©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Post-transcriptional Regulation of Human Interleukin-2 Gene Expression at Processing of Precursor Transcripts (*)

(Received for publication, March 8, 1995; and in revised form, May 22, 1995)

Lisya Gerez Gila Arad Shimon Efrat (§) Mali Ketzinel Raymond Kaempfer (¶)

From the Department of Molecular Virology, The Hebrew University, Hadassah Medical School, 91120 Jerusalem, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Interleukin-2 (IL-2) regulates the clonal expansion of activated T cells and is produced in limited amounts during an immune response. Mitogenic induction of human IL-2 gene expression elicits a transient wave of unstable mRNA. We show here that transcription continues unabated during and well beyond the time when the wave is subsiding, yet few, if any, new mRNA molecules are generated once the wave has reached its maximum. Instead, IL-2 precursor transcripts accumulate, becoming the majority of expressed IL-2 RNA molecules. The flow of precursor transcripts into mature mRNA becomes inhibited in the course of induction. When translation is blocked (e.g. by cycloheximide), expression of IL-2 mRNA can be superinduced by 2 orders of magnitude. This superinduction is completely dependent upon transcription, yet is not accompanied by any significant increase in the rate of primary transcription or in mRNA stability. Instead, the processing of nuclear IL-2 precursor transcripts is greatly facilitated, resulting in pronounced superinduction of cytoplasmic mRNA. Once its transcription has been induced, therefore, expression of the IL-2 gene is down-regulated extensively at the level of precursor RNA processing.


INTRODUCTION

Interleukin-2 (IL-2) (^1)plays a central role in the cellular immune response, for it regulates the clonal expansion of activated T cells and is a powerful immunomodulator(1, 2) . The strength of an immune reaction is regulated largely by the amount of IL-2 produced in response to a stimulus(1) . These properties underscore the importance of understanding mechanisms that regulate IL-2 gene expression.

Induction of IL-2 gene expression in human lymphoid cell populations, elicited upon mitogenic stimulation, yields a transient wave of mRNA(3, 4) . This induced expression is sensitive to cell-mediated suppression. Levels of IL-2 mRNA rise an order of magnitude upon depletion of CD8 cells or after low doses of -irradiation(5) , a treatment considered to prevent the activation of cells with suppressive capacity(6, 7) . Concomitant with the induction of IL-2 gene expression, mitogenic stimulation induces a transient activation of cells, including the CD8 subset, possessing the ability to effectively inhibit the expression of these genes(5, 8) . Induction of IL-2 mRNA largely precedes the appearance of inhibitory cell activity, allowing expression to occur before a dominant state of suppression is established(5) .

In addition to being sensitive to suppression by CD8 cells, expression of IL-2 mRNA can be superinduced extensively by inhibitors of translation such as cycloheximide (CHX) (4, 9) without any increase in the rate of primary transcription(10, 11) . These results show that during induction, the strength of the mRNA signal is greatly reduced by a post-transcriptional mechanism. Together with cell-mediated suppression, this control mechanism causes the IL-2 gene to be expressed to only a small proportion of its full potential, thereby rendering expression particularly sensitive to regulation by external signals.

Stability of IL-2 mRNA is sensitive to the nature of the inducer, increasing upon exposure of cells to CD28 monoclonal antibodies(12) . AUUUA motifs in the 3`-untranslated region of a number of cytokine and proto-oncogene mRNA species contribute to their instability(13, 14) ; inhibition of translation led to stabilization of these mRNAs. Such sequences are also found in IL-2 mRNA. Bovine IL-2 mRNA was stable in extract from fibroblasts yet unstable in extract from bovine lymphocytes where stability could be increased by pretreatment of cells with CHX(15) . Stability of IL-2 mRNA thus may be controlled in a cell-specific manner. Destabilization of c-fos, c-myc, and granulocyte-macrophage colony-stimulating factor mRNA requires translation of their open reading frames(16, 17, 18) . CD28, moreover, caused stabilization of IL-2, interferon-, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor mRNA but not of c-fos and c-myc mRNA despite the presence of AUUUA motifs in each (12) . These results show that sequences other than the AUUUA motif are involved in regulating mRNA stability(19) .

