In this work we present a general strategy for generating mutants of a specific mRNA degradation pathway. To our knowledge, this is the first report where somatic cell genetics has been used to study mRNA turnover in a mammalian system allowing the definition of complementation groups. With the help of a reporter gene containing the ARE of the IL-3 3′UTR, changes in mRNA stability could be translated into different steady-state expression levels (Fig. A and B). To obtain posttranscriptional mutants, it was important to use GFP as a reporter gene instead of a selectable “all-or-nothing” resistance marker, because alteration of mRNA stability only modulates the levels of gene expression but does not completely turn it on or off. Using an efficient mutagenesis protocol adopted from that of McKendry et al. (28
), four pools of 2 × 107
cells each were exposed to 8 to 12 rounds of treatment with the frameshift mutagen ICR191. The extent of random genomic mutations was estimated by the frequency of cells that had lost the monoallelic (X-linked) HPRT function, which reached a plateau value at about 0.8 in 103
cells. Based on this frequency, the probability of knocking out both alleles of any gene could be estimated to (0.8 × 10−3
, or 0.64 × 10−6
. Hence, each pool of 2 × 107
cells should contain roughly 13 mutants for an autosomal gene promoting mRNA decay. We thus could believe that isolating a mutant should be feasible, barring unexpected complications such as redundancy of the putative regulatory gene or a lethal effect of the homozygous mutation.
From the mutagenized cells, GFP-overexpressing clones were selected by a multistep procedure. Automated sorting by flow cytometry allowed us to enrich for highly fluorescent cells. These cells were immediately subcloned in order to prevent the formation of many siblings and to protect slowly dividing cells from being overgrown. An advantage of using GFP was that the clones could be directly analyzed by eye with a fluorescent microscope. Promising candidates were expanded, and GFP overexpression could finally be confirmed for 156 clones by FACS analysis. Altogether, selection was rather time-consuming, and a considerable number of clones were lost at each step (see Table ). Increasing the cloning efficiency would certainly help to reduce the amount of work in future attempts and could perhaps be achieved by the use of conditioned medium.
The GFP-overexpressing clones were first characterized by performing actinomycin D chase experiments and Northern blot analysis. As expected, all 156 candidate clones displayed increased steady-state levels of the reporter mRNA. Surprisingly, stabilization of the mRNA was found in only three clones, whereas other events, apparently leading to transcriptional activation, were more frequent. This class of mutants, exemplified by clone 12-2 (Fig. C), might have undergone amplification of the reporter gene, although it is difficult to imagine how a frameshift mutagen could achieve this. Another, more likely possibility is that loss of a transcriptional repressor has occurred in these mutants. Interestingly, a repressor element, termed the nuclear inhibitory protein (NIP) region, has been identified at position −260 in the human IL-3 promoter (27
). One of three complexes that bind to the NIP region in vitro is believed to represent the repressor activity (9
), but the protein has not been identified so far. Although it is not known whether the NIP region, or a second repressor mapped further downstream (42
), is also functional in the murine IL-3 promoter, it would be worthwhile to analyze the transcriptional mutants for promoter binding proteins by DNA footprinting or electromobility shift assays.
Our interest, however, focused on the three posttranscriptional mutants termed slowA, slowB, and slowC (Fig. D through F). First, the mutants were shown to have a trans-acting defect, as degradation of mRNA from two genomic IL-3 constructs was also impeded, similar to the stabilization observed with the GFP reporter transcript (Fig. A and B). Reporter transcripts with the ARE-containing 3′UTRs of IL-2 and TNF-α, however, were not stabilized in either of the mutants (Fig. C and D). This might indicate that the AREs of these two cytokines are recognized by a decay pathway different from the one which promotes IL-3 mRNA degradation. Alternatively, the (class II) AREs are targeted by one mechanism—which is defective in the mutants—yet IL-2 and TNF-α, in contrast to IL-3, harbor an additional destabilizing element in their 3′UTRs. A more extensive analysis using reporter constructs containing different AREs with and without flanking 3′UTR sequences will enable this issue to be resolved.
