INTRODUCTION

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Tristetraprolin (TTP), also known as Nup475, TIS11, or G0S24 (11, 17, 22, 27, 49), is the product of the immediate-early response genes Zfp-36 in mouse cells and ZFP36 in human cells, which map to chromosomes 7 and 19q13.1, respectively (43). TTP is the prototype of a family of zinc finger proteins of the unusual Cys-Cys-Cys-His (CCCH) class; a structure for zinc fingers of this type was recently described, and the finger was shown to bind zinc with high affinity (52). Proteins containing zinc fingers of this class have since been identified in organisms ranging from humans to yeasts (10, 13, 28-30, 35, 40, 43, 47, 50).

TTP is widely distributed, with particularly high levels of expression in spleen, lymph nodes, and thymus (22). Although it was originally described as a nuclear protein in both quiescent and serum-stimulated fibroblasts (11), it was later shown to rapidly translocate from the nucleus to the cytosol upon stimulation with serum or other mitogens (45). This translocation was accompanied by rapid serine phosphorylation of the protein (44). More recently, it has been shown to be almost exclusively cytosolic in macrophage cell lines (45) and in primary mouse macrophages (7).

A link between TTP and the cytokine tumor necrosis factor alpha (TNF-) was first suggested by studies of TTP-deficient mice (46). These animals appeared normal at birth but then rapidly developed a complex syndrome consisting of dermatitis, alopecia, conjunctivitis, cachexia, myeloid hyperplasia accompanied by extramedullary hematopoiesis, autoimmunity, and erosive polyarticular arthritis (46). Since some aspects of this phenotype resembled earlier mouse models of TNF- excess (9, 19, 48), we attempted to prevent the development of the TTP deficiency syndrome with weekly injections of anti-TNF- antibodies. This treatment prevented the development of essentially all aspects of the phenotype (46). Bone marrow transplantation from TTP-deficient mice into RAG-2-immunodeficient mice reproduced the TTP deficiency phenotype after a lag period of several months (6), suggesting that the phenotype might be due to the slow reconstitution of one or more populations of hematopoietic cells. Macrophages from the TTP-deficient mice (derived from bone marrow, fetal liver, or resident peritoneal cells) secreted approximately fivefold-more TNF- into the medium, accompanied by a twofold increase in cellular levels of TNF- mRNA, compared to the wild-type macrophages (6). These findings indicated that overexpression of TNF- from macrophages and perhaps other cells was likely to be important in the development of the TTP deficiency phenotype.

More recently, we showed that the stability of TNF- mRNA was increased by more than twofold in bone marrow-derived macrophages from TTP-deficient mice compared to cells derived from wild-type mice (half-life = 39 min in the wild type versus 85 min in the TTP-deficient cells) (7). We also demonstrated that both TTP mRNA and protein were induced by both lipopolysaccharide (LPS) and TNF- itself in normal macrophages (7). These results suggested that TTP might participate in an autoregulatory loop stimulated by TNF-, so that in the absence of TTP, TNF- could positively feed back to stimulate and maintain its own expression, resulting in the state of chronic TNF- excess seen in the TTP-deficient mice (7, 46).

To begin to investigate the apparent ability of TTP to destabilize TNF- mRNA, we focused on the AU-rich element (ARE) found in the 3' untranslated region (UTR) of TNF- from various species (5) as well as in other cytokine mRNAs such as granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) (36, 41). These AREs have long been known to confer instability on their respective mRNAs (1, 41, 42, 53). We first showed that cotransfection of TTP into HEK (human embryonic kidney) 293 cells with constructs in which the AREs from TNF-, IL-3, or GM-CSF mRNAs (53) were inserted into the 3' UTR of -globin led to the rapid degradation of -globin mRNA in all three cases (7). We next demonstrated that TTP could bind directly to the TNF- mRNA, specifically in the ARE region (7). These data indicated that, in some way, TTP could destabilize TNF- mRNA by binding to its ARE. This was the first demonstration of a direct binding partner for TTP and suggested the possibility that the CCCH family of proteins in general might be RNA binding proteins.

In the present study, we asked whether the integrity of TTP's zinc fingers was necessary for its mRNA-destabilizing and/or direct binding effect and explored the nature of the cleavage of TNF- mRNA that resulted from TTP binding to its ARE in intact cells. Our data indicate that TTP exhibits zinc finger-dependent ARE-binding activity, as well as a zinc finger-dependent ability to promote TNF- mRNA deadenylation and degradation. Through regulation of its cellular, subcellular, and tissue-specific expression; induction kinetics; and posttranslational modification, this protein offers a myriad of potential mechanisms for regulating the stability of ARE-containing mRNAs.

	MATERIALS AND METHODS

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Plasmid construction. (i) Parent plasmids. The human TTP (hTTP) cDNA (43) and an hTTP genomic clone were obtained as described elsewhere (23). Plasmid H6E was made by inserting a 3.7-kb EcoRI-XbaI fragment from the human genomic clone into the plasmid vector pBS+ (Stratagene, La Jolla, Calif.). This insert contained ~1 kb of promoter, the first exon, the single intron, the second exon, and 30 bp of the 3' flanking region.

(ii) Expression constructs. H6E.HGH3' was constructed as follows: a 597-bp NsiI-XbaI fragment in the 3' UTR of H6E that contained five rapid degradation signal sequences was replaced by 110 bp of human growth hormone (HGH) sequence that encodes the entire HGH 3' UTR (GenBank accession no. M13438). The template used to amplify this fragment was pGH (Nichols Institute Diagnostics, San Juan, Calif.). The PCR primers were 5'GTGGCTTCTAGatgcatGGGTGGCATC3' (5') and 5'GAAGGACACCtctagaGACAAAATGATGC3' (3'), where the capital letters correspond to the HGH sequences and the lowercase letters correspond to the recognition sites for NsiI (5' primer) and XbaI (3' primer).

Transfection of HEK 293 cells, Northern blot analysis, RNase H assay, and cytosolic extract preparation. HEK 293 cells were maintained in minimal essential medium (Life Technologies, Inc., Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 100 U of penicillin per ml, and 100 µg of streptomycin per ml. Transient transfection of 2 × 10⁶ cells with CMV.mTNF- or other constructs in calcium-phosphate precipitates was performed as described previously (23, 24), except that the transfection mixture was allowed to stay on the cells for 16 to 20 h and the glycerol shock step was omitted. In some experiments, pXGH5 (Nichols Institute Diagnostics) was also cotransfected to monitor transfection efficiency. Assays of released HGH were performed as described previously (23, 24).

Analysis of RNA-protein complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic mobility shift assay, and immunoprecipitation. (i) Preparation of RNA probes. Plasmid p3'mTNF- containing the mTNF- 3' UTR (bp 1110 to 1627 of GenBank accession no. X02611) was constructed by reverse transcription-PCR with RNA from RAW 264.7 cells treated for 4 h with 1 µg of LPS (Sigma, St. Louis, Mo.) per ml as a template for reverse transcription. The 5' primer for PCR amplification was 5'CTTTCCgaattcACTGGAGCCTC3', and the 3' primer was 5'TAGAtctagaAGCGATCTTTATTTCTCTC3', where the lowercase letters indicate the restriction sites for EcoRI and XbaI, respectively. The resulting PCR product was digested with these enzymes and cloned into the EcoRI and XbaI sites of the vector pSK (Stratagene).

(ii) Cross-linking of proteins to RNA. Cytosolic extracts prepared from HEK 293 cells transfected with CMV.hTTP.tag or vector (20 µg of protein) were incubated with 2 × 10⁶ cpm of RNA probe in a 96-well plate at room temperature for 20 min in 20 µl of lysis buffer (without protease inhibitors). Heparin and yeast tRNA were added to final concentrations of 0.5 µg/µl and 50 ng/µl, respectively, for an additional 10 min. The 96-well plate was then placed on ice and irradiated with 254-nm UV light in a Stratalinker (Stratagene) for 30 min at a distance of 5 cm from the light source. RNA not associated with protein was digested with 100 U of RNase T₁ (Life Technologies, Inc.) for 20 min at room temperature and further digested with 1 µg of RNase A (Pharmacia Biotech, Piscataway, N.J.) per µl at 37°C for 15 min. The RNase-resistant RNA-protein complexes were analyzed by SDS-PAGE (10% acrylamide gel) followed by autoradiography.

(iii) Western blotting. Cell extracts (5 to 50 µg of protein) were mixed with a 1/5 volume of 5× SDS sample buffer (2), boiled for 5 min, and then loaded onto SDS-10% PAGE gels. Western blotting was performed by standard techniques. Membranes were incubated in Tris-buffered saline-0.5% Tween 20 with either polyclonal antiserum HA.11 (1:2,500); a rabbit antiserum to mTTP, 2640 (1:100 [38]); or a rabbit antiserum to hTTP, DU88 (1:100 [32]). Incubation of the membranes with secondary antibody and development were performed as described elsewhere (6).

(iv) RNA electrophoretic mobility shift assay. Cytosolic extracts prepared from HEK 293 cells transfected with either vector alone, H6E.HGH3', or expression constructs driven by the CMV promoter (10 µg of protein) were incubated with 10⁵ cpm of RNA probe at room temperature for 20 min in 20 µl of lysis buffer (without protease inhibitors). Heparin and yeast tRNA were added to final concentrations of 0.5 µg/µl and 50 ng/µl, respectively, for an additional 10 min. RNA not associated with protein was digested with 100 U of RNase T₁ for 20 min at room temperature; the reaction mixture was then loaded onto a 6% nondenaturing acrylamide gel and subjected to electrophoresis at 250 V for 90 min, in 0.4× Tris-borate-EDTA buffer.

Green fluorescent protein (GFP) assays. Cells were plated onto 100-mm-diameter dishes and transfected with hTTP-EGFP fusion constructs as described above. Twenty four hours after the removal of the transfection mixture, the cells were transferred into four-well Titertek slides (Fisher Scientific, Pittsburgh, Pa.) and incubated at 37°C overnight. The cells were washed once in phosphate-buffered saline, fixed with 3.7% (vol/vol) formaldehyde for 5 min, and washed again with phosphate-buffered saline. Glass coverslips were mounted with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, Calif.) and sealed with nail polish. Fluorescence microscopy was performed with a Zeiss confocal microscope model LSM 410 UV (Carl Zeiss, Inc., Thornwood, N.Y.). Images were collected under 488-nm excitation with a 515- to 565-nm emission filter and a 100 × 1.4 numerical aperture oil immersion lens. Photographs were taken with a 16.1-s scan.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of TTP on TNF- mRNA species. In most of the expression studies in 293 cells described below, we used a TNF- expression construct, CMV.mTNF, that did not contain the entire native 3' UTR; instead, the TNF- sequence ended in the middle of the fourth AUUUA motif within the ARE (bp 1325 of GenBank accession no. X02611) (Fig. 1) and was immediately followed by 33 adenylate residues encoded by the vector. To test whether this shortened ARE exhibited TTP binding activity, we compared TTP binding to a 3'-truncated RNA probe, comprising bases 1110 to 1325 of GenBank accession no. X02611, to its binding to a nontruncated probe, comprising bases 1281 to 1350. This nontruncated probe contained the seven natural AUUUA motifs, five of them in clustered nanomers (Fig. 1). We recently demonstrated that TTP could bind directly to probe 1197-1350 probe (7). UV cross-linking of these probes to proteins in extracts from CMV.hTTP.tag-transfected cells indicated that TTP bound to the truncated probe 1110-1325 almost as well as to the probe containing all of the native AUUUA motifs (probe 1281-1350) (Fig. 1). A probe spanning bases 1197 to 1300, which contained only one AUUUA motif, exhibited barely detectable TTP binding activity under these conditions (Fig. 1).

View larger version (70K): [in this window] [in a new window]   FIG. 1.   UV cross-linking of hTTP to TNF- mRNA ARE probes. Cytosolic extracts were prepared from 293 cells transfected with 5 µg of either CMV.hTTP.tag or vector alone as described in Materials and Methods. Extract (20 µg of protein) was incubated with the indicated 32P-labeled TNF- RNA probes (2 × 106 cpm). The numbers at the top of each set refer to the base numbers in the mTNF- mRNA, as shown at the bottom of the figure. Probe 1110-1325 contained approximately 35% U residues, probe 1197-1300 contained 40% U residues, and probe 1281-1350 contained 62% U residues. Heparin and yeast tRNA were then added to decrease nonspecific binding. After UV cross-linking of the probes to cellular proteins, RNases T1 and A were added to digest probe not cross-linked to protein. The RNase-resistant RNA-protein complexes were resolved by SDS-10% PAGE followed by autoradiography. Lanes 1, probe alone (5,000 cpm); lanes 2, probe (2 × 106 cpm) treated with RNases T1 and A; lanes 3, extract (20 µg of protein) from 293 cells transfected with vector alone (5 µg of DNA); lanes 4, extract (20 µg of protein) from 293 cells transfected with CMV.hTTP.tag (5 µg). The position of TTP cross-linked to 32P-labeled RNA is indicated by the arrow. The positions of protein molecular weight standards are indicated on the left. Shown at the bottom is a portion of the mTNF- mRNA 3' UTR (GenBank accession no. X02611), from which the probes were derived. The five AU-rich nanomers are underlined. The five flanking A's within the ARE that were mutated to form a nonbinding probe are indicated in boldface.

View larger version (59K): [in this window] [in a new window]   FIG. 2.   Effect of TTP on TNF- mRNA stability. CMV.mTNF- was cotransfected into 293 cells with either TTP expression constructs or vector alone. After addition of actinomycin D to a final concentration of 10 µg/ml (+ActD) or buffer alone (ActD) for 4 h, total cellular RNA was harvested. Each lane was loaded with 10 µg of total RNA. Electrophoresis and Northern blot hybridization were performed as described in Materials and Methods. Lane 0, RNA from mock-transfected 293 cells. Lanes 1 to 8, RNA from 293 cells cotransfected with CMV.mTNF- (1 µg) and vector or CMV.hTTP.tag as follows: lanes 1, vector alone (BS+; 5 µg/plate); lanes 2 to 8, CMV.hTTP.tag (0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µg/plate, respectively). Vector was also added in lanes 2 to 7 to make the total amount of cotransfected plasmids 5 µg/plate. The Northern blots were probed with either a 32P-labeled mTNF- cDNA or a 32P-labeled mTTP cDNA. The two arrows indicate the two species of TNF- mRNA discussed in the text. The position of transfected-cell-expressed TTP mRNA is indicated by an arrow. Film exposure was 7 h for the filters hybridized with mTNF- and TTP cDNAs, as indicated; portions of the same blot probed with the TTP cDNA were also exposed to film for 18 h to show the expression of TTP mRNA in 293 cells transfected with low concentrations of CMV.hTTP.tag. The positions of the 18S rRNA are indicated. The blot hybridized with the mTNF- cDNA was stripped and reprobed with a GAPDH cDNA probe; the filter was then exposed to film for 8 h and is shown at the bottom to demonstrate equivalent loading.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Effect of low amounts of transfected CMV.hTTP.tag on the accumulation of TNF- mRNA. 293 cells were cotransfected with CMV.mTNF- and pXGH5 (1 µg each per plate) and either vector alone (5 µg/plate) or CMV.hTTP.tag (0.01, 0.05, and 0.1 µg/plate). Vector was also added to make the total amount of cotransfected plasmids 5 µg/plate. One day after replacement of the transfection medium, the medium was collected from each plate for the assay of released HGH. Total cellular RNA was then harvested. Each lane of the gel was loaded with 10 µg of total RNA. Electrophoresis and Northern blot hybridization were performed as described in Materials and Methods. The Northern blots were probed with either an mTNF- cDNA probe or an mTTP cDNA probe and exposed to film. The blot that hybridized with the mTNF- cDNA was stripped and reprobed with a GAPDH cDNA probe. The film showing the expressed mTNF- mRNA was scanned with a laser scanning densitometer, and the results were normalized to HGH expression as well as to the PhosphorImager value for GAPDH mRNA. The graph shows the average results (± standard errors) from four such experiments; the inset shows the Northern blots from a representative experiment. The two species of TNF- mRNA discussed in the text are indicated with two arrows; the positions of TTP mRNA and GAPDH mRNA are also indicated by arrows.

View larger version (62K): [in this window] [in a new window]   FIG. 4.   Effect of TTP expression on the expression of TNF- constructs containing or lacking the ARE. CMV.mTNF- (1 µg/plate) or CMV.mTNF- (dARE) (1 µg/plate) was cotransfected into 293 cells with either vector alone (BS+, 5 µg/plate), H6E.HGH3' (5 or 10 µg/plate), or CMV.hTTP.tag (0.01, 0.1, or 1 µg/plate). Vector was also added to make the total amount of cotransfected plasmids 5 µg/plate. Preparation of total cellular RNA, electrophoresis, and Northern blot analysis were performed as described in Materials and Methods. Each lane was loaded with 10 µg of total RNA. The Northern blots were probed with an mTNF- cDNA, together with probes for cyclophilin (Cyclo) or mTTP as indicated. On the left, the arrows indicate the two species of mTNF- mRNA formed in the presence of TTP; on the right, the arrow indicates the single band of mTNF- mRNA expressed from the plasmid lacking the ARE. TTP mRNA expressed from the cotransfected plasmid and the endogenous cyclophilin mRNA are also indicated by arrows.

Evidence that TTP promoted deadenylation of TNF- mRNA. The effect of TTP expression in causing shortening of the TNF- mRNA suggested that TTP was promoting deadenylation of the TNF- mRNA poly(A) tail. To evaluate this possibility, oligo(dT)_12-18 (P1) was added to total cellular RNA, and RNase H was used to remove the poly(A) tail (31). When this technique was used on RNA samples from cells cotransfected with CMV.mTNF- and either vector alone or TTP expression constructs (H6E.HGH3' in Fig. 5A and CMV.hTTP.tag in Fig. 5B and C), only the smaller of the two TNF- mRNA species remained (Fig. 5A and B; P1, lanes 1 and 2). The smaller of the two mRNA species seen in the cells transfected with TTP constructs did not further decrease in size with the RNase treatment; this fact, and its identity in size to the deadenylated TNF- mRNA from the control cells, indicated that the smaller form of the TNF- mRNA was deadenylated mRNA.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   Evidence that the smaller species of TNF- mRNA formed in the presence of TTP is a deadenylated intermediate. 293 cells were cotransfected with CMV.mTNF- (1 µg/plate) and either vector alone or TTP expression constructs as follows: lanes 1, vector alone (10 µg/plate); lanes 2, H6E.HGH3' (10 µg/plate); lanes 3, vector alone (5 µg/plate); lanes 4, CMV.hTTP.tag (5 µg/plate). As indicated, the RNA samples were treated with 0.8 U of RNase H or not treated () as described in Materials and Methods. In panels A and B, 5 µg of RNA was loaded into each lane; in panel C, 3 µg of RNA was loaded into each lane when no RNase H () was used. P1, oligonucleotide poly(dT)12-18 (0.5 µg) was added to 10 µg of 293 cell RNA. P2, an oligonucleotide (0.6 µg) complementary to bases 506 to 528 of the TNF- mRNA was added to 10 µg of 293 cell RNA. P1 + P2, both oligonucleotides were added to 15 µg of 293 cell RNA (C). The Northern blots were probed with an mTNF- cDNA. The position of the 18S rRNA is indicated. The two pairs of arrows in each panel indicate TNF- mRNA species that contained (top arrow of each pair) or did not contain (bottom arrow of each pair) their poly(A) tails. The top pair of arrows in each panel points to full-length and deadenylated TNF- mRNA; the bottom pair of arrows in each panel (3') points to the 810-bp 3' fragment of TNF- mRNA and its deadenylated form in lanes P2-2 and P2-4. The single arrow at the bottom (5') indicates the ~400-bp 5' fragment of TNF- mRNA, which is the same size in control and in TTP-expressing cells.

Evidence for a precursor-product relationship between the upper and lower forms of TNF- mRNA. In order to demonstrate that the larger, presumably polyadenylated form of TNF- mRNA could be converted to the smaller, deadenylated form by the presence of TTP, we analyzed the patterns of TNF- mRNA expression in cells cotransfected with small amounts of TTP expression constructs, before and after 4 h of exposure to actinomycin D (10 µg/ml). As shown in the cells transfected with vector alone (BS+, 10 µg; BS+, 5 µg), there was no conversion of the larger form of TNF- mRNA to a stable, smaller form in the absence of TTP, although the total amount of full-length mRNA decreased modestly after 4 h of actinomycin D exposure (Fig. 6A). However, in the presence of TTP (10 µg of H6E.HGH3' or 0.05 to 0.5 µg of CMV.hTTP.tag), actinomycin D exposure clearly led to the disappearance of the larger band, so that only the smaller band remained (Fig. 6A). Two additional experiments also examined intermediate time points. Figure 6B shows the time course of disappearance of the upper band in the presence of TTP (10 µg of H6E.HGH3' transfected) after 0, 2, and 3 h of actinomycin D treatment. Figure 6C shows a longer time course after expression of CMV.hTTP.tag (0.1 µg) with 0, 4, and 8 h of actinomycin D treatment. In both cases, the expression of TTP resulted in both forms of TNF- mRNA; the upper form then gradually disappeared after actinomycin D treatment.

View larger version (47K): [in this window] [in a new window]   FIG. 6.   Effect of TTP on the formation of the two species of TNF- mRNA in the presence and absence of actinomycin D. CMV.mTNF- (1 µg/plate) was cotransfected into 293 cells with either TTP expression constructs, as indicated, or vector alone (BS+). After addition of buffer alone () or actinomycin D (ActD) to a final concentration of 10 µg/ml (+), total cellular RNA was harvested. Each lane was loaded with 10 µg of total RNA. Electrophoresis and Northern blot hybridization were performed as described in Materials and Methods. (A) (Left) TNF- and GAPDH mRNA from mock-transfected 293 cells or from cells cotransfected with CMV.mTNF- and vector alone (10 µg/plate) or CMV.mTNF- with TTP expression construct H6E.HGH3' (10 µg/plate). (Right) TNF- and GAPDH mRNA from 293 cells cotransfected with CMV.mTNF- and vector alone (5 µg/plate) or CMV.mTNF- with CMV.hTTP.tag (0.05, 0.1, and 0.5 µg/plate). Vector was also added to make the total amount of cotransfected plasmids 5 µg/plate. Actinomycin D (+) was added for 4 h as indicated. The two arrows labeled TNF- indicate the two species of TNF- mRNA formed in the presence of TTP. (B) H6E.HGH3' (T) (10 µg/plate) or an equivalent amount of vector (V) was used in the cotransfection, and actinomycin D (10 µg/ml) was added for 0, 2, and 3 h as indicated. (C) CMV.hTTP.tag (T) (0.1 µg/plate) or an equivalent amount of vector (V) was used, and actinomycin D (10 µg/ml) was added for 0, 4, and 8 h as indicated. The two arrows indicate the two forms of TNF- mRNA in panels B and C.

Evidence that the ARE-binding protein in 293 and macrophage extracts is TTP. We next examined TNF- ARE-binding activity in cytosolic extracts from bone marrow-derived macrophages from wild-type and TTP^/ mice that had been stimulated with LPS. After the cell extracts were UV cross-linked to the TNF- ARE probe and treated with RNases, an RNase-resistant RNA-protein complex was immunoprecipitated by an anti-TTP antibody but not by preimmune serum (Fig. 7). The macrophage TTP that was immunoprecipitated from the LPS-treated TTP^+/+ cells, but not from untreated TTP^+/+ cells or from the treated or untreated TTP^/ cells, appeared as a smear with an average size of ~50 kDa, compared to the apparent 40 to 44 kDa of mTTP expressed from CMV.mTTP in 293 cells (Fig. 7). In our earlier studies, TTP migrated as a smear or multiple bands of ~35 to ~55 kDa (7, 44, 45). The difference in apparent molecular masses seen in the present experiment may have been due to differences in posttranslational modification of the TTP protein, since, for example, its apparent molecular mass is known to increase after mitogen-stimulated phosphorylation (44). Despite these differences in apparent M_r, the identity of the immunoprecipitated protein as TTP cross-linked to ³²P-labeled TNF- ARE was confirmed by the facts that the complex was precipitated from 293 cells that were transfected with TTP-expressing plasmids but not from cells transfected with vector alone, it was precipitated from 293 cells by three different antibodies including an antibody to the epitope tag (7), and it was specifically immunoprecipitated from LPS-stimulated wild-type macrophages but not from unstimulated wild-type cells or from stimulated or unstimulated TTP-deficient cells.