Here, we have analyzed the nature of the CHX-sensitive mechanism that inhibits IL-2 gene expression in human lymphoid cell populations. This inhibition, we show, is based primarily on a block in post-transcriptional processing of nuclear precursor transcripts that develops shortly after the onset of induction. Long before transcription of the IL-2 gene begins to decline, further generation of new IL-2 mRNA molecules is thus prevented. The induced mRNA is unstable and in the absence of further formation, its decay generates a transient wave of mRNA. In the presence of CHX, little, if any, stabilization of IL-2 mRNA is observed. Instead, processing of IL-2 precursor transcripts is greatly facilitated, resulting in extensive superinduction of cytoplasmic mRNA.


EXPERIMENTAL PROCEDURES

Cell Culture and Induction

Human tonsil primary cells were prepared as described(4) . PBMC were separated from buffy coat from healthy donors on Ficoll Paque (Pharmacia) and washed three times with 50 ml of RPMI 1640 medium. Cells were resuspended at a density of 4 10^6/ml and preincubated overnight at 37 °C in RPMI 1640 medium containing 2% fetal calf serum, 2 mM glutamine, 10 mM minimum Eagle's medium non-essential amino acids, 100 mM sodium pyruvate, 10 mM Hepes, pH 7.2, 5 10M 2-mercaptoethanol, 100 units/ml penicillin, 100 µg/ml streptomycin, 40 µg/ml gentamycin, and 5 µg/ml nystatin. Induction was with 4% PHA (PHA-P, Difco); where indicated, CHX (Sigma) was added to a final concentration of 20 µg/ml, emetine (Sigma) was added to 10 µg/ml, actinomycin D (Merck) was added to 10 µg/ml, or cyclosporin A (Sandoz) was added to 1 µg/ml. Cell viability was followed by trypan blue exclusion.

Plasmids and Hybridization Probes

A 588-base pair XbaI-MscI fragment of IL-2 genomic DNA (20) consisting of adjoining segments of exon 3 and intron 3 was inserted into pBS (Stratagene) under the T7 promoter. Plasmid DNA was digested with XbaI and transcribed using [alpha-P]UTP to generate labeled antisense RNA transcript SC1. A 415-nt beta-actin probe was transcribed from cDNA inserted into pBS under the T3 promoter. A 650-base pair fragment of IL-2 cDNA excised from plasmid p-316 (21) was labeled by nick translation to generate cDNA probe.

Quantitation of mRNA in Northern Blots

Extraction of total RNA, agarose/formaldehyde gel electrophoresis, and hybridization analysis were done as described(22) .

Ribonuclease Protection Analysis

Total RNA was isolated by the guanidinium isothiocyanate/CsCl method (23) or by guanidinium isothiocyanate/phenol extraction(24) . To prepare nuclear and cytoplasmic RNA fractions, nuclei and cytoplasm were separated (25) before RNA extraction by the guanidinium isothiocyanate/CsCl method. RNase protection analysis (26) was performed using genomic riboprobe SC1. Aliquots of 5 µg of extracted RNA were hybridized at 42 °C with 10^6 cpm of P-labeled RNA probe in 30 µl of 80% formamide, 40 mM Pipes, pH 6.7, 1 mM EDTA, and 0.4 M NaCl. After 18 h, 300 µl of 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 300 mM NaCl containing 1.4 µg/ml of RNase A and 0.07 µg/ml of RNase T1 were added, and incubation was continued for 15 min at 30 °C. The mixture was then brought to 0.075% SDS and digested for 15 min at 37 °C with 20 µg of proteinase K. RNA was extracted with phenol/chloroform and ethanol precipitated in the presence of 20 µg of Escherichia coli MRE600 tRNA. Protected RNA fragments were separated by electrophoresis in 8 M urea/5% polyacrylamide gels. Size markers were generated by MspI or HinfI digestion of pGEM-3 DNA and filling in with [alpha-P]dCTP and [alpha-P]dATP, respectively.

Nuclear Run-on Transcription Analysis

Nuclei from 2.8 10^8 PBMC were isolated from cells washed with RPMI 1640 medium by preincubating for 10 min on ice in hypotonic buffer (10 mM Hepes-KOH, pH 7.6, 25 mM KCl, 5 mM MgCl(2), 1 mM dithiothreitol, and 0.1 mM EDTA) before cell disruption by 10 strokes in a Dounce homogenizer. Nuclei were pelleted by centrifugation, resuspended in hypotonic buffer, layered over hypotonic buffer/25% glycerol, pelleted by centrifugation, resuspended into 0.5 ml of hypotonic buffer/25% glycerol, counted, and diluted to a final concentration of 2 10^8/ml. Elongation of nascent RNA was for 30 min at 30 °C in reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), 300 mM KCl, 3 mM dithiothreitol, 1 mM spermidine, 35 units of RNasin, and 1 mM each of CTP, ATP, GTP) in the presence of 160 µCi of [alpha-P]UTP. After treatment with DNase I (0.02 units/ml) for 15 min at 30 °C and proteinase K (0.3 mg/ml) for 30 min at 42 °C, labeled RNA was extracted with phenol/chloroform and precipitated with 5% trichloroacetic acid. The RNA was applied to a nitrocellulose filter that was dried and then incubated for 30 min at 30 °C in a solution of 5 units/ml of DNase I in 20 mM Hepes, pH 7.6, 5 mM MgCl(2), and 1 mM CaCl(2). After the addition of SDS to a final concentration of 0.03% and the addition of EDTA to 0.5 mM, incubation of the RNA filter was continued for 10 min at 65 °C. RNA was extracted in 5 mM EDTA, 1% SDS, and 10 mM Tris-HCl, pH 7.5, made 0.1 M in NaCl, and precipitated with ethanol. Aliquots of 10^6 cpm of labeled RNA were hybridized to cDNAs dot-blotted on nitrocellulose filters.


RESULTS

Superinduction of IL-2 mRNA

Addition of CHX to a culture of human tonsil cells 4 h after the onset of induction by PHA led to extensive superinduction of IL-2 mRNA, by at least 30-fold (Fig. 1A). Kinetics of a similar superinduction experiment are shown in Fig. 1, B and C. The effect of CHX was to increase the amplitude of the induced wave of IL-2 mRNA. Even in the presence of CHX, however, the level of IL-2 mRNA declined promptly after reaching its maximum. The rate of decline during superinduction was similar to that seen during normal induction (Fig. 1C).


Figure 1: Superinduction of IL-2 mRNA by CHX. Human tonsil cells were induced with PHA (bullet). Where indicated, CHX was included from 4 h onwards (). Cell viability remained constant. Total RNA was extracted at times indicated, subjected to formaldehyde/agarose gel electrophoresis and blot-hybridized with nick-translated IL-2 cDNA. Data for A and that for B and C represent 2 different experiments. Autoradiogram of B was quantitated by microdensitometry C; the amount of RNA is shown in arbitrary units. Size markers denote nucleotide length of single-stranded X174 RF DNA (HaeIII digest).



In Fig. 2A, induction of IL-2 mRNA was followed by RNase protection analysis using a 608-nt antisense RNA probe in which 117 nt are complementary to a portion of exon 3 and are thus protected by mature mRNA (cf. Fig. 9D). Induction of a culture of PBMC yielded a wave of IL-2 mRNA sequences that reached its maximum by 6-8 h, considerably earlier than in tonsil cells (Fig. 1), and then declined promptly (Fig. 2, A and C). Addition of CHX at 4 h yielded a pronounced superinduction of mRNA. Unlike for tonsil cells, however, CHX-mediated superinduction in PBMC was sustained; high levels of mRNA persisted up to 24 h (Fig. 2, A and C). A similar result was obtained when the translation inhibitor, emetine, was used (Fig. 2, B and D). When CHX (4) or emetine (Fig. 2B) was added at the start of induction together with PHA, expression of IL-2 mRNA was prevented.


Figure 2: Superinduction of IL-2 mRNA by CHX or emetine. PBMC were induced with PHA (). CHX (bullet) or emetine (▪) were included from 4 h onwards. Total RNA was extracted at times indicated and subjected to RNase protection analysis using IL-2 probe SC1 (see Fig. 9D) to detect mRNA (117-nt band). Data of A and B, representing 2 different experiments, were quantitated by microdensitometry; the amount of RNA is shown in arbitrary units in C and D, respectively. Analysis of beta-actin mRNA is shown for A. The size of protected RNA was calibrated with MspI-digested pGEM3 DNA (not shown).




Figure 9: Accumulation of IL-2 precursor transcripts during induction. PBMC were induced with PHA. Total RNA, extracted at times shown, was subjected to RNase protection analysis using IL-2 probe SC1 (D) to measure precursor transcripts (pre-mRNA) (▴) and mRNA () (A-C). Autoradiogram (A) was quantitated by microdensitometry (B). In C, the amount of IL-2 precursor transcripts and mRNA, corrected for content of U residues, is plotted as the percentage of total IL-2 RNA present.



Lack of Correlation between Accumulation of IL-2 mRNA and Rate of Transcription

The primary transcription rate of the IL-2 gene in the cell population that showed a strong superinduction response to CHX in Fig. 2A was studied by nuclear run-on analysis (Fig. 3). In agreement with earlier reports(10, 11) , there was no perceptible change in the rate of transcription in the presence of CHX. Transcription of beta-actin and rRNA genes, though induced by PHA, also did not respond to CHX.


Figure 3: Nuclear run-on analysis. The PBMC population studied in Fig. 2A was incubated with PHA for the times indicated. Where indicated, CHX was included from 4 h onwards. Intact nuclei were isolated and incubated to allow extension of nascent primary transcripts in the presence of [alpha-P]UTP. Nuclear RNA was extracted, and aliquots of 4 10^6 cpm were hybridized to dot-immobilized pGEM-3 DNA carrying cDNA inserts of the indicated genes. Hybridization controls were pGEM3 DNA without insert or containing either beta-actin cDNA or a human 18 S rDNA fragment.



In the cell culture that did not receive CHX, moreover, the rate of transcription increased after the onset of induction and then was sustained up to at least 24 h, even though by this time expression of IL-2 mRNA had returned to basal levels (Fig. 2A). Indeed, while transcription occurred at a high rate at 8 h (Fig. 3), IL-2 mRNA declined strongly by 9 h (Fig. 2A). These results show that transcription continues well beyond the time when the mRNA wave has reached its maximum, yet does not result in further accumulation of mRNA. Thus, a post-transcriptional mechanism prevents expression of IL-2 mRNA late in induction. This control is sensitive to CHX.

Indeed, the addition of CHX throughout induction, from 4 h onwards, elicited a prompt and significant superinduction of IL-2 mRNA at essentially the same rate both during the transient wave of mRNA and well after it had subsided (Fig. 4, B and D). Superinduction lasted for up to 55 h (Fig. 4, A and C). This prolonged responsiveness to CHX is explained by the sustained transcription of the IL-2 gene shown in Fig. 3.


Figure 4: Response to CHX throughout the course of induction. PBMC were induced with PHA (). CHX was included from times indicated (bullet, ▪). Total RNA, extracted at times shown, was subjected to RNase protection analysis using probe SC1. Autoradiograms of A and B represent two different experiments; microdensitometry is shown in C and D, respectively.



Dependence of Superinduction on Transcription

In the experiment of Fig. 5A, CHX-mediated superinduction of IL-2 mRNA (117-nt band) was especially pronounced at 24 h. This superinduction was prevented when actinomycin D was added simultaneously with CHX (Fig. 5, A and C). Addition of CHX after the wave of IL-2 mRNA had subsided (at 24 h) elicited extensive expression of IL-2 mRNA (Fig. 5, B and D; cf. Fig. 4); this rise in mRNA was blocked likewise by actinomycin D. Cyclosporin A, a transcription inhibitor specific for IL-2 and certain other cytokine genes, strongly reduced the superinduction response to CHX but did not block it completely (Fig. 6). Thus, superinduction of IL-2 mRNA by CHX depends on synthesis of new RNA molecules.


Figure 5: Dependence of superinduction on transcription. PBMC were induced with PHA (). CHX was added at 4 h (A, C) or 24 h (B, D) (bullet, ▪). Actinomycin D (ActD) was added at 4 or 24 h as shown in the absence () or the presence (▪) of CHX or at 0 h (). Cell viability remained constant. Total RNA, extracted at times shown, was subjected to RNase protection analysis using probe SC1 (see Fig. 9D) to detect mRNA (117-nt band) and pre-mRNA (588-nt band). In A and B, only those portions of the autoradiogram are shown that contain these 2 bands, but they derive from the same exposure. Autoradiograms of A and B represent 2 different experiments; microdensitometry of the 117-nt band is shown in C and D, respectively.




Figure 6: Inhibition of superinduction of IL-2 mRNA by cyclosporin A. PBMC were induced with PHA (). CHX was added at 4 h (bullet, ▪). Cyclosporin A (CsA) (100 ng/ml) was added at 0 h () or 4 h (▪) as shown. Cell viability remained constant. Total RNA, extracted at times shown, was subjected to RNase protection analysis using probe SC1. Autoradiogram (A) was quantitated by microdensitometry (B).



IL-2 mRNA Remains Unstable during Superinduction

The results of Fig. 5and Fig. 6show that superinduction elicited by CHX requires continuing transcription. Late in induction, vigorous transcription of the IL-2 gene takes place (Fig. 3) but does not yield mRNA (Fig. 2A). Because the rate of transcription is unaffected by CHX (Fig. 3), the dependence of superinduction on transcription could suggest that CHX acts to stabilize newly synthesized mRNA molecules, as shown for c-fos, c-myc, and granulocyte-macrophage colony-stimulating factor mRNA(13, 27, 28) . However, the observation in Fig. 1C that in tonsil cells, superinduction of IL-2 mRNA by CHX is followed by a prompt decline of the mRNA wave suggests that stabilization of mRNA cannot be the sole basis of superinduction. As seen in Fig. 5C, the rate of decay of IL-2 mRNA in the presence of actinomycin D was not affected perceptibly by CHX. To examine this point further, we studied the effect of actinomycin D on the decay of IL-2 mRNA during normal induction (Fig. 7). Addition of actinomycin D together with PHA prevented the appearance of the mRNA wave. Addition at a later time (just before the mRNA wave declined) did not elicit significantly more rapid decay than in the untreated control (Fig. 7B).


Figure 7: Effect of actinomycin D on IL-2 mRNA induction. PBMC were induced with PHA (). Actinomycin D (ActD) was added at 0 h () or 4 h (▪) as shown. Cell viability remained constant. Total RNA, extracted at times shown, was subjected to RNase protection analysis using probe SC1. Autoradiogram (A) was quantitated by microdensitometry (B).



This result shows that IL-2 mRNA is unstable during induction and that the decline of the mRNA wave reflects mRNA decay. It lends independent support to the conclusion from Fig. 2A and 3 that few, if any, new mRNA molecules are generated once the wave has reached its maximum. Addition of actinomycin D also induced a decline in IL-2 mRNA in a cell population that was first superinduced with CHX and that expressed high levels of mRNA (Fig. 8). Together with the data from Fig. 1and 5C, these results indicate that stabilization of IL-2 mRNA by CHX, if it occurs, is insufficient to explain the extent of superinduction.


Figure 8: Lack of IL-2 mRNA stabilization in superinduced cells. PBMC were induced with PHA (). CHX was added at 4 h (bullet, ▪). Actinomycin D (ActD) was added at 7 h (▪). Cell viability remained constant. Total RNA, extracted at times shown, was subjected to RNase protection analysis using probe SC1. Autoradiogram (A) was quantitated by microdensitometry (B).



Precursor Transcripts Accumulate during Induction

In Fig. 9, induction of both IL-2 mRNA and precursor transcripts was followed by RNase protection analysis using the 608-nt antisense RNA probe shown in Fig. 9D, complementary to 117 nt from exon 3 and 471 nt from intron 3. Precursor transcripts containing intron 3 protect a 588-nt labeled RNA fragment, whereas spliced RNA (mRNA) protects a 117-nt band (Fig. 9A).

Induction of PBMC yielded a wave of IL-2 mRNA that reached its maximum at 6 h and then declined rapidly, returning to almost basal levels by 10 h (Fig. 9A). Precursor transcripts reached their maximum earlier (by 3 h), as would be expected for a precursor-product relationship; their level declined between 3 and 6 h concomitant with a rise in mRNA (Fig. 9, A and B). In contrast to the highly transient expression of mRNA, however, the expression of precursor transcripts was more sustained, continuing well beyond the time when the mRNA level had declined completely. Indeed, even though their level declined gradually, precursor transcripts strongly predominated over mRNA from 10 h onwards (Fig. 9C). Expression of precursor transcripts persisted up to 25 h, in good agreement with the conclusions from Fig. 3-6 that active transcription was still ongoing 24 h after induction yet did not result in accumulation of mRNA. These results suggest that processing of precursor transcripts occurs during the early phases of induction but then becomes inhibited.

