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Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice and cells derived from them showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target mRNAs for TTP include tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-2β. Here we used microarray analysis of RNA from wild-type and TTP-deficient fibroblast cell lines to identify transcripts with different decay rates, after serum stimulation and actinomycin D treatment. Of 250 mRNAs apparently stabilized in the absence of TTP, 23 contained two or more conserved TTP binding sites; nine of these appeared to be stabilized on Northern blots. The most dramatically affected transcript encoded the protein Ier3, recently implicated in the physiological control of blood pressure. The Ier3 transcript contained several conserved TTP binding sites that could bind TTP directly and conferred TTP sensitivity to the mRNA in cell transfection studies. These studies have identified several new, physiologically relevant TTP target transcripts in fibroblasts; these target mRNAs encode proteins from a variety of functional classes.
Rates of mRNA decay, along with rates of gene transcription, mRNA splicing, and nuclear export, are critical for the ultimate steady-state levels of mRNA in cells. Numerous mechanisms for regulating mRNA decay have been identified and are subject to regulation by extracellular factors. One cis-acting factor known to confer instability on mRNAs is the AU-rich element (ARE), originally identified as a component of the granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA 3′-untranslated region (3′-UTR) (29). Since then, a large volume of literature has attempted to classify these elements, documented their ability to confer instability on otherwise stable mRNAs, and identified binding proteins that are thought to either promote or inhibit ARE-mediated mRNA instability (2, 3, 15, 36, 38).
One such group of proteins is the tristatraprolin (TTP) family of CCCH tandem zinc finger proteins (5). The four members of this family in rodents, and three in humans, can bind to AREs in mRNA at a consensus nonamer site, UUAUUUAUU, although variations of this sequence can still permit relatively high-affinity binding (8). The tandem zinc finger domain of the proteins is necessary and sufficient for RNA binding (19). The RNA binding step is the first of a poorly understood series of events in which removal of the poly(A) tail is stimulated, a process known as deadenylation, and the overall degradation of the mRNA is accelerated (19).
Although all members of the mammalian TTP family can stimulate the deadenylation and breakdown of ARE-containing mRNAs in transfection and cell-free assays (7, 21), to date physiological mRNA substrates have been identified only for TTP. These identifications were based on the creation of TTP knockout (KO) mice, which developed a systemic syndrome of arthritis, weight loss, skin lesions, autoimmunity, and myeloid hyperplasia (33). Subsequent studies of macrophages derived from these mice demonstrated that one physiological TTP target was the mRNA encoding tumor necrosis factor alpha (TNF-α), an mRNA long known to be unstable, and whose instability was dependent on the presence of a highly conserved ARE in its 3′-UTR (11, 13). Confirmation that TNF was a critical component of the TTP-KO phenotype came from studies in which monoclonal anti-TNF antibodies were injected into newborn TTP-KO mice or the mice were interbred with mice lacking both TNF receptors; in both cases, the systemic inflammatory syndrome was almost completely abrogated (10, 33).
A second physiological substrate was the GM-CSF mRNA, which was studied on the basis of its known ARE and the contribution of that ARE to mRNA instability. In bone marrow-derived stromal cells from TTP-KO mice, the GM-CSF mRNA was also stabilized when compared to the mRNA from wild-type (WT) cells, indicating that the GM-CSF mRNA was a physiologically significant target in this cell type (12). Whether GM-CSF plays a role in the myeloid hyperplasia characteristic of the TTP-KO phenotype remains to be determined. Most recently, interleukin-2 (IL-2) mRNA has been shown to be stabilized in primary T lymphocytes derived from the TTP-KO mice and is another example of an ARE-containing mRNA whose ARE is known to confer instability (26). It is not clear how this target relates to the original phenotype of the TTP-KO mice.
To our knowledge, no additional physiological TTP substrates have been identified, using the strict criterion that mRNA stabilization needs to be demonstrated in cells derived from the TTP-KO mice. However, small interfering RNA knockdown experiments have been used recently to identify β-1,4-galactosyltransferase 1 as a TTP target in human umbilical cord cells (17). In addition, there are many examples in the literature of “forced” TTP-promoted mRNA degradation, in which overexpression of TTP in transfected cells has been shown to stimulate the breakdown of potential target mRNAs. This is not surprising, given that the major determinant of TTP binding is the primary sequence of the ARE binding site (6, 22, 37) and that relatively minor decreases in TTP's ARE binding affinity that occur with sequence variations of the optimal binding nonamer might still permit binding and TTP-stimulated mRNA decay if the TTP concentration is high enough (8).
To avoid some of the potential artifacts associated with overexpression experiments, we have performed a global analysis of mRNA turnover in stable fibroblast cell lines derived from littermate TTP-KO and WT mice. These cell lines have been cultured for more than 200 passages and are well matched in terms of growth rates, morphology, and responses of immediate-early genes such as c-fos to serum deprivation and serum stimulation. We used an experimental paradigm based on the original identification of TTP mRNA as a rapidly induced, labile transcript following stimulation of serum-deprived fibroblasts with insulin, growth factors, serum, or phorbol esters (24). We found more than 250 transcripts whose mRNA was significantly stabilized in the TTP-deficient cells 90 min after stimulation of serum-deprived cells with serum. We selected 23 of these for further analysis, based on the presence in the 3′-UTR of two or more copies of the core heptamer of the ideal TTP binding site, UAUUUAU, that were conserved in mice and humans. Using Northern blotting of similar time courses, we confirmed that at least seven of these were clearly stabilized in the TTP-deficient cells and an additional two were stabilized to a lesser extent. These novel potential physiological TTP targets included the mRNAs encoding secreted proteins, protein kinases, enzymes, and proteins of poorly understood function. These data suggest that TTP can promote the decay of many transcripts in this cell type. They also support this type of global transcript analysis for the identification of binding partners for TTP in other cell types and to identify partners for the other TTP family members in physiologically appropriate cells and conditions.
