Synergistic roles of tristetraprolin family members in myeloid cells in the control of inflammation.

Members of the tristetraprolin (TTP) family of RNA-binding proteins can bind to and promote the decay of specific transcripts containing AU-rich motifs. ZFP36 (TTP) is best known for regulating pro-inflammatory cytokine expression in myeloid cells; however, its mammalian paralogues ZFP36L1 and ZFP36L2 have not been viewed as important in controlling inflammation. We knocked out these genes in myeloid cells in mice, singly and together. Single-gene myeloid-specific knockouts resulted in almost no spontaneous phenotypes. In contrast, mice with myeloid cell deficiency of all three genes developed severe inflammation, with a median survival of 8 wk. Macrophages from these mice expressed many more stabilized transcripts than cells from myeloid-specific TTP knockout mice; many of these encoded pro-inflammatory cytokines and chemokines. The failure of weight gain, arthritis, and early death could be prevented completely by two normal alleles of any of the three paralogues, and even one normal allele of Zfp36 or Zfp36l2 was enough to prevent the inflammatory phenotype. Our findings emphasize the importance of all three family members, acting in concert, in myeloid cell function.

Beta-hydroxybutyrate dampens adipose progenitors' profibrotic activation through canonical Tgfß signaling and non-canonical ZFP36-dependent mechanisms.

BACKGROUND/PURPOSE: Adipose tissue contains progenitor cells that contribute to beneficial tissue expansion when needed by de novo adipocyte formation (classical white or beige fat cells with thermogenic potential). However, in chronic obesity, they can exhibit an activated pro-fibrotic, extracellular matrix (ECM)-depositing phenotype that highly aggravates obesity-related adipose tissue dysfunction. METHODS: Given that progenitors' fibrotic activation and fat cell browning appear to be antagonistic cell fates, we have examined the anti-fibrotic potential of pro-browning agents in an obesogenic condition. RESULTS: In obese mice fed a high fat diet, thermoneutral housing, which induces brown fat cell dormancy, increases the expression of ECM gene programs compared to conventionally raised animals, indicating aggravation of obesity-related tissue fibrosis at thermoneutrality. In a model of primary cultured murine adipose progenitors, we found that exposure to ß-hydroxybutyrate selectively reduced Tgfß-dependent profibrotic responses of ECM genes like Ctgf, Loxl2 and Fn1. This effect is observed in both subcutaneous and visceral-derived adipose progenitors, as well as in 3T3-L1 fibroblasts. In 30 patients with obesity eligible for bariatric surgery, those with higher circulating ß-hydroxybutyrate levels have lower subcutaneous adipose tissue fibrotic scores. Mechanistically, ß-hydroxybutyrate limits Tgfß-dependent collagen accumulation and reduces Smad2-3 protein expression and phosphorylation in visceral progenitors. Moreover, ß-hydroxybutyrate induces the expression of the ZFP36 gene, encoding a post-transcriptional regulator that promotes the degradation of mRNA by binding to AU-rich sites within 3'UTRs. Importantly, complete ZFP36 deficiency in a mouse embryonic fibroblast line from null mice, or siRNA knock-down in primary progenitors, indicate that ZFP36 is required for ß-hydroxybutyrate anti-fibrotic effects. CONCLUSION: These data unravel the potential of ß-hydroxybutyrate to limit adipose tissue matrix deposition, a finding that might exploited in an obesogenic context.

The tandem zinc finger RNA binding domain of members of the tristetraprolin protein family.

Tristetraprolin (TTP), the prototype member of the protein family of the same name, was originally discovered as the product of a rapidly inducible gene in mouse cells. Development of a knockout (KO) mouse established that absence of the protein led to a severe inflammatory syndrome, due in part to elevated levels of tumor necrosis factor (TNF). TTP was found to bind directly and with high affinity to specific AU-rich sequences in the 3'-untranslated region of the TNF mRNA. This initial binding led to promotion of TNF mRNA decay and inhibition of its translation. Many additional TTP target mRNAs have since been identified, some of which are cytokines and chemokines involved in the inflammatory response. There are three other proteins in the mouse with similar activities and domain structures, but whose KO phenotypes are remarkably different. Moreover, proteins with similar domain structures and activities have been found throughout eukaryotes, demonstrating that this protein family arose from an ancient ancestor. The defining characteristic of this protein family is the tandem zinc finger (TZF) domain, a 64 amino acid sequence with many conserved residues that is responsible for the direct RNA binding. We discuss here many aspects of this protein domain that have been elucidated since the original discovery of TTP, including its sequence conservation throughout eukarya; its apparent continued evolution in some lineages; its functional dependence on many key conserved residues; its "interchangeability" among evolutionarily distant species; and the evidence that RNA binding is required for the physiological functions of the proteins. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Inhibiting transcription in cultured metazoan cells with actinomycin D to monitor mRNA turnover.

Decay of transcribed mRNA is a key determinant of steady state mRNA levels in cells. Global analysis of mRNA decay in cultured cells has revealed amazing heterogeneity in rates of decay under normal growth conditions, with calculated half-lives ranging from several minutes to many days. The factors that are responsible for this wide range of decay rates are largely unknown, although our knowledge of trans-acting RNA binding proteins and non-coding RNAs that can control decay rates is increasing. Many methods have been used to try to determine mRNA decay rates under various experimental conditions in cultured cells, and transcription inhibitors like actinomycin D have probably the longest history of any technique for this purpose. Despite this long history of use, the actinomycin D method has been criticized as prone to artifacts, and as ineffective for some promoters. With appropriate guidelines and controls, however, it can be a versatile, effective technique for measuring endogenous mRNA decay in cultured mammalian and insect cells, as well as the decay of exogenously-expressed transcripts. It can be used readily on a genome-wide level, and is remarkably cost-effective. In this short review, we will discuss our utilization of this approach in these cells; we hope that these methods will allow more investigators to apply this useful technique to study mRNA decay under the appropriate conditions.

A Knock-In Tristetraprolin (TTP) Zinc Finger Point Mutation in Mice: Comparison with Complete TTP Deficiency.

Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that can bind to AU-rich element-containing mRNAs and promote their decay. TTP knockout mice develop a severe inflammatory syndrome, largely due to excess tumor necrosis factor (TNF), whose mRNA is a direct target of TTP binding and destabilization. TTP's RNA binding activity and its ability to promote mRNA decay are lost when one of the zinc-coordinating residues of either zinc finger is mutated. To address several long-standing questions about TTP activity in intact animals, we developed a knock-in mouse with a cysteine-to-arginine mutation within the first zinc finger. Homozygous knock-in mice developed a severe inflammatory syndrome that was essentially identical to that of complete TTP deficiency, suggesting that TTP's critical anti-inflammatory role in mammalian physiology is secondary to its ability to bind RNA. In addition, there was no evidence for a "dominant-negative" effect of the mutant allele in heterozygotes, as suggested by previous experiments. Finally, mRNA decay experiments in mutant macrophages demonstrated that TTP can regulate the stability of its own mRNA, albeit to a minor extent. These studies suggest that RNA binding is an essential first step in the physiological activities of members of this protein family.

Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA.

The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3' UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.

Measurement of mRNA Decay in Mouse Embryonic Fibroblasts.

mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real time RT-PCR. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in the former case. This protocol is suitