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(The FASEB Journal. 1998;12:299-306.)
© 1998 FASEB

RESEARCH COMMUNICATION

Translational control: a general mechanism for gene regulation during T cell activation

Jose A. Garcia-Sanza,b,1, Wolfgang Mikulitsc, Alexandra Livingstone2,a, Ivan Lefkovitsa, and Ernst W. Müllnerc

a Basel Institute for Immunology, CH-4005 Basel, Switzerland
b Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Universidad Autónoma, Campus de Cantoblanco, E-28049 Madrid, Spain
c Institute of Molecular Biology, Vienna Biocenter, University of Vienna,A-1030 Vienna, Austria


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Distributional changes of individual mRNAs between free ribonucleoprotein particles (mRNP) and ribosome-bound transcripts are used to assess translational control. Simultaneous analysis of many mRNA species is required to estimate the overall contribution of translation to the regulation of gene expression. To this purpose, total cytoplasmic RNA was fractionated in sucrose step gradients and poly(A)+ RNA was prepared from mRNP and ribosome-bound fractions. Since direct, simultaneous analysis of a profusion of mRNAs is not feasible, distribution of their in vitro translation products was examined after separation in 2-dimensional gels, followed by computer-based analysis of autoradiographs. When this analysis was applied to antigenically stimulated T cells, 36% of in vitro translation products showed a greater than 10-fold increase in intensity, suggesting transcriptional activation of the corresponding mRNAs. In comparison, 7.9% of individual mRNAs (54 of 685 species) were translationally activated. They were redistributed from free mRNP to ribosome-associated fractions; 4.7% (32 species) were translationally repressed, as indicated by the opposite pattern. The differential recruitment of 12.6% of mRNA species demonstrates specificity and the general significance of translational control during T cell activation, which implies that translation may play a similar role in regulating gene expression in a variety of physiological processes.—Garcia-Sanz, J. A., Mikulits, W., Livingstone, A., Lefkovits, I., Müllner, E. W. Translational control: a general mechanism for gene regulation during T cell activation. FASEB J. 12, 299–306 (1998)


Key Words: translation initiation • mRNP particles • polysome-bound mRNA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRANSLATIONAL CONTROL is critical in regulating a variety of physiological processes in eukaryotic cells (1, 2), including development (3–6), cell differentiation and proliferation (7), protection of cells from external damage (8), and regulation of metabolic pathways (9). Translational control is characterized by the differential utilization of preexisting mRNAs, which can ensure rapid, efficient production of critical gene products without the time lag resulting from RNA synthesis and processing. Changes either in pool size or the phosphorylation state of translation initiation factors (eIFs)3 (10) leads to general variations in translation efficiency (9, 11, 12). Specific translational control is achieved for the vast majority of known examples by changes in the translation initiation rate (13, 14). Other mechanisms responsible for specific translational control include alterations in translation elongation (15, 16) or poly(A) tail-length modulation (17). Initiation rate changes involve mainly the interaction of trans-acting factors (proteins or occasionally RNAs) with cis-acting elements located in the 5' and/or 3' untranslated regions of a particular mRNA (1, 2, 18).

Translational regulation has been described in many individual genes, but the most important parameter—the overall contribution of translational control to a specific physiological process—remains unknown. A strategy was devised to analyze the complexity of translationally regulated mRNAs by using a combination of subcellular RNA fractionation into polysome-bound and free ribonucleoprotein particle (mRNP) fractions, followed by in vitro translation of these fractions, separation of the reaction products by 2-dimensional (2D) gels, and computer-based analysis.