View larger version (46K): [in this window] [in a new window]   FIG. 7.   UV cross-linking and immunoprecipitation of TTP-RNA complexes from 293 cells or TTP+/+ or TTP/ macrophages. Cytosolic extracts from 293 cells transfected either with CMV.mTTP (5 µg) or with vector (BS) alone (5 µg) were prepared as described in Materials and Methods. Cytosolic extracts from TTP+/+ or TTP/ macrophages untreated () or treated with (+) 1 µg of LPS per ml for 4 h were prepared as described elsewhere (7). Incubation of extracts (each sample contained 20 µg of protein from 293 cells or 40 µg of macrophage protein) with the 32P-labeled TNF- probe (1281-1350), UV cross-linking, and RNase digestion were performed as described in Materials and Methods. The samples were then precleared with preimmune serum and divided into two portions, which were then incubated with preimmune serum (P), a polyclonal antibody to an mTTP-glutathione S-transferase fusion protein (I), a polyclonal antibody raised against an amino-terminal peptide of mTTP (248), or a polyclonal antibody to an hTTP-glutathione S-transferase fusion protein (DU88). The immunoprecipitated complexes were resolved by SDS-PAGE (10% polyacrylamide gel) and autoradiography. Film exposure was 8 h for the 293 cell gel and 6 days for the macrophage gel. The positions of radiolabeled transfected-cell-expressed TTP (293 cells) and endogenous macrophage TTP are indicated by the arrows. The positions of protein molecular weight standards are indicated.

Involvement of the TTP zinc fingers in the binding of TTP to the ARE of TNF- mRNA. We next evaluated the possible involvement of each of the two CCCH zinc fingers in the ARE-binding activity of TTP, by using the TNF- probe 1197-1350 (Fig. 1). When cell extracts prepared from 293 cells that had been transfected with vector alone were used in UV cross-linking experiments, a major radioactive band of ~80,000 in M_r and several minor species were noted (Fig. 8A, lane 1). When cell extracts prepared from 293 cells that had been transfected with hTTP expression constructs were used in UV cross-linking experiments, the extracts from cells transfected with either the wild-type CMV.hTTP.tag (lane 3) or the S228A mutant (a point mutation at a MAP kinase phosphorylation site in the protein [44]) (lane 6) formed readily detectable RNase-resistant RNA-protein complexes of ~43,000 in M_r with the ³²P-labeled TNF- RNA probe, while simultaneously decreasing binding of the ARE probe to the endogenous cellular ~80,000-M_r protein. However, extracts from cells transfected with 10 µg of H6E.HGH3' (hTTP driven by its native promoter and intron) (lane 2), with the C124R mutation in the CMV.hTTP.tag construct (the third C in the first zinc finger mutated to an R; lane 4), or the C147R mutation in CMV.hTTP.tag (the first C in the second zinc finger mutated to an R; lane 5) exhibited no detectable ARE-binding activity. This indicates that single cysteine-to-arginine mutations in each of the TTP zinc fingers completely prevented TTP binding to the TNF- ARE.

View larger version (40K): [in this window] [in a new window]   FIG. 8.   UV cross-linking assays of TTP-mTNF--ARE complexes. Cytosolic extracts of 293 cells transfected with either vector alone or constructs expressing hTTP were prepared as described in Materials and Methods. (A) Lanes 1, extracts from 293 cells transfected with 5 µg of vector plasmid; lanes 2, extracts from 293 cells transfected with 10 µg of plasmid H6E.HGH3'; lanes 3 to 6, extracts from 293 cells transfected with 5 µg of wild-type CMV.hTTP.tag (lane 3), zinc finger mutant C124R (lane 4), zinc finger mutant C147R (lane 5), or phosphorylation site mutant S228A (lane 6); lane 7, TNF- probe (1197-1350) alone (5,000 cpm). UV cross-linking and RNase digestion were performed as described in Materials and Methods. The RNA-protein complexes were resolved by SDS-PAGE (10% polyacrylamide gel) followed by autoradiography. The bands at ~42,000 Mr in lanes 3 and 6 (arrow) represent radiolabeled TTP. Note the endogenous cellular protein of ~80,000 in Mr that is cross-linked to the TNF- ARE in the absence of expressed TTP; this cross-linking was markedly decreased in the presence of TTP (lanes 3 and 6). (B) UV cross-linked and RNase-digested samples as described for panel A (lanes 1 to 3) were precleared with preimmune serum and divided into three portions that were incubated with preimmune serum (P), a polyclonal antibody to an hTTP-glutathione S-transferase fusion protein (DU88), or a polyclonal antibody to an mTTP-glutathione S-transferase fusion protein (2640). The immunoprecipitated complexes were resolved by SDS-PAGE (10% polyacrylamide gel) and autoradiography. The higher-molecular-weight immunoprecipitated complex is indicated by the upper arrow, and the position of TTP is indicated by the lower one. (C) UV cross-linked and RNase-digested samples as described for panel A (lanes 1 to 6) were precleared with preimmune serum and divided into two portions that were incubated with preimmune serum (P) or a polyclonal anti-epitope tag antibody (I). The immunoprecipitated complexes were resolved by SDS-PAGE (10% polyacrylamide gel) and autoradiography. A higher-molecular-weight immunoprecipitated complex is indicated by the upper arrow, and the position of TTP is indicated by the lower one. (D) Five micrograms of protein extract prepared from 293 cells as described above (lanes 1 and 3 to 6) was analyzed by Western blotting with a polyclonal antibody to the epitope tag of the fusion protein hTTG.tag. The positions of molecular weight standards (in thousands) are indicated to the left of each gel. The position of TTP is indicated by the lower arrow; the position of the ~100,000-Mr complex that contains TTP is indicated by the upper arrow.