It was crucial to establish that the mutants displayed recessive defects, since this determines the strategy for later identification of the defective genes, as outlined below. A dominant mutation would require a different strategy, as the defective gene could be cloned directly by expressing a DNA library from the mutant in wt cells. Fusion of the parental cell line with the three mutants revealed that they are genetically recessive, since their phenotype was corrected in the hybrids (Fig. A). Two complementation groups could be defined by analyzing mRNA decay patterns of intermutant hybrids (Fig. B). slowA belongs to one group, while slowB and slowC form a second group. The two groups could not be distinguished further, either with respect to decay of β-globin–IL2 or β-globin–TNF-α mRNA or by their response to expression of TTP. In this context it would be worthwhile to generate mutants of different mRNA decay pathways, using the technique described here with GFP reporter constructs containing AREs of other cytokines or proto-oncogenes, particularly IL-2 and TNF-α, as our mutants failed to stabilize these transcripts. This should allow definition of more complementation groups and help to identify the common as well as the specific trans-acting factors that participate in these pathways.
Once recessive IL-3 mRNA turnover mutants had been obtained, it was possible to test whether expression of known regulators of mRNA degradation could rescue the mutant phenotype. With AUF1 (p37) and VHL, no effect on GFP levels or reporter mRNA stability could be observed in either complementation group. In the case of VHL, this is perhaps not surprising, since VHL is mainly involved in the regulation of hypoxia-induced genes such as the VEGF gene. Interestingly, the p37 isoform of AUF1 also had no effect on reporter mRNA decay. Recently, expression of the p42 isoform of AUF1 in K562 erythroleukemia cells has been shown to antagonize hemin-induced stabilization of β-globin transcripts containing the AREs of c-fos
and GM-CSF, the latter being almost identical to the ARE of IL-3. The p37 isoform of AUF1 is also an effective destabilizer, as was demonstrated with the ARE of c-fos
). The gene product missing in our mutants is obviously not AUF1, but perhaps a component downstream, or an essential upstream activator of AUF1. Alternatively, the destabilizing function of AUF1 could be restricted to certain cell lineages and might not be active in HT1080 fibrosarcoma cells.
TTP, on the other hand, could correct the defect in slowA and slowC, as its expression reinstalled rapid decay of GFPIL3 mRNA (Fig. ). Originally, the mRNA turnover-promoting function of TTP was discovered in macrophages from TTP knockout mice, which overexpress TNF-α due to enhanced mRNA stability (6
). Upon reintroduction of TTP into TTP−/−
macrophages, reduced levels of β-globin reporter transcripts containing AREs from TNF-α, IL-3, and GM-CSF were observed. This suggested a broader role for TTP in regulating mRNA degradation for various cytokines, but destabilizing activity had been shown only with TNF-α mRNA. Our data establish that TTP is also a component of the IL-3 mRNA degradation pathway. Since it fully restored rapid decay in both complementation groups, TTP appears to be a potent mRNA destabilizer that acts downstream in the degradation pathway. The observation that TTP can directly bind to class II AREs (6
) supports this idea.
On the basis of this finding with TTP, experiments are in progress to investigate whether TTP can antagonize IL-3 overproduction in autocrine tumors which express abnormally stable IL-3 mRNA (32
). This can be tested at the levels of mRNA stability, IL-3 secretion, autocrine growth in vitro, and tumor formation in vivo. Should TTP have tumor suppressor activity, this would further emphasize its role as an important biological regulator.
More than one hypothesis could explain the capacity of ectopically expressed TTP to revert the mutant phenotypes. First, both alleles of the TTP gene itself could be mutated. This was ruled out, as wild-type cDNA sequences of the human TTP coding region were obtained from both mutants. Second, a gene dosage effect might have occurred following a TTP promoter mutation or by loss of a transcription factor. Similar levels of hTTP mRNA were found in slowA and the parental cell line (Fig. ). Whether the modest but consistent reduction of hTTP mRNA observed in unstimulated slowC (Fig. and data not shown) is of any significance for the mutant phenotype remains to be investigated. Northern blot analysis also largely excluded the possibility of incorrect splicing or 3′-end processing, since hTTP mRNA had the same size in the parental cell and the mutants. Taken together, these data indicate that the TTP gene itself is intact in both complementation groups. Therefore, we favor a model where the missing component in the IL-3 mRNA degradation pathway is an activator of decay, located upstream of TTP, which can be overruled by ectopic expression of high levels of TTP. The precise relationship between TTP and the two functions A and B/C, however, will become clear only after cloning of the corresponding genes. Taking advantage of the GFP reporter system, we plan to perform complementation by transfer of a cDNA library into the mutants and selection of revertant clones. This should hopefully allow identification of the defective genes in slowA and slowB/C, and eventually help us understand how upstream factors function in concert with known regulators of ARE-dependent mRNA degradation.