CHX Enhances Processing of Precursor Transcripts into mRNA

In Fig. 10A, changes in levels of precursor transcripts and of mRNA were measured concurrently during exposure to CHX. In the absence of CHX, the induced wave of mRNA subsided after 8 h. Although in this experiment precursor transcripts were not detected during the mRNA wave, they had become prominent by 24 h, at which time little, if any, mRNA remained. Addition of CHX at 4 h led to a prompt and extensive superinduction of mRNA lasting at least 24 h. This increase in mRNA was accompanied first by an increase in precursor transcripts observed at 8 h and then by their decline. By 24 h, precursor transcripts were clearly present in the control, yet they were no longer observed in superinduced cells. Thus, superinduction of mRNA by CHX is accompanied by a disappearance of precursor transcripts.


Figure 10: Chase of IL-2 precursor transcripts into mRNA in the presence of CHX. PBMC were induced with PHA. Where indicated, CHX was present from 4 h. Total (A), nuclear (B) and cytoplasmic RNA (C) were extracted at times shown and subjected to RNase protection analysis using IL-2 SC1 and beta-actin probes as indicated. In A, the 117-nt band is shown for two different analyses of the same RNA samples; upper and lowergels were exposed for 3 and 1 days, respectively. The probe (P) was also subjected to RNase protection analysis in the absence of cellular RNA (P*). Data of A and that of B and C represent 2 different experiments.



This point is reinforced by analysis of the data on precursor transcripts shown in Fig. 5. In Fig. 5A, precursors (588-nt band) are seen early (before the peak of mRNA), as in Fig. 9A, and again late (at 24 h) after expression of mRNA has ceased, as in Fig. 10A. Addition of CHX at 4 h (Fig. 5A, 3 lanes on the right) led to enhanced processing of precursors into mRNA. In Fig. 5B, precursors were prominent at 24 h, whereas mRNA had declined by that time, consistent with the data of Fig. 5A, 9A, and 10A. In the experiment of Fig. 5B, precursors declined strongly between 24 and 28 h, showing that when they are not processed into mRNA, precursors are degraded. In the presence of CHX, however, precursor transcripts, most of them newly synthesized, were converted efficiently into spliced mRNA. Thus, although in Fig. 5B CHX was added late (at 24 h), the same course of events is observed as when CHX was added at 4 h: an enhanced flow of precursor transcripts into mRNA.

In Fig. 10B, the induction and the fate of precursor transcripts were studied in the nuclear compartment. Concomitant with the superinduction of cytoplasmic mRNA in the presence of CHX (Fig. 10C), there was a pronounced decline in nuclear precursor transcripts (588-nt band) at 9 h (Fig. 10B), as would be expected if processing were enhanced. Indeed, superinduction of mRNA in the cytoplasm was accompanied by a complete disappearance of precursor transcripts from the nucleus, indicating that post-transcriptional processing of precursor transcripts into mRNA was greatly facilitated.

During induction in the absence of CHX, abundant amounts of shorter RNA fragments accumulated in nuclei between 4 and 9 h (Fig. 10B). Consistent with the data in Fig. 5B and 9A, this result shows that when they are not processed into mature mRNA, precursor transcripts are degraded in the nucleus. In the presence of CHX, these fragments were altogether absent (Fig. 10B). This result indicates that during superinduction, the amount of precursor transcripts that was converted into mRNA actually exceeded the sum total of protected 588-nt RNA and shorter fragments remaining in the untreated control by 9 h. Such extensive processing of precursor transcripts upon the addition of CHX is also reflected by the extent of concomitant superinduction of cytoplasmic mRNA (Fig. 10C).

In the control induced for 9 h, nuclear RNA was composed mainly of precursor transcripts and degradation products, but in the presence of CHX, the predominant species was mature mRNA (117-nt band in Fig. 10B). This shift into mRNA within the nucleus is consistent with the conclusion that enhanced processing of precursor transcripts had occurred during superinduction.