Cell lines were derived from mouse embryonic fibroblast (MEF) cultures from littermate E14.5 embryos (for Zfp36+/+ and Zfp36−/− cells ) or E9.5 embryos (for Zfp36l1+/+ and Zfp36l1−/− cells ). MEFs were maintained at 37°C (5% CO2) in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine. When the cells had reached approximately 70 to 90% confluence, they were trypsinized and diluted 2- to 50-fold in the same medium, based on morphology and growth rate, such that they would reach near confluence in 3 to 4 days when subcultured. By approximately passage 30, these cell lines had developed a homogeneous fibroblast morphology and similar rapid growth rates. They were maintained with similar subculturing techniques. These cell lines do not express TNF, GM-CSF, or IL-2 mRNAs at levels readily detectable by our routine Northern blotting techniques.
For serum deprivation experiments, cells at approximately 70 to 80% confluence were washed once in serum-free DMEM and then incubated for 16 h in DMEM containing 0.5% (vol/vol) FBS. The cells were then stimulated with 10% (vol/vol) FBS (HyClone, Logan, UT) or 0.1 μg/ml epidermal growth factor (EGF; R&D Systems, Inc., Minneapolis, MN) for various lengths of time. In some cases, actinomycin D (Sigma Chemical Co., St. Louis, MO) was added to a final concentration of 5 μg/ml, and cells were harvested at intervals for RNA or protein preparation.
Total cellular RNA and protein extracts for Western blotting were prepared as described previously (23, 24). For microarray analysis, five identical experiments involving sequential serum deprivation, serum stimulation, and actinomycin D treatment were performed on 5 different days; each experiment involved simultaneous identical experiments with the WT and KO cell lines. In each experiment, the cells were serum deprived for 16 h and a sample was taken at that point as a serum-deprived control. The cells were then stimulated by 10% FBS for 90 min and another sample was taken, after which actinomycin D was added; samples for RNA analysis were then removed at 30, 60, 90, and 120 min after actinomycin D treatment. In some of the subsequent validation experiments, actinomycin D was added 45 min after serum stimulation, as noted in the text. In all experiments, each sample represented three combined 100-mm dishes of cells.
For Northern blotting, probes for mouse TTP (24) and c-fos (31) were described previously. For the 23 mRNAs tested by Northern blotting as part of the microarray validation studies, mouse expressed sequence tags (ESTs) were identified by blasting the sequences of the microarray “hits” against the GenBank mouse EST database, and appropriate EST clones were ordered from the IMAGE consortium through either Invitrogen or American Type Culture Collection (ATCC; Manassas, VA). The GenBank accession numbers and IMAGE clone numbers for each of the probes used in these Northern blots are listed in Table Table1.1. Northern blotting was performed as described previously (24). Northern blots were quantitated using a PhosphorImager (Typhoon; Molecular Dynamics). For Western blotting, an antiserum was used that was directed against a recombinant mouse TTP-maltose binding protein fusion protein, as described previously (9). An antiserum to mouse Zfp36l1 was generated by similar techniques.
Gene expression analysis was conducted using Affymetrix Mouse Genome 430 2.0 GeneChip arrays (Affymetrix, Santa Clara, CA). This whole mouse genome array interrogates 34,000 variants and 39,000 transcripts from well-characterized mouse genes using 45,000 probe sets. Total RNA (1 μg) was amplified using the Affymetrix one-cycle cDNA synthesis protocol. For each array, 15 μg of amplified biotin-cRNAs was fragmented and hybridized to the array for 16 h at 45°C in a rotating hybridization oven using the Affymetrix eukaryotic target hybridization controls and protocol. Slides were stained with streptavidin/phycoerythrin using a double-antibody staining procedure and washed using the EukGE-WS2v5 protocol of the Affymetrix fluidics station FS450 for antibody amplification. Arrays were scanned with an Affymetrix scanner 3000, and data were obtained using the GeneChip operating software (GCOS; version 1.2.0.037).
Normalization of the image files was accomplished with quantile normalization, and signal was estimated with the positional dependent nearest neighbor (PDNN) algorithm (39). To assess quality, principal component analysis (28) was used; plots were generated and points were colored according to experimental condition (WT or KO), time (0 to 120 min), and replicate (samples 1 to 5). The second and third principal components clearly distinguish experimental condition and time, respectively (see Fig. S1 in the supplemental material). No segregation by replicate was evident in these components of variability.
The significance analysis of microarrays (SAM) algorithm (35) was used to identify RNAs that demonstrated differential rates of decay between the WT and KO cell lines. Specifically, the two-class unpaired time course algorithm was used to test for differences in slope (i.e., relative degradation rate) between the two groups. This algorithm performs a least-squares fit of the expression values within each. The slopes are then compared, along with the error of the fit, to form a score for each probe set. Permutation was utilized to arrive at a q-value. Probe sets exhibiting a q-value of less than 0.01 were called significant, resulting in 306 significance calls.
The SAM algorithm was also used to identify transcripts whose mean levels 90 min after serum stimulation but immediately before actinomycin D addition demonstrated a consistent difference. This analysis employed the two-class unpaired variant of SAM, which is similar to the algorithm described above except that the group means (instead of slopes) are compared. Probe sets exhibiting a q-value of less than 1% were called significant, resulting in 3,788 significance calls (179 of these overlapped with the 306 from the RNA decay experiment).
The nonamer TTP binding site, UUAUUUAUU, has been characterized previously (6). We searched the mouse 3′-UTR sequences of all mouse 430 2.0 probe sets using the Motif Alignment and Search Tool (MAST) (1), using several variations of this optimal binding site. In this paper, we have focused on transcripts differing significantly in slope (with the slope from the WT cells being steeper than that from the KO cells) that contained two or more perfect matches with the core heptamer UAUUUAU. Transcripts meeting those criteria were then used in BLAST searches of the GenBank human nr and EST databases for evidence of conservation; in some cases, sequences from other mammals were evaluated as well.
Plasmid pTNFα 1309-1332 (bp 1309 to 1332 of GenBank accession no. X02611) was constructed as described previously (20). To make plasmids Ier3 ARE (bp 671 to 783 in GenBank accession no. NM_133662), or its mutants Mu8 and Mu6 (see Fig. Fig.5A5A for the sequences), a double-stranded oligonucleotide including Asp718I and XbaI sequences at its 5′ or 3′ ends, respectively, was inserted between the Asp718I and XbaI sites of vector SK− (Stratagene). DNA inserts were confirmed by dRhodamine terminator cycle sequencing (Perkin-Elmer).
The templates for RNA probes were prepared by linearizing plasmids with XbaI followed by gel purification of the linearized DNA. The RNA probes were transcribed in the presence of [α-32P]UTP (800 Ci/mmol) using the Promega Riboprobe in vitro transcription systems protocol. The resulting probes were separated from the free nucleotides using G50 columns. Transfection of HEK 293 cells with expression plasmids CMV.hTTP.tag and its zinc finger mutant, C124R, and preparation of cell extracts were performed as described previously (21).
Extracts prepared from 293 cells (5 μg of protein) were incubated with 5 × 105 cpm of RNA probe TNF ARE (RNase TI treated, 10 U/reaction; Epicenter), or Ier3 ARE, or Mu8, or Mu6 (without RNase T1 treatment) in 25 μl of a buffer that contained 10 mM HEPES (pH 7.6), 40 mM KCl, 3 mM MgCl2, 50 μg heparin, and 1.2 μg yeast tRNA, as described previously (20) and the protein-RNA complexes formed were resolved on 6% nondenaturing acrylamide (37.5:1) gels.
The Ier3 expression vector CMV.Ier3.Flag was prepared by reverse transcription-PCR (RT-PCR) using total cellular RNA from the KO 66−/− and WT 67+/+ cell lines stimulated with 10% FBS for 90 min as the template (separately) for RT. The 5′ primer for PCR amplification was 5′-gtcgacTGCACTCCTCTACACTCTCTGCACAACGTC -3′, and the 3′ primer was 5′-tctagaGACAGGCAAATCAAGTTTATTCGTGTTCACAG-3′. The uppercase letters in the 5′ and 3′ primers contained bp 1 to 30 and bp 1058 to 1090 of GenBank accession no. AY168443 (containing 30 bp more 5′-UTR than GenBank accession no. NM_133662), respectively. The lowercase letters in the primers indicate the restriction sites for SalI and XbaI, respectively. The resulting PCR product was a 1,120-bp mouse Ier3 cDNA and was digested with SalI and XbaI and cloned into the SalI and XbaI sites of the vector pSK−, which contains the hCMV promoter-enhancer (21). Mutants carrying mutations in the ARE region of the Ier3 mRNA were made using the PCR primer-overlapping mutagenesis technique (25) (see Fig. Fig.6B6B for the sequences). The correct sequences of the inserts were confirmed by dRhodamine terminator cycle sequencing.
Cell transfection assays in 293 cells were performed exactly as described previously (20). The cells were cotransfected with the vector alone plasmid (BS+; Stratagene); the wild-type human TTP-expressing plasmid CMV.hTTP.tag, or its zinc finger mutant C124R; and the wild-type or ARE mutants of CMV.Ier3 constructs. The amount of transfected DNA was adjusted so that each plate was transfected with the same amount of DNA (5 μg); this was made up of 2 to 10 ng of CMV.hTTP.tag or 5 to 20 ng of C124R and 0.5 μg (or otherwise indicated) of CMV.Ier3 plasmids; and the total of 5 μg was made up by BS+. The cells were harvested, and total cellular RNA was prepared using RNeasy (QIAGEN). For Northern blotting, the probes consisted of either a mouse Ier3 cDNA probe (GenBank accession no. BI904834) or a mouse TTP cDNA probe (20). Northern blotting was performed as described previously (20).
After 16 h of incubation in 0.5% FBS, both cell lines (the KO cell line 66−/− and the WT cell line 67+/+) had achieved apparent quiescence, as evidenced by undetectable levels of c-fos mRNA (Fig. (Fig.1A,1A, lower panels, lanes 1 and 8). After stimulation with 10% FBS, both lines exhibited a typical and similar c-fos mRNA induction response, with new mRNA being readily detectable by 10 min, peaking of mRNA levels at 30 to 45 min, and virtually complete disappearance by 90 min (Fig. (Fig.1A,1A, lower panel). TTP mRNA showed a similar induction curve in the WT cells that is characteristic of the TTP response in fibroblasts (24): i.e., undetectable levels at time zero, followed by readily detectable mRNA levels at 10 min, peak levels at 45 min, and near baseline levels by 90 min (Fig. (Fig.1A,1A, upper panels, lanes 8 to 14). In the KO cell line, a fusion mRNA was detected with the TTP probe that contains the Neo transcript interrupting the normal mRNA (33). This fusion mRNA was barely detectable at time zero, but was clearly increased by 10 min, peaked at 30 to 45 min, and was nearly back to baseline by 90 min (Fig. (Fig.1A,1A, upper panel, lanes 1 to 7, single line). The induction pattern of the TTP fusion RNA was very similar to that of the native TTP mRNA, suggesting that its induction and decay are under similar control to those of the native mRNA.
We analyzed the expression patterns of TTP protein in a similar experiment, in order to choose a time point at which TTP protein would be maximal for the later microarray analysis. As in the case of the mRNA, TTP protein was not detected at time zero in the WT cell line (Fig. (Fig.1B,1B, lane 8). However, after 15 min of serum stimulation, a trace of immunoreactive protein could be detected by Western blotting (Fig. (Fig.1B,1B, lane 9), and protein was obvious by 30 min (lane 10). Protein levels appeared to reach near maximal values by 45 min, with little change between 45 and 120 min (lanes 11 to 14). As seen in other systems, there was a gradual increase in the apparent Mr of the various immunoreactive species, consistent with increased phosphorylation (9). We chose 90 min, a time when TTP protein levels would be essentially maximal, as the time for the actinomycin D treatment in the microarray studies (see below). As expected, no immunoreactive TTP was detected in the samples from the KO cell line processed in parallel, although the nonspecific bands recognized by the antiserum were very similar between the two cell lines (Fig. (Fig.1B,1B, lanes 1 to 7).
The mRNAs for both TTP and c-fos are known to be quite labile, and AREs in the 3′-UTRs of both mRNAs have been described as possible targets for TTP binding and/or stimulated mRNA decay (27, 34). For these studies, we chose 45 min after serum stimulation, a time at which both TTP and c-fos mRNA levels were near maximal (Fig. (Fig.1A,1A, lane 12) and the TTP protein level was also near maximal (Fig. (Fig.1B,1B, lane 11). In the case of c-fos mRNA, there was again none detectable at time zero, i.e., after overnight serum deprivation, but this reached readily detectable levels by 45 min (Fig. (Fig.1C,1C, lower panel, lanes 2 and 8). Actinomycin D was added at that point, and mRNA decay was measured at intervals thereafter. As shown in Fig. Fig.1C,1C, the c-fos mRNA decay rates appeared to be very similar in both the WT and KO cell lines (lower panel, lanes 2 to 6 and 8 to 12, respectively).
Analysis of five similar experiments demonstrated that there was no stabilization of the c-fos mRNA in the TTP-deficient cells, at a time when both c-fos mRNA and TTP protein should be near maximal (Fig. (Fig.1D).1D). These results demonstrate that TTP did not promote the destabilization of this rapidly and transiently inducible and very labile mRNA under these conditions, despite the presence of an ARE in its 3′-UTR.
We also examined the effect of actinomycin D on TTP mRNA turnover. In the WT cells, the mRNA decayed rapidly when actinomycin D was added 45 min after FBS stimulation (Fig. (Fig.1C,1C, upper panel, lanes 8 to 12); several identical experiments yielded an average half-life of 17.4 min under these conditions. In the case of the fusion mRNA in the KO cells, the decay rate was also very rapid (Fig. (Fig.1C,1C, upper panel, lanes 2 to 6), with an average half-life of 14.5 min. These very similar decay patterns, along with the similar induction kinetics described in Fig. Fig.1A,1A, suggest that the TTP fusion mRNA is not abnormally stabilized in the TTP-deficient cells, arguing against a destabilizing effect of TTP on its own mRNA under these conditions.
The experimental protocol for the microarray samples involved five identical experiments, performed on separate days, in which serum-deprived fibroblasts of the WT and KO cell lines were stimulated with 10% FBS for 90 min, a time at which TTP protein levels were shown to be maximal, then treated with actinomycin D, with samples being obtained at intervals thereafter. A total of 50 RNA samples were prepared for microarray analysis, but only 48 could be labeled adequately. The relative mRNA abundances were estimated for all ~42,000 transcripts on these chips.
These microarray data were used to estimate relative transcript decay rates in the two cell lines, with a decrease in relative decay rate in the KO cell line suggesting the absence of TTP action. Decay rates for 306 transcripts were significantly different; of these, 250 transcripts exhibited increased stability in the TTP-KO cells. Of these, 33 contained two or more perfect 7-mer TTP binding site core sequences (UAUUUAU). These were then aligned with their human orthologues to test for conservation, yielding 23 transcripts in which the putative binding sites were conserved between mice and humans. The decay curves from the microarray data of RNA samples from the WT and KO samples for these 23 transcripts are shown in Fig. S2 in the supplemental material; other data concerning the names of the 23 encoded proteins, and the corresponding RefSeq or other GenBank accession numbers of the respective cDNAs are detailed in Table Table1,1, in descending order of their SAM scores.
The 23 transcripts with significantly different decay rates and two or more conserved UAUUUAU heptamers in the 3′-UTR were analyzed further, initially by Northern blotting. The top score belonged to the transcript containing immediate-early response gene 3 (Ier3), also known as IEX-1 or gly96 (see reference 30 and references therein). We examined the behavior of this transcript in some detail in this system to confirm the microarray results and to serve as an example of the other potentially stabilized transcripts. We first characterized the Ier3 transcript expression pattern after serum stimulation of the two fibroblast lines, derived initially from littermate mice. As shown in Fig. Fig.2A,2A, the patterns of induction of the Ier3 mRNA were virtually identical in the two cell lines: In both cases, the mRNA levels were essentially undetectable at time zero (after overnight serum starvation) (lanes 1 and 8), detectable within 10 min (lanes 2 and 9), and then gradually increased to reach apparently maximal levels by 90 min (lanes 7 and 14). Over a longer time course (Fig. (Fig.2B),2B), the peak mRNA levels in both cell lines appeared to occur at approximately 2 h (lanes 4 and 12). Note that transcript levels were virtually undetectable in both cell lines after 24 h, which corresponded to cells growing in normal growth medium (albeit at near confluence).
We then performed actinomycin D time course experiments beginning at both 45 and 90 min after serum stimulation, since these were the times of peak TTP protein accumulation (see Fig. Fig.1B,1B, lanes 11 and 13) and also times at which Ier3 mRNA was highly expressed (Fig. (Fig.2A,2A, lanes 5, 7, 12, and 14). When actinomycin D was added after 90 min of serum stimulation, there was an obvious difference in the decay rates of the Ier3 mRNA in the two cell lines (Fig. (Fig.2C).2C). In the WT cell line, the mRNA levels decreased rapidly after actinomycin D addition and were almost undetectable by 60 min (Fig. (Fig.2C,2C, lower panel, lanes 2 to 8). In contrast, the mRNA levels decreased much more slowly in the KO cell line, with mRNA levels still readily detectable by 120 min after actinomycin D addition (Fig. (Fig.2C,2C, upper panel, lanes 2 to 8). When the results from several identical experiments were averaged, there were obvious and statistically significant differences between the Ier3 mRNA decay rates in the two cell lines, with apparent half-lives of 21 min for the WT cells and 55 min for the KO cells (Fig. 2C1). In parallel decay experiments in which actinomycin was added 45 min after serum stimulation, the Ier3 mRNA again decayed rapidly thereafter in the WT line (Fig. (Fig.2D,2D, lower panel, lanes 2 to 9). In marked contrast, there was barely detectable degradation of the mRNA in the KO cell line during this time course (Fig. (Fig.2D,2D, upper panel, lanes 2 to 9). The averages from several identical experiments confirmed the results from the Northern blots shown, with apparent half-lives of 27 min (WT) compared to 83 min (KO) (Fig. 2D1). Similar differences in the decay of Ier3 were observed in a separate pair of WT and KO cell lines derived from primary MEFs taken from another pair of littermate mice (Fig. 2C2 and D2). These experiments were performed when the cells were in relatively early passages (20 to 30 passages) with apparent half-lives of 21 min in the WT cells and 66 min in the KO cells (actinomycin D added after 90 min of serum stimulation; Fig. 2C2), and apparent half-lives in the WT cells of 18 min and in the KO cells of 70 min (actinomycin D added at after 45 min of serum stimulation; Fig. 2D2). These results demonstrated that the differences in the decay of the Ier3 transcript in WT and TTP-KO cells were present in a second, independently derived pair of cell lines and were not dependent on the age (or passage numbers) of the cell lines. We have recently obtained similar results in a third independent pair of cell lines (not shown).
To examine whether the Ier3 transcript was stabilized in cells deficient in other TTP family members, we tested three pairs of fibroblast cell lines derived from WT and Zfp36l1 knockout embryos (32). In WT cells, Zfp36l1 mRNA was readily detectable in serum-starved cells (Fig. (Fig.3A,3A, upper panel, lane 8), with minimal if any increases seen after serum stimulation (lanes 9 to 12), returning to basal levels by 8 h (lanes 13 and 14). As expected from previous analyses (32), there was no detectable Zfp36l1 mRNA in the Zfp36l1-KO cells (Fig. (Fig.3A,3A, lanes 1 to 7). Levels of TTP mRNA expression in these cells were essentially identical in the two cell lines (Fig. (Fig.3A,3A, lower panel) and were similar to those seen in the previous experiments (Fig. (Fig.1A).1A). Similarly, there was no difference in the TTP mRNA degradation pattern found by averaging results from three pairs of fibroblast cell lines, after treatment of cells with actinomycin D 90 min after serum stimulation (data not shown), with apparent half-lives of 16 ± 3 min (mean ± standard deviation) for the WT cells and 17 ± 5 min for the KO cells. These results suggest that the stability of the TTP mRNA was not influenced by Zfp36l1 under these conditions.
Western analysis demonstrated that Zfp36l1 protein was readily detected after overnight serum starvation in the WT cell line (Fig. (Fig.3B,3B, lane 6), and changed minimally after either EGF or serum stimulation (lanes 6 to 10). No immunoreactive Zfp36l1 was detected in the KO cells processed in parallel, although the nonspecific bands recognized by the antiserum were very similar in the two cell lines (Fig. (Fig.3B,3B, lanes 1 to 5).
The Ier3 transcript expression pattern after serum stimulation of these fibroblast lines was essentially identical to that seen in the WT cells described in Fig. Fig.22 (not shown). We then performed actinomycin D time course experiments beginning 90 min after serum stimulation. There were no differences in the decay patterns of the Ier3 mRNA in the two cell lines (Fig. (Fig.3C).3C). When identical experiments were quantitated in three pairs of Zfp36l1-KO and WT cell lines, there were no differences between the normalized mean PhosphorImager values from the KO and WT cell lines (Fig. (Fig.3D),3D), with apparent half-lives of 26.6 ± 8.8 min (mean ± standard deviation) for the WT cells and 27.4 ± 6.9 min for the KO cells.
When the mouse Ier3 mRNA 3′-UTR sequence was examined for potential TTP binding sites and aligned with the orthologous sequences from other mammals, it was apparent that there were four canonical UAUUUAU heptamers in the mouse sequence that were present in all other mammals for which sequence was available (except for the dog, which had three such heptamers) (Fig. (Fig.4).4). An additional AUUUAU hexamer was present in all species except the cow, but not in the same location (Fig. (Fig.4,4, underlined).
We next tested the ability of TTP, expressed in 293 cells, to bind to the putative ARE binding sites within the 3′-UTR of the Ier3 mRNA. In each of the conserved heptameric binding sites shown in Fig. Fig.4,4, we mutated the core A residues to C's; similar mutations abrogate binding of TTP to the TNF ARE (21a). In one case, Mu8, all A residues in all four putative binding sites were mutated to C's; in the case of Mu6, the first three binding sites were mutated (Fig. (Fig.5A5A).
These mutant probes, and their 113-base WT counterpart, were then used in RNA gel shift experiments. Shown in Fig. Fig.5B5B is the typical pattern of TNF ARE probe binding to expressed TTP when cytosolic extracts were used from cells transfected with vector alone (BS+; lane 2) or cells expressing wild-type TTP (lane 3) or a mutant TTP (C124R) that does not bind ARE sequences with significant affinity under these conditions (lane 4) (19). The migration position of free probe alone (FP) is shown in lane 1. These extracts were also used in binding with the WT (lanes 5 to 8), Mu8 (lanes 9 to 12), and Mu6 (lanes 13 to 16) Ier3 probes; the positions of the free Ier3 probes are shown in lanes 5, 12, and 16.
It is apparent that the intact WT probe migration was greatly retarded: i.e., that its position in the gel was shifted very well by WT TTP (lane 7) but not by the nonbinding mutant form of TTP (lane 8). Mu8 was still able to bind to a small amount of WT TTP protein under these conditions, but its migration in the gel was greatly increased compared to that of the WT probe (lane 10); again, there was no apparent binding to the mutant TTP (lane 11). When mutant Mu6 with a single ARE was used, there was less retardation of migration than seen with the WT probe (compare lanes 14 and 7), but there was still a moderate decrease in complex migration compared to that of the Mu8 probe (compare lanes 14 and 10). Again, no binding was seen with the mutant TTP protein (lane 15) expressed at comparable levels (Fig. (Fig.5B5B).
We next evaluated a similar set of mutations for their susceptibility to TTP-induced destabilization of the Ier3 mRNA, using a cotransfection assay in 293 cells that we have described previously (20). The full-length Ier3 cDNA used to generate the expression construct was obtained by RT-PCR from total RNA prepared from fibroblasts stimulated with serum for 90 min. The sequences of the mouse Ier3 cDNAs made from the two cell lines (TTP-KO and WT) described here were identical. This conservation of the UAUUUAU sequences in the 3′-UTR of Ier3 mRNA from the two cell types strongly supported the concept that the differences in Ier3 mRNA decay in the two cell types were due to the presence or absence of TTP protein expression, rather than a change in the sequence of the Ier3 transcript.
When a full-length Ier3 mRNA was used as the TTP “target,” there was the expected effect of TTP to decrease steady-state mRNA levels at very small amounts of transfected TTP plasmid DNA (2 to 10 ng; Fig. Fig.6A,6A, upper panel, lanes 3 to 5). The nonbinding TTP mutant C124R did not decrease Ier3 mRNA levels, even at higher concentrations of transfected DNA (Fig. (Fig.6A,6A, upper panel, lanes 6 to 8), which resulted in slightly higher levels of expressed TTP mRNA (Fig. (Fig.6A,6A, lower panel, lanes 1 to 8). If anything, there was increased accumulation of the Ier3 mRNA in the presence of the TTP mutant, as has been seen previously with other TTP target mRNAs (22). These studies suggested that TTP was promoting the instability of the Ier3 mRNA in a zinc finger-dependent fashion, as seen with other physiological TTP target mRNAs (22).
Similar experiments were then performed with a more extensive set of Ier3 mutants. The sequences of each of the single-binding-site mutants is shown in Fig. Fig.6B;6B; in addition, we used a set of mutants in which there were various combinations of the single-site mutants, labeled mutants AB, AC, BC, and ABC. These were then used in cotransfection studies with WT TTP. The results of a representative experiment are shown in Fig. Fig.6C,6C, with the Ier3 mRNA levels being shown in the upper panel and the expressed TTP mRNA levels shown in the lower panel. The results of five to seven similar experiments are shown in Fig. Fig.6D,6D, in which the effect of TTP on each Ier3 “target” mRNA is compared to the effect of equal amounts of transfected vector DNA. As shown in Fig. Fig.6D,6D, TTP caused an average decrease of WT Ier3 mRNA levels to 31.7% ± 7.5% of the control. Mutants A and C were still affected by TTP, but to a lesser extent than the WT; however, mutant B was affected as strongly as the WT transcript (30.4% ± 12.5% of the control). Transcripts AB, AC, and BC were all affected to some extent by TTP, with decreases ranging from about 60% of the control to about 40% of the control. In marked contrast, the transcript with all apparent binding sites mutated actually increased to 136% ± 34% of the control in the presence of TTP, a finding that was consistently seen in all five experiments. These data suggest that all of the putative TTP binding sites present in the Ier3 ARE contribute to some extent to the susceptibility of the mRNA to TTP-induced destabilization. However, note that there are two potential binding sites in both the A and C locations in the transcript; the total number of TTP molecules that can bind to this overall ARE therefore remains to be determined.
Of the 23 transcripts whose decay in this system was further tested by Northern blotting, six transcripts in addition to Ier3 exhibited clear stabilization in the TTP-KO cells, and two other transcripts appeared to be stabilized to a lesser extent (Fig. (Fig.7).7). The identities of all nine stabilized targets (including Ier3) and the qualitative extents of their stabilization in the KO cells are listed in Table Table1.1. Of the remaining 14 transcripts from the original 23, one (transcript 4) yielded no apparent hybridizing bands on the Northern blots (either of fibroblasts or lipopolysaccharide-stimulated macrophages), and 13 exhibited transcripts that were not apparently stabilized by the absence of TTP. These findings are also summarized in Table Table11.
In addition to the Ier3 transcript described above, the seven additional transcripts that appeared to be stabilized in the TTP-deficient cells included myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila, translocated to 11 [Mllt11], transcript 3 in Fig. Fig.77 and Table Table1);1); leukemia inhibitory factor (Lif) (transcript 5); B double prime 1, a subunit of RNA polymerase III transcription initiation factor IIIB, transcript variant 1 (Bdp1) (transcript 7); proviral integration site 3 (Pim3) (transcript 9); a protein kinase, polo-like kinase 3 (Plk3) (transcript 12); and RUN- and SH3 domain-containing 2 (Rusc2; transcript 13). The two slightly affected transcripts shown in Fig. Fig.77 were those encoding polo-like kinase 2 (Plk2) (transcript 6) and pleckstrin homology-like domain, family A, member 1 (Phlda1) (transcript 14).
We also evaluated the potential TTP binding sites within the 3′-UTRs of the six additional transcripts that were stabilized in the TTP-deficient cells; these sequences, and their alignment with all available mammalian sequences from the GenBank nr, EST, and genomic databases are shown in Fig. S3 in the supplemental material. It is apparent from this figure that all six contain two or more conserved heptameric TTP binding sites in their 3′-UTRs.
As described above, we focused our attention on those transcripts that were significantly stabilized in the TTP-KO cells, as suggested by the microarray data, and that contained two or more potential TTP binding heptamers in their 3′-UTRs. However, we also checked by Northern blotting three of the transcripts that were reported to be significantly stabilized in the TTP-KO cells, with the highest SAM scores, but that did not appear to contain potential classical TTP binding sites. There was no evidence by Northern blotting that TTP deficiency resulted in the stabilization of any of these three transcripts, corresponding to GenBank accession no. BB079486, NM_024244, and BB705689 (data not shown).
The TNF mRNA was the first-identified and remains the best-characterized target for TTP-induced destabilization (13). That it is a physiologically relevant target was demonstrated by the systemic inflammatory phenotype seen in mice deficient in TTP, which could be prevented almost entirely by repeated injections of anti-TNF antibodies or by interbreeding the TTP-KO mice with mice deficient in both TNF receptors (10, 33). Nonetheless, the subsequent identification of GM-CSF mRNA as a physiological target for TTP demonstrated that there was more than one target for TTP-induced mRNA destabilization and that this interaction could occur in bone marrow-derived stromal cells instead of in the macrophages thought to be responsible for most TNF secretion (12). This indicated that TTP might be a more general regulator of ARE-containing mRNA stability, with some targets presumably cell type and stimulus specific.
In an attempt to identify other mRNA members that might be regulated by TTP, we returned to the cell type and stimuli that were used in the initial identification of TTP: cultured mouse fibroblasts, serum deprived and then stimulated with insulin, serum, or phorbol esters (24). In these cells, TTP mRNA levels are essentially undetectable in the serum-deprived cells, but increase dramatically after serum stimulation to reach maxima at approximately 45 min. We found that the corresponding protein levels were also undetectable at time zero, but reached a more stable plateau after approximately 90 min of serum stimulation. Therefore, we chose 90 min after serum treatment as a time when maximal concentrations of TTP protein would be available for target mRNA binding, while mRNAs encoded by genes rapidly but transiently induced by serum would still be available as potential TTP targets. We anticipated that the single known genetic difference between the two stable fibroblast cell lines used (i.e., the presence or absence of TTP) might permit the identification of novel TTP targets whose mRNAs were abnormally stabilized in the specific absence of TTP. Nonetheless, one limitation of our study is that only a single cell type, a single stimulus, and a single time after stimulation were used; we anticipate that other physiologically relevant TTP targets will be uncovered by using similar approaches with other cell types, stimuli, and timing paradigms.
Since we were simultaneously measuring levels of some 40,000 transcripts in these studies, one might anticipate that the mRNA decay rates for some transcripts might differ based on statistical expectations alone. We attempted to minimize this effect by purifying cellular RNA from several time points after actinomycin D addition, with multiple biological replicates at each time point. Nonetheless, it was critical to confirm any differences in decay rates from the microarray experiments by a second measurement technique: in this case, Northern blotting. In addition, since the conversion from primary embryonic fibroblast cultures to stable cell lines is undoubtedly associated with one or more genetic changes, it was important to revalidate the results with a different pair of cell lines derived from other littermate mice.