A mouse T cell clone, before and after antigenic stimulation, was used as a source of free mRNP and polysome-bound mRNAs; unstimulated T lymphocytes (naive or memory) have undetectable DNA synthesis rates and low rates of RNA and protein synthesis. After antigenic or mitogenic stimulation, there is a burst of protein synthesis (19, 20) that correlates with elevated {epsilon}IF expression and phosphorylation (21–23). These changes are followed by the induction of more than 100 genes (24) and entry into the cell cycle, leading to a limited number of cell divisions (24, 25). Our analysis clearly indicates that a subset of individual mRNA species are translationally controlled. The percentage of translationally controlled mRNA species is comparable to the percentage of transcriptionally activated mRNAs, showing that translational control contributes significantly to the changes in gene expression that result in T cell activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and culture conditions
The murine CD4+ T cell clone 11.3.7 recognizing horse myoglobin (26) was cultured at 1 x 106 cells/ml in Iscove's modified Dulbecco's medium (containing 7% heat-inactivated fetal calf serum, 2 mM L-glutamine, 0.05 mM ß-mercaptoethanol, and antibiotics) and maintained by restimulation every 2 wk with antigen. Two weeks after the last stimulation, live cells were purified on Ficoll (Pharmacia, Stockholm, Sweden) and rested for 3 additional days in medium (resting cells), or restimulated (1x107 cells in 10 ml) with 5 µM horse myoglobin (Sigma, St. Louis, Mo.) in the presence 2.5 x 107 irradiated (3300 rads) syngeneic spleen cells. Twenty-four hours later, 15 ml culture medium and 0.25 ml of recombinant mouse interleukin 2 (IL-2) supernatant (27) were added. Cells were cultured for an additional 48 h (activated cells), as described (26). Effective stimulation (increase of S-phase-specific cells) was monitored by cytofluorometry after propidium iodine staining of DNA. Resting and activated cells were Ficoll purified, washed, and used to prepare RNA.

RNA fractionation in sucrose step gradients
Harvesting, cell lysis, and the preparation of sucrose step gradients were performed as described for polysome gradients (28). In brief, cells were lysed in 1 ml of extraction buffer [10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.5, 3 mM MgCl2, 40 mM KCl, 5% glycerol, 0.5% (v/v) NP-40, and 400 U/ml RNasin (Promega, Madison, Wis.)]. After removal of nuclei, the cytosolic supernatant was supplemented with 150 mg/ml cycloheximide, 1 mM phenyl-methyl-sulfonyl- fluoride, 5 mg/ml heparin, and 20 mM dithiothreitol. Mitochondria and membrane debris were removed by centrifugation, and 1 ml of cytosolic extract was layered on a 4 ml sucrose cushion (30% w/v) in extraction buffer supplemented with 0.5 mg/ml heparin, 10 mM dithiothreitol, and 100 µg/ml cycloheximide. The samples were centrifuged (130,000xg, 2 h, 4°C in a TST-55 rotor) and 1 ml fractions were collected and deproteinized with 250 µg/ml proteinase K in the presence of 1% (w/v) sodium dodecyl sulfate. After phenol-chloroform extraction and ethanol precipitation, samples were dissolved in 100 µl H2O and the amount of RNA in each fraction was determined photometrically.

Northern blot analysis and polyadenylated mRNA preparation
Aliquots of the cytoplasmic RNA samples were separated on denaturing formaldehyde-agarose gels, followed by RNA staining and transfer to nylon membranes. Northern hybridization, using random-primed 32P-labeled cDNA probes specific for mouse ß-actin (29), eIF1{alpha} mRNA, and mouse ribosomal protein L32 (rpL32) cDNA (obtained from K. Campbell, Basel Institute for Immunology), and washing were both performed as described (30). The bulk of the RNA was used for quantitative preparation of polyadenylated mRNA [poly(A)+ mRNA] from ribosome-free and ribosome-bound fractions, from both resting and activated T cells, using oligotex(dT)tm beads (Qiagen, Hilden, Germany) as described (31).

In vitro translation reactions and computer analysis of the 2D gels
Ribosome-free and ribosome-bound poly(A)+ mRNA samples from resting and activated T cells were translated in vitro using nuclease-treated rabbit reticulocyte lysates (Promega), in the presence of 35S-methionine (Amersham, Little Chalfont, U.K.) (32). Reaction products were separated by 2D gel electrophoresis according to O'Farrell (33), using the ISODALT system (34). The resulting radiofluorographs were scanned and subjected to image analysis with the Kepler software system (Large Scale Biology Corporation, Rockville, Md.) as described (35). Briefly, image files were processed for noise and streak removal as well as background correction. The processed image files were converted into spot files by spot modeling and fitting. This transformation reduced data size by three orders of magnitude. In the abstracted image files or `spot lists', each spot was defined by five parameters: x and y coordinates, spot volume parameters sx, sy, and amplitude. One spot list pattern was chosen, assigned a numbering system, and termed the master pattern, to which all others were subsequently compared. All spot list patterns were then matched to the master pattern and individual spots received numbers, called master spot numbers, congruent with the master spots. Spots on individual patterns that did not occur on the master pattern were transferred into it with a utility provided by the program. At the end of the matching process, the master pattern contained all spots occurring in any of the patterns. Information on each and all spots was stored in the database, which kept track of all images, spot lists, and spot identities and maintained congruence within the system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Translation initiation is thought to be the rate-limiting step in protein synthesis (13). Potential targets for translational control during T cell activation should show changes in ribosome binding. Thus, in one T cell state (resting or activated), these transcripts would not be associated to ribosomes, but would become ribosome bound in the contrary state. RNA from resting and activated cells of the mouse CD4+ T cell clone 11.3.7 was fractionated into ribosome-free and ribosome-bound compartments using 30% sucrose step gradients. Efficient separation was proven by the presence of 28S and 18S rRNAs in fractions 2 to 5 (ribosome-bound) and by their complete absence in fraction 1 (free ribonucleoprotein particles, RNPs) ( Fig. 1). In resting T cells, 85% of total cytoplasmic mRNA was associated with ribosomes, whereas the remaining 15% was recovered as free mRNPs. In activated T cells, these percentages were 90% and 10%, respectively ( Table 1). Quantitative poly(A)+ purification revealed the same relative mRNA abundance in both free mRNP (fraction 1) and ribosome-bound samples (pool of fractions 3, 4, and 5) (3.0–3.7%), indicating that mRNAs are not preferentially associated with polysomes ( Table 1). Similar data were obtained with different T cell lines and primary human T lymphocytes under various conditions of growth or activation (J. A. Garcia-Sanz, and E. W. Müllner, data not shown). To monitor the distribution of individual mRNA species upon lymphocyte activation, we used cDNA probes for eIF1{alpha} and rpL32 mRNA, two known targets for translational regulation (36), and ß-actin mRNA, a gene whose expression is controlled mainly at the transcriptional level. As predicted, there was a clear redistribution upon T cell activation of free eIF1{alpha} and rpL32 transcripts from the free mRNP fraction toward polysome-bound fractions, whereas ß-actin mRNA remained exclusively ribosome associated ( Fig. 1).



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  		Figure 1. Sucrose step gradient fractionation of cytoplasmic RNA allows the detection of translationally regulated mRNAs. RNAs from resting and activated T cells were fractionated into ribosome-free and ribosome-bound compartments by using 30% sucrose cushions. A) As a control for cell status, DNA profiles of T cells before and after activation were measured by flow cytometry after propidium iodine staining. B) The integrity of RNA and the quality of the fractionation were demonstrated by ethidium bromide staining of samples after electrophoresis in denaturing formaldehyde agarose gels. C) After transfer to nylon membranes, Northern blots were sequentially hybridzed with mouse ß-actin, eF1{alpha}, and rpL32 cDNA probes. Fraction 2 was discarded from further processing to avoid possible cross-contamination with RNA from the ribosome-free fraction.


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  		Table 1. Analysis of the ribosome-free and ribosome-bound fractionated RNA and in vitro translated products

Since the degree of ribosome association cannot be simultaneously determined for many mRNA species, as an alternative we analyzed the distribution of the corresponding in vitro translation products after separating the samples in 2D gel electrophoresis. The processed images of the resulting radiofluorographs from an experiment using the 11.3.7 mouse T cell clone before and after antigenic stimulation are represented in Fig. 2. The mRNAs from the free mRNP fraction were not associated with ribosomes and obviously not translated in vivo. They were, however, efficiently translated in vitro ( Fig. 2, top panels). 2D gel coordinates for the majority of in vitro translation products from the free mRNP fractions could be precisely matched to corresponding products from the ribosome-bound fractions (65% in resting and 79% in activated T cells) ( Fig. 2, Fig. 3B). These data demonstrate that mRNAs in the free mRNP fraction are functional transcripts, indicate that the cells possess a means of actively suppressing translation of a subset of transcripts, and show the specificity of translational control as verified by the differential distribution of ß-actin and rpL32 mRNAs ( Fig. 1). In addition, the average amount of individual in vitro translation product from ribosome-free and ribosome-bound fractions was similar ( Fig. 3A). Thus, the presence of free mRNAs could not be explained by a limitation in the capacity of the cellular translation machinery.



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  		Figure 2. Analysis of mRNAs translationally regulated upon T cell activation poly(A)+ RNAs from ribosome-free (fraction 1, Fig. 1) and ribosome-bound (fractions 3–5, Fig. 1) samples, derived from resting and activated T cells, were used as substrates for in vitro translation reactions in the presence of 35S-methionine, using nuclease-treated rabbit reticulocyte lysates. Reaction products were separated by 2D gel electrophoresis, using the ISODALT system (see Materials and Methods). The resulting radiofluorographs were scanned and subjected to image analysis with the Kepler software system (see Material and Methods). Spot size corresponds to area in the autoradiograms. After T cell activation, distributional changes bigger than twofold from the ribosome-free toward the ribosome-bound fractions were identified as products of translationally activated mRNAs (red spots), whereas changes of the same magnitude from the ribosome-bound toward the ribosome-free fractions were identified as products of translationally repressed mRNAs (blue spots). Black spots represent an arbitrary subset of proteins present in all four 2D gels and were used as landmarks.



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  		Figure 3. A) Relative abundance of in vitro translation products from ribosome-free and ribosome-bound fractions. Quantitation of translation products after 2D gel electrophoresis was performed as described in the legend to Fig. 2. The spots were ordered by decreasing intensity and plotted against a running index on two independent x-axes: on the top for products from the ribosome-free mRNAs, on the bottom for the ribosome-bound transcripts. The relative intensity profiles for the spots were similar for ribosome-free and ribosome-bound mRNAs in both resting and activated T cells. B) Spot intensity ratio (ribosome-free/ribosome-bound) for each individual species present in the ribosome-free compartment. For 85 spots (19.1%) in resting T cells and 67 (12.1%) in activated T cells, the spot intensity ratio was >= 0.5, indicating that at least 33% of the respective mRNA were present in the free mRNP fraction. Spots with a ratio >= 100 represent transcripts detected exclusively in the ribosome-free fraction.

To detect transcripts that may undergo translational activation or repression upon T lymphocyte activation, the 2D gels were analyzed quantitatively. Breakdown of the raw data revealed that 394 of 445 spots from resting T cells and 529 of 554 spots from activated T cells were ribosome associated, indicating that the large majority of mRNA species were actively engaged in protein synthesis. However, 96 spots (in both resting and activated T cells) corresponded to mRNAs present simultaneously in both fractions. An additional 51 spots (11.5%) from resting and 25 spots (4.5%) from activated T cells corresponded to mRNAs found exclusively in the free mRNP fraction ( Fig. 3B). The apparent translational inhibition for these transcripts was therefore complete, since the products were unique to the free mRNP fraction.

During T cell activation, translationally controlled mRNAs should redistribute between free mRNP and ribosome-associated compartments. This, in turn, should be mirrored by relative intensity changes in the in vitro translation products. In this study, species were considered translationally activated when the spot intensity ratio (free/bound) of resting cells divided by the (free/bound) ratio of activated cells was >= 2; translational repression was assumed when this ratio was <= 0.5. Spots were excluded from this analysis if their absolute intensity was close to the detection limit. Using this criterion, 86 mRNA species of a total of 685 (12.6%) appeared to be translationally regulated ( Fig. 2, Fig. 4). Of these, 54 (7.9%) corresponded to translationally activated transcripts ( Fig. 4, top). Despite a general increase in translation efficiency after T cell activation (20, 36), another 32 species (4.7%) represented translationally repressed mRNA species ( Fig. 4, bottom). This demonstrates the specificity of the process, since the increased translation rate does not simply cause more effective ribosome engagement by a subset of poorly translated mRNAs; rather, a group of transcripts is translationally repressed under these conditions.



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  		Figure 4. Translational control of individual mRNAs after T cell activation. An identification number was assigned to each spot from the 2D gels during the matching process. The relative spot intensities from mRNAs with changes in translational efficiency are shown in the bar graphs. In the top panel, each bar graph corresponds to one of the red spots in Fig. 2. In the bottom panel, each bar graph corresponds to one of the blue spots on Fig. 2. Each individual graph displays the relative amount of a translation product in the ribosome-free (open bars) and ribosome-bound fractions (filled bars) in both resting (R) and activated (A) T cells. The highest value in each graph was normalized to 100% to facilitate comparison. Spots were excluded from this analysis if their absolute intensity was close to the limit of detection.

It is well established that many genes are transcriptionally activated upon T cell activation. From these 2D gels, we could also estimate the complexity of mRNAs that appear or disappear after T lymphocyte activation. Upon T cell activation, the intensity of 249 species (36%) increased by at least 10-fold; another 96 species (14%) showed a decrease of the same magnitude. The vast majority of these products may represent transcriptionally regulated mRNAs, although we cannot formally exclude the possibility that some correspond to trancripts exhibiting changes in mRNA stability, processing, or export efficiency ( Fig. 5). This combination of techniques thus allowed direct comparison of the transcript fraction regulated by changes in the total amount and the percentage of translationally controlled transcripts in a particular physiological transition.



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  		Figure 5. Analysis of mRNAs regulated by changes in total amount upon T cell activation. As in Fig. 2, the redistribution of individual in vitro translation products was taken as an indication of redistribution of their corresponding mRNAs. Red indicates in vitro translation products with an increase of intensities of more than 10-fold in intensity upon stimulation. Blue spots correspond to products with more than a 10-fold decrease. The majority of their corresponding mRNAs most likely represent transcriptionally activated and transcriptionally repressed mRNAs, respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results indicate that a significant fraction of the cytoplasmic mRNA is not ribosome bound, and therefore not translated, in both resting and activated T cells. The differential distribution of ß-actin, eIF1{alpha}, and rPL32 mRNAs between free mRNP and polysome-bound fractions directly demonstrated the specificity of the distribution. The mRNAs present in the free mRNP fraction were functional, since they contained poly(A)-tail, and after deproteinization, could be translated in vitro with rabbit reticulocyte lysates, probably into full-length proteins, as confirmed by analysis of the 2D gels ( Fig. 2, Fig. 4).

The presence of apparently intact mRNA species in the free mRNP fraction was unlikely to be explained by any limitation of the cellular translation machinery, since 1) individual mRNA species were affected differently, and 2) despite the general increase in translation efficiency that takes place upon T cell activation, translation of 5.4% of the mRNA species was repressed. Rather, it indicates the existence of physiological mechanisms regulating the translation of single mRNA species or whole mRNA subclasses. This interpretation was strengthened by the observation that a significant fraction of untranslated mRNAs was mobilized into polysomes after T cell activation, whereas others that were previously actively engaged in translation became repressed. Although this regulation would take place at the level of translational initiation, which might involve whole classes of mRNAs with common structural determinants, we cannot distinguish whether it involves specific mRNA binding proteins such as the one involved in the control of ferritin mRNA (37) or a more general mechanism such as signal-dependent phosphorylation of translation factors (38).

Since the rate-limiting step in protein synthesis is thought to be translation initiation (13), we hypothesized that most translationally repressed mRNAs should be devoid of ribosomes. After physiological changes in the cell (i.e., T cell activation), some of these mRNAs would become ribosome bound. A strategy was hence devised to combine a series of well-established techniques to fractionate free mRNPs from polysome-associated mRNAs and to identify their particular protein products on a large scale, allowing statistical analysis. The results indicate that translational control has a prominent role in regulating gene expression during T cell activation. The percentage of mRNA species affected is, indeed, of the same order of magnitude as transcriptionally controlled ones. Translational regulation may be helpful in inducing rapid de novo synthesis of critical regulatory proteins, and would bypass the requirement for extracellular signal transduction pathways to the nucleus, transcription, processing, and nuclear RNA export.

If specific regulation at the translational level is an important step, it would be expected that deregulation produces a discernible phenotype. It has been described that alterations in translation mechanisms have the potential to form the molecular basis of certain diseases. For instance, overexpression of a dominant negative mutant of the {gamma}-interferon-induced p65 kinase or mutation of its substrate, eIF2{alpha}, gives rise to malignant transformation (39–41). Overexpression of initiation factors such as the cap binding protein eIF4E also leads to malignant transformation (42), suggesting that increased translation efficiency, and not the mere mutation of these factors, is responsible for the malignant transformation. Translational control of c-myc and p53 proto-oncogenes had also been implicated in human diseases such as multiple myeloma and acute myelogenous leukemia, respectively (43, 44). In T lymphocytes, translational control of the growth factor IL-2 has been described as one of the molecular mechanisms responsible for the induction of T cell clonal anergy, a phenomenon characterized by the lack of response of the anergic T cells to subsequent stimulation (45). Our data allow us to hypothesize that the prominent role of translational control regulating gene expression is not restricted to T cell activation, but is a general phenomenon involved in a variety of other physiological transitions including macrophage activation, lactogenesis, tumorigenesis, or apoptosis. The approach described here can be applied extensively to determine the complexity of translationally controlled mRNAs in these and other situations.

The nature of mRNAs translationally regulated after T cell activation is still unknown. Experiments are currently under way to isolate their cDNAs, which will enable identification and characterization of the trans-acting factors that modulate translation initiation of individual transcripts or structurally related mRNA subfamilies.


   ACKNOWLEDGMENTS
 
We thank Dr. Pierre Cosson for continuous encouragement, critical and helpful comments, and Dr. Kerry Campbell for critical reading of the manuscript and providing the rPL32 cDNA probe. We are grateful to Lotte Kuhn, Marina Kuhn, and Danielle Lenig for excellent technical assistance, Mr. J. Widmer and Dr. J. R. Frey for their help with data handling and analysis, Ms. Catherine Mark for editorial assistance, and Ms. B. Pfeiffer and I. Poveda for artwork. The Basel Institute for Immunology was founded and is supported by F. Hoffman-La Roche Ltd. Basel, Switzerland. The Department of Immunology and Oncology was founded and is supported by the C.S.I.C., and Pharmacia Upjohn. This research was partially supported by the Jubiläumsfonds der Österreichischen Nationalbank and the Fonds zur Förderung der Wissenschaftlichen Forschung, Austria (grants to E.W.M.).


   FOOTNOTES
 
2 Present address: Department of Biology, Imperial College of Science, Technology and Medicine, London, U.K.

1 Correspondence: Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Universidad Autónoma, Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail: jasanz{at}cnb.uam.es

3 Abbreviations: 2D gels, 2-dimensional gels; eIF, eukaryotic translation initiation factors; IL-2, interleukin 2; mRNPs, free ribonucleoprotein particles; poly(A)+ mRNA, polyadenylated mRNA; rpL32, ribosomal protein L32.

Received for publication May 12, 1997. Accepted for publication October 30, 1997.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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