DISCUSSION

Induction of human IL-2 gene expression results in the appearance of a transient wave of mRNA. Our results show that few, if any, new mRNA molecules are generated once the wave has reached its maximum. Yet transcription continues unabated well beyond this point but fails to yield more IL-2 mRNA. Instead, IL-2 precursor transcripts accumulate. During induction, precursor transcripts are expressed to low levels, well below those of mature mRNA. Late in induction, however, after the wave of mRNA has subsided, precursor transcripts constitute the majority of expressed IL-2 RNA molecules (Fig. 9C). The decline of the wave reflects the subsequent decay of unstable IL-2 mRNA.

The accumulation of precursor transcripts is surprising. These molecules should either be processed into mRNA or be degraded. Premature termination of transcription cannot account for the accumulation, because the probe used in this study covers a 3`-proximal portion of the IL-2 gene (exon 3-intron 3). Instead, our results demonstrate that processing of functional IL-2 precursor transcripts becomes inhibited in the course of induction and that this inhibition constitutes a major element of control.

Expression of IL-2 mRNA can be superinduced extensively in the presence of the inhibitors of translation(4, 9) , as shown here for CHX and emetine. Superinduction results in an increase in the amplitude and duration of the mRNA wave. In principle, this superinduction could result from increased transcription, from stabilization of precursor transcripts and/or mRNA, or from enhanced processing of precursor transcripts. The superinduction of IL-2 mRNA is completely dependent upon de novo transcription (Fig. 5, 6, and 8), yet the rate of transcription does not change perceptibly when CHX is present (Fig. 3). Addition of CHX late in induction, after the wave of IL-2 mRNA has returned to basal levels, also results in a strong increase in mRNA; this process equally requires synthesis of new RNA molecules (Fig. 5). On the other hand, significant stabilization of IL-2 mRNA was not detected (Fig. 1, 5-8). Even if it occurs, stabilization of mRNA by CHX is too limited to account for the observed extent of superinduction.

These observations indicate that the rate of synthesis of new IL-2 RNA molecules is not changed in the presence of CHX but that the formation of IL-2 mRNA is greatly enhanced. Indeed, we show here that superinduction of IL-2 mRNA by CHX results from enhanced processing of precursor transcripts. Concomitant with a strong increase in mature mRNA, addition of CHX causes a disappearance of precursors ( Fig. 5and 10). Although precursor transcripts are stable enough to accumulate during normal induction (Fig. 5A and 9A), it cannot be argued that they are degraded more rapidly in conditions of superinduction, because levels of mature IL-2 mRNA increase extensively when CHX is present. Indeed, given that AUUUA motifs found in the 3`-untranslated region of IL-2 mRNA are also present in IL-2 precursor transcripts, greater not lesser stability might be expected in the presence of CHX. The decline in precursor transcripts observed in the presence of CHX thus must be based on more efficient processing into mRNA. The same response, enhanced processing, is observed whether CHX is added early or late during induction. Apparently, the flow of newly synthesized precursor transcripts into mature IL-2 mRNA becomes inhibited in the course of induction by a mechanism involving a labile protein. This post-transcriptional block is relieved when translation is inhibited.

The level of precursor transcripts in the cell is determined by a dynamic balance between synthesis, processing, and degradation. Because of this transient nature, the pool size of precursor transcripts remains limited. Yet when a block in processing develops, a significant increase in precursor transcripts can be observed (Fig. 5, A and B, and 10, A and B). Accumulation of precursor transcripts is, however, not sustained owing to the instability of these molecules (Fig. 5B). When they are not processed into mature mRNA, precursor transcripts are degraded (Fig. 5B and 9A) in the nucleus (Fig. 10B). During superinduction, a transient rise in precursor transcripts precedes their disappearance (Fig. 10A), suggesting that the stability of these RNA molecules may be increased in conditions that permit their processing.

The demonstration that excision of intron 3 from IL-2 precursor transcripts is highly regulated does not necessarily imply that excision of introns 1 and 2 is controlled in parallel. This question will require further study. Clearly, regulation of the excision of intron 3 is sufficient to effectively control the conversion of precursor transcripts into IL-2 mRNA.