Electrophoretic mobility shift assays. The specificity of TTP binding to the TNF- ARE was also analyzed by electrophoretic mobility shift assays with TNF- 3' UTR probes. Incubation of probe 1197-1350 (containing the seven AUUUA motifs and some sequence 5' to them [Fig. 1]) with a cytosolic extract prepared from 293 cells transfected with vector alone resulted in three major RNA-protein complexes, labeled I, II, and III (Fig. 9A, lane 1). When extracts from cells transfected with hTTP expression constructs were used, there were changes in the mobility of RNA-protein complexes I and II, while complex III disappeared (Fig. 9A, lanes 3, 2, and 6). In a separate experiment, the extract from control cells was incubated with probe 1197-1350, and RNA-protein complexes were separated in a mobility shift assay. After the gel was exposed to UV light, complexes I, II, and III were eluted and analyzed by SDS-PAGE. Complexes I and II corresponded to an ~80-kDa protein, and complex III corresponded to a ~55-kDa protein (data not shown). In the mobility shift assays, the TTP-probe complex migrated in approximately the same positions as did complexes I and II (as noted above for the UV cross-linking assays, the binding of TTP to the TNF- mRNA ARE simultaneously decreased the binding of the ARE probe to the endogenous cellular ~80,000-M_r protein). The difference in appearance between lanes 3 and 2 was probably due to the amounts of TTP protein expressed by the different expression vectors. In lane 3, the extract was from 293 cells transfected with 10 µg of H6E.GHG3', a construct that uses the weaker native promoter and produces the native TTP protein; in lanes 2 and 6, epitope-tagged TTP was expressed with the stronger CMV promoter. The same changes in protein-probe complex formation were seen when probes 1110-1325 (containing four AUUUA motifs [Fig. 1]), 1281-1350 (containing seven AUUUA motifs), and 1309-1332 (containing only four clustered UUAUUUAUU nanomers) were used in the same assay (data not shown). In order to demonstrate that the binding of complexes I and II, and TTP, to the TNF- ARE probes was specific, we also used a 54-nucleotide region from the c-fos 3' UTR that has a 62% AU content without any AUUUA motifs (53) in the mobility shift assay. This 54-nucleotide probe did not form complexes I and II with cytosolic extracts prepared from 293 cells transfected with vector alone, nor did it form a binding complex with extracts from TTP-expressing cells (data not shown).

View larger version (34K): [in this window] [in a new window]   FIG. 9.   Electrophoretic mobility shift assays of TTP-TNF--ARE complexes. Cytosolic extracts of 293 cells transfected with either vector alone or constructs expressing hTTP were prepared as described in Materials and Methods. Ten micrograms of protein extract was used in the incubation with 105 cpm of a TNF- ARE probe, and RNA mobility shift assays were performed as described in Materials and Methods. Lanes 1, extracts from 293 cells transfected with 5 µg of vector plasmid; lanes 3, extracts from 293 cells transfected with 10 µg of plasmid H6E.HGH3'; lanes 2 and 4 to 6, extracts from 293 cells transfected with 5 µg of wild-type CMV.hTTP.tag (lane 2), zinc finger mutant C124R (lane 4), zinc finger mutant C147R (lane 5), or phosphorylation site mutant S228A (lane 6). (A) RNA-protein complex migration patterns were compared in the presence of wild-type TTP (lanes 2 and 3) and its zinc finger mutants (lanes 4 and 5) or its phosphorylation site mutant (lane 6). P, mTNF- probe alone (1197-1350) after digestion with RNase T1. (B) RNA-protein complex migration patterns were compared as described for panel A, in the absence (no Ab) or presence (HA.11) of a polyclonal anti-epitope tag antibody. The supershifted RNA-protein complexes (SS) are indicated by the arrow. RNA-protein complexes I, II, and III are indicated in both panels. P, mTNF- probe alone (1197-1350) after digestion with RNase T1.

View larger version (57K): [in this window] [in a new window]   FIG. 10.   TTP zinc finger mutants are ineffective at promoting the size shift of TNF- mRNA. CMV.mTNF- (1 µg/plate) was cotransfected into 293 cells with 5 µg of vector alone per plate (lanes 1) or 5 µg of wild-type CMV.hTTP.tag (lane 2), phosphorylation site mutant S228A (lane 3), zinc finger mutant C124R (lane 4), or zinc finger mutant C147R (lane 5). For lanes 6 to 8, 293 cells were transfected with 10 µg of wild-type plasmid H6E.HGH3' (lane 6) or its zinc finger mutants (C124R, lane 7; C147R, lane 8). Preparation of total cellular RNA, electrophoresis, and Northern blot analysis were performed as described in Materials and Methods. Each lane was loaded with 10 µg of total RNA. The Northern blots were probed with a 32P-labeled mTNF- cDNA (upper panel) or mTTP cDNA (lower panel). In the upper panel, the arrows indicate the two species of mTNF- mRNA. In the lower panel, the arrow indicates the position of transfected-cell-expressed TTP mRNA. The position of the 18S rRNA is indicated.

