(Received for publication, March 8, 1995; and in revised form, May 22, 1995)
From the
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.
Interleukin-2 (IL-2) ()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-
,
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.
Figure 1:
Superinduction of IL-2 mRNA by CHX.
Human tonsil cells were induced with PHA (). 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 (
) 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
-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.
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
[-
P]UTP. Nuclear RNA was extracted, and
aliquots of 4
10
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
-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 (
, ▪). 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.
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) (
,
▪). 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 (
, ▪). 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).
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 (
, ▪). 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).
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.
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
-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.
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 -actin gene (Fig. 3), levels
of
-actin mRNA did not change perceptibly during induction or
superinduction ( Fig. 2and Fig. 10). In PHA-induced PBMC,
the steady state level of
-actin mRNA greatly exceeds that of IL-2
mRNA, yet the increase in transcription of the
-actin gene during
induction remains below that for the IL-2 gene. The additional
-actin gene transcription induced by PHA may be too low to affect
the pool size of stable
-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/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-1
gene(31) . Most IL-1
gene transcription
fails to yield mature mRNA. When translation is blocked, e.g. by CHX, processing of unstable IL-1
precursor transcripts
into mature mRNA is greatly facilitated, resulting in a superinduction
of IL-1
mRNA by 2 orders of magnitude. Pre-mRNA processing thus is
a limiting step in retinoic acid-induced IL-1
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-1
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.