Despite the fact that PHA induces transcription of the beta-actin gene (Fig. 3), levels of beta-actin mRNA did not change perceptibly during induction or superinduction ( Fig. 2and Fig. 10). In PHA-induced PBMC, the steady state level of beta-actin mRNA greatly exceeds that of IL-2 mRNA, yet the increase in transcription of the beta-actin gene during induction remains below that for the IL-2 gene. The additional beta-actin gene transcription induced by PHA may be too low to affect the pool size of stable beta-actin mRNA.

Our results permit analysis of the question why mitogenic induction results in the appearance of a transient wave of IL-2 mRNA, a phenomenon generally observed for cytokine genes. Transcriptional shutoff of the IL-2 gene cannot be the explanation of transient expression, since transcription is sustained at a high rate during and well beyond the time when the wave is subsiding ( Fig. 2and Fig. 3). The possibility that IL-2 mRNA becomes unstable during induction near the time when the mRNA wave has reached its maximum, with the rate of decay exceeding the rate of mRNA formation, can be rejected on the basis of our results showing that in the presence of CHX, no significant stabilization of IL-2 mRNA can be detected. This is seen especially well in tonsil cells (Fig. 1), where the wave of mRNA develops more slowly than in PBMC, apparently reaching its maximum only a short time before transcription of the IL-2 gene ceases. This leaves explanations based on destabilization of precursor transcripts or inhibition of post-transcriptional processing. The results of Fig. 5, 9, and 10 demonstrate the accumulation of precursor transcripts at and beyond the time when the wave of mRNA is declining. These results do not support destabilization of precursor transcripts. Instead, precursor transcripts disappear in the presence of CHX, when mRNA levels increase greatly ( Fig. 5and Fig. 10), showing that processing is facilitated. Our results demonstrate that a block in processing develops in the course of normal induction, resulting in the accumulation of precursor transcripts that are subsequently degraded (Fig. 5B, 9, and 10B). Due to its intrinsic instability, the existing pool of IL-2 mRNA decays and a wave of mRNA is generated. Once transcription has been induced, the primary mechanism regulating IL-2 gene expression thus is at the level of precursor RNA processing.

There are few good examples of this type of regulation. Facilitation of post-transcriptional processing was suggested to be the basis for increased expression of thymidine kinase mRNA at the onset of DNA synthesis during the cell cycle. Quiescent cells, accordingly, would lack a factor required for processing of precursor transcripts that becomes available at the G(1)/S boundary(29) . Suppression of class I major histocompatibility gene expression after adenovirus 12 transformation is apparently caused by an inhibition of processing of the mRNA in the nucleus; as in the case of the IL-2 gene studied here, no changes in transcription rate or stability of mRNA were detected(30) . Processing of precursor transcripts was shown to regulate retinoic acid-induced expression of the IL-1beta gene(31) . Most IL-1beta gene transcription fails to yield mature mRNA. When translation is blocked, e.g. by CHX, processing of unstable IL-1beta precursor transcripts into mature mRNA is greatly facilitated, resulting in a superinduction of IL-1beta mRNA by 2 orders of magnitude. Pre-mRNA processing thus is a limiting step in retinoic acid-induced IL-1beta gene expression(31) .

Studies of processing of precursor transcripts have largely been done in in vitro systems. As shown here, however, for the IL-2 gene and elsewhere for the IL-1beta gene(31) , nuclear processing of precursor transcripts is a dominant element of post-transcriptional control in intact cells. Our observations support the concept that pre-mRNA processing may constitute, more widely than hitherto thought, an essential step for regulation of cytokine gene expression. Only after mature mRNA has been generated can the control of its stability begin to serve as a secondary target for post-transcriptional regulation.


FOOTNOTES

*
This work was supported in part by a grant from the Volkswagen Foundation, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.

To whom correspondence should be addressed. Tel.: 972-275-8389; Fax: 972-278-4010; kaempfer{at}md2.huji.ac.il.

(^1)
The abbreviations used are: IL, interleukin; PBMC, peripheral blood mononuclear cell(s); PHA, phytohemagglutinin; CHX, cycloheximide; nt, nucleotide; Pipes, 1,4-piperazinediethanesulfonic acid.


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