It should be noted that our intention was to use microarray and bioinformatics approaches to focus on transcripts that, in our view, had the greatest likelihood of being physiological TTP targets, rather than on discovering systematically the entire population of TTP targets. This approach meant that many interesting candidates had to be set aside while the highest-probability targets were interrogated further. For example, we focused on transcripts containing two or more heptamer TTP binding sites that were conserved between mice and humans. However, we have shown previously that the internal UUU sequence in the heptamer can be expanded to four or contracted to two U's with only twofold decreases in binding affinity (8), making it possible that sequences with those types of loosened consensus sequence could still be true binding partners. Similarly, in the 250 transcripts significantly stabilized in the TTP-KO cells, there were approximately 42 that contained only a single TTP binding heptamer; we have demonstrated previously that even a single binding site can confer TTP-dependent destabilization on a transcript (21a). In addition, there were many transcripts that had two or more binding sites that were not perfectly conserved in humans. These could well be important targets in the mouse, or the apparent failure of conservation could have been due to imperfect consensus, inadequate database information, etc. In all of these cases, likely targets that were bypassed because of our focus on high-probability targets will need to be evaluated in future follow-up studies.
The microarray studies also suggested that there were 56 transcripts that were significantly destabilized in the absence of TTP. Some of these may have been a statistical by-product of the large number of comparisons being made. However, there are precedents in the literature for ARE binding proteins that stabilize transcripts upon binding, and we have previously demonstrated that TTP, at high concentrations, can actually protect ARE-containing transcripts from accelerated decay (20). Thus, it remains theoretically possible that among these 56 transcripts will be some whose physiological decay is actually prevented by TTP binding, a possibility that will also require follow-up experiments.
The top candidate transcript in this study was that encoding Ier3, also known as IEX-1 or gly96 (see reference 30 and references therein). Its 3′-UTR contains several highly conserved potential TTP binding sites, and we showed by direct binding and cotransfection studies that these sites were important in TTP binding as well as in mediating TTP-dependent transcript decay. The Ier3 transcript has in common with the TNF, GM-CSF, and IL-2 mRNAs that it can be rapidly induced in the specific cell type of interest, in this case by serum, and it exhibited a short half-life in this situation. Most of the other validated TTP targets exhibited similar rapid induction and subsequent rapid decay kinetics, suggesting that this type of “immediate-early response” might be characteristic of most if not all physiological TTP targets. Nonetheless, the lack of effect of TTP deficiency on the prototypical immediate-early response transcript, that of c-fos, as well as its apparent lack of effect on its own fusion transcript, argues that TTP is not involved in the regulation of many other rapidly inducible, rapidly turning over transcripts. It is possible that the other TTP family members, which are more constitutively expressed in this cell type, might be responsible for the decay of mRNAs that might also be more constitutively expressed.
We studied the TTP regulation of the Ier3 transcript in some detail as an example of the studies that will be necessary to establish each of the top nine hits as physiological TTP binding partners and “targets.” This point deserves further emphasis: The other eight “top hits” were evaluated by only one or two Northern blotting experiments, and each will have to be subjected to the same battery of tests as the Ier3 transcript in order to be considered a true, physiological TTP target transcript. The Ier3 transcript fulfilled the same types of experimental criteria that were applied to previous studies of the TNF mRNA: i.e., the mRNA was markedly stabilized in KO cell lines derived from three different pairs of mice; TTP could bind to several ARE binding sites in the transcript; and TTP could be used to promote decay of the Ier3 transcript in cell transfection experiments, in a manner dependent on the presence of the characteristic TTP binding sequences in the transcript and key zinc finger residues in the TTP protein. Interestingly, the Ier3 transcript was not stabilized in analogous experiments using fibroblasts deficient in Zfp36l1 (32), a TTP family member that is expressed more constitutively in these cells. Considerable further research will be required to determine the effect of the TTP-induced destabilization of this transcript in normal physiology; it remains possible that such an effect will only be observed during some abnormal perturbation of normal physiology.
The Ier3 transcript and protein have been the subject of considerable study since their original cloning as a serum-induced gene (14). Mice in which the Ier3 gene was disrupted were recently shown to exhibit elevated blood pressure and cardiac hypertrophy (30), while earlier studies of overexpression in lymphocytes in intact mice demonstrated impaired T-cell apoptosis and increased susceptibility to a lupus-like syndrome (40). It will be of interest to determine whether the increased Ier3 mRNA stability demonstrated in the TTP-KO cells will be reflected in a contribution to the TTP deficiency phenotype in mice, which, among other characteristics, exhibit a systemic autoimmune syndrome with some lupus-like features (33). To our knowledge, there have been no reports of systemic overexpression of Ier3 in mice that might help to identify potentially relevant aspects of the TTP deficiency phenotype. One approach might be to remove the ARE, resulting in a stabilized mRNA and enhanced expression; this approach was used successfully in the case of the TNF mRNA, when removal of the ARE resulted in a profound inflammatory state that resembled a more severe form of the TTP deficiency syndrome (18). We anticipate that a similar approach, applied to the AREs of Ier3 and, possibly, the other transcripts described here, should reveal the physiological significance of these sequence elements in the intact animal.
We are very grateful to Betsy Kennington for technical assistance, Heping Cao for preparing the Zfp36l1 antiserum, and Anton Jetten and Farhad Imani for careful review of the manuscript.
This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.
Published ahead of print on 9 October 2006.
†Supplemental material for this article may be found at http://mcb.asm.org/.