TTP is largely nonnuclear in these experiments. We previously demonstrated by differential centrifugation techniques that TTP was almost exclusively cytosolic in normal mouse macrophages (7) and in the macrophage cell line RAW 264.7 (45), although it had previously been localized to the nucleus of both quiescent (11, 45) and serum-stimulated (11) fibroblasts. For the present study, we constructed plasmids that expressed human TTP as a fusion protein with a modified GFP, which normally is distributed throughout the cytoplasm and nucleus; this modified GFP localizes within the cell based on the peptides fused to it (12, 39). When 293 cells were transfected with EGFP-N1 (GFP alone driven by the CMV promoter), fluorescence was present in both the nucleus and the cytoplasm (Fig. 11A). However, when the TTP-GFP fusion construct was transfected into 293 cells, the fluorescence was somewhat heterogeneous and appeared to be largely nonnuclear. This was true in cells transfected with both CMV.hTTP.EGFP (Fig. 11C to F) and the H6E.EGFP construct (Fig. 11G and H) in which the hTTP-GFP fusion protein expression was driven by the native hTTP promoter and intron. Both the promoter and the single intron of TTP play important roles in its expression (23, 24). Similar predominantly cytosolic distribution was seen in HeLa cells transfected with the same constructs (data not shown).

View larger version (54K): [in this window] [in a new window]   FIG. 11.   Expression of hTTP-GFP fusion protein in 293 cells. 293 cells were transfected with either EGFP-N1 or hTTP-GFP fusion constructs, as described in Materials and Methods. Twenty-four hours after the transfection, cells were analyzed for GFP expression by confocal fluorescence microscopy with a fluorescein isothiocyanate filter. (A) Cells transfected with EGFP-N1 (5 µg); (B) the same field as in panel A under phase-contrast microscopy showing several nontransfected cells that were nonfluorescent; (C and D) cells transfected with CMV.hTTP.EGFP (10 µg); (E and F) cells transfected with CMV.hTTP.EGFP (5 µg); (G and H) cells transfected with H6E.EGFP (10 µg).

View larger version (51K): [in this window] [in a new window]   FIG. 12.   Effect of GFP-TTP on TNF- mRNA stability and binding to the ARE of mTNF- mRNA. (A and B) CMV.mTNF- (1 µg/plate) was cotransfected into 293 cells with 5 µg of either vector alone (lane 1), H6E.EGFP (lane 2), CMV.hTTP.EGFP (lane 3), or the zinc finger mutants (C124R, lane 4; C147R, lane 5) of CMV.hTTP.EGFP per plate. Preparation of total cellular RNA, electrophoresis, and Northern blot analysis were performed as described in Materials and Methods. Each lane was loaded with 10 µg of total RNA. The Northern blots were probed with 32P-labeled mTNF- cDNA (A) or mTTP cDNA (B). In panel A, the arrows indicate the two species of mTNF- mRNA; the position of the 18S rRNA is also indicated. In panel B, the positions of TTP-GFP (arrow) and the 18S rRNA are indicated. (C) 293 cells were transfected with the same plasmids as described for panels A and B but without the CMV.mTNF-. Cytosolic extracts were prepared, and 20 µg of total cytosolic protein per sample was used in UV cross-linking assays with a 32P-labeled mTNF- RNA (1281-1350) probe, followed by SDS-PAGE (10% polyacrylamide gel) and autoradiography. The top arrow indicates the position of the radiolabeled endogenous cellular protein with an Mr of ~80,000; the bottom arrow indicates the radiolabeled TTP-GFP fusion protein. The position of the 108-kDa protein standard is indicated.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The experiments reported here identify a probable function for TTP, the prototype of a subclass of vertebrate CCCH proteins characterized by two closely spaced zinc fingers with a characteristic lead-in sequence for each finger.

This protein exhibited several activities in our assays. In a cell-free system, TTP bound directly to the ARE from the TNF- mRNA. This binding was dependent upon the integrity of both zinc fingers, in that mutation of a single cysteine to arginine in either zinc finger almost totally abrogated binding to the ARE, as determined by both UV light cross-linking and gel mobility shift experiments. In intact cells, the protein caused decreases in the apparent size of TNF- mRNA in cotransfection experiments; again, this effect was not seen with cotransfection of either single amino acid zinc finger mutant. The cotransfection experiments coupled with RNase H analysis pointed to an action of TTP in stimulating removal of the poly(A) tail of the mRNA, leading to the formation of a deadenylated intermediate. Under appropriate conditions, i.e., at low concentrations of expressed TTP, this species was also degraded to yield a net decrease in total hybridizable TNF- mRNA accumulation.

We postulate, therefore, that TTP can participate in the series of steps comprising the initial deadenylation followed by the ultimate degradation of at least some of those mRNAs containing so-called type II AREs (8, 37), exemplified by TNF-, GM-CSF, and IL-3 (53). It seems likely that an early or possibly the first step in th