of both the nuclear matrix [30] and the microfilament/stress fiber system of the cytoskeleton. Quite obviously, prosomes have the capacity to interact via their outer rings constituted by seven a-type subunits (in variable combination) with RNA, in view of their RNase activity. Furthermore, they interact with a vast spectrum of proteins including actin [10] and RNP proteins (discussion in [4]). In view of the molecular size of the 20S particles (12 3 17 nm), they could constitute bifunctional selective linkers of RNP to the nuclear matrix and the cytoskeleton (discussion in [10]). The other consideration relating to the surprising observations reported here concerns (globin) premRNA metabolism in erythroblasts. There are three distinct metabolic classes of nuclear pre-mRNAs with half-lives of 20 min and 3 and 12 h [31]; the least stable nuclear RNA has also the highest M r corresponding, hence, to the “giant” primary transcripts. In steady state, the largest fraction of accumulating nuclear RNA is processed, therefore, and of relatively low M r compared to primary transcripts. The one or two spots observed here by in situ hybridization where globin transcripts accumulate must, hence, be interpreted as being PCs rather than sites of transcription. The somewhat peripheral localization of the 23Kprosomes in the PCs and their extension into the nucleoplasm (fat arrows in Fig. 3) might thus be quite significant. Indeed, prosomes were previously shown to be present on primary transcripts of (lampbrush) chromosome loops [32] as well as in the RNA-based nuclear matrix of myoblasts (Pilotti et al., submitted). Their accumulation in the PCs may thus be interpreted as being due either to the block of productive globin premRNA processing, in these uninduced or pseudoinduced AEV cells, or to a steady-state of pre-mRNA flow through the PCs, in a manner analogous to that of the preribosomes in the granular area of nucleoli [33]. The quite obvious question arose if the PCs identified here might correspond to the well known “speckles” of the nucleus which were interpreted as centers of accumulation of splicing factors [34]. The size of our PCs (about 10 mm in diameter) seems to exceed largely the size of classical speckles (about 1 mm in diameter) by at least one order of magnitude. In preliminary experiments we found by double labeling that, indeed, the staining pattern obtained with the anti-SC-35 antibody (which recognizes the splicing factor SC-35 [35]) differs significantly from that of the PCs; however, interestingly, some speckles seem to align at the periphery of the PCs (De Conto, unpublished observa-

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tion). This is in line with data about pre-mRNA processing occurring at the periphery of speckles [36]. In the cytoplasm, the observation of specific ACs for the p23K prosomes in association with globin mRNA is most intriguing. This new finding contrasts with that of the ubiquitous distribution of (translated) bulk globin mRNA in induced AEV cells (De Conto et al., in preparation; Sjakste et al., in preparation). Several types of mRNA had already been found to be highly localized in the cytoplasm of animal cells, e.g., vimentin, desmin, vinculin, titin mRNA in myofibrils [37– 39], and, particularly intriguingly, histone mRNA [40]. As globin mRNA, the latter does not code for a structural protein, possibly assembled in a cotranslational manner. It was shown right from the beginning of the prosome story that prosomes are absent from polyribosomes [1] (discussion in [4]). The apparent contradiction in ubiquitous globin mRNA localization (see Fig. 2a– c) and sectorization of p23K-prosome contiguous to globin mRNA may be explained by assuming that the ACs contain only the untranslated globin mRNA. Support for this interpretation stems from the fact that the AEV cells shown in Figs. 2f, 2i, and 3 have few globin transcripts in the cytoplasm and are, thus, obviously in very early stages of mRNA transfer. In conclusion, it seems appropriate to say that the presence of specific types of prosomes in the specific globin mRNA compartments observed here may lead to new investigations of interest concerning both transcript processing and transport, as well as prosome function at mRNP and matrix/cytoskeleton levels. This research was supported by grants from the Centre National de la Recherche Franc¸aise (CNRS); the Association pour la Recherche contre le Cancer (ARC); the Russian Foundation for Support of Fundamental Science (Grant 99-04-49204); Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST), FIN’97; and MURST, FIL’97. S. V. Razin was a Visiting Professor of the CNRS and the University Paris 7 “Denis Diderot,” the recipient of a shortterm EMBO Fellowship, and the recipient of a fellowship from the Fondation pour la Recherche Me´dicale Franc¸aise. F. De Conto was supported by the fellowship “Premio di Studio Rodolphe Me´rieux” 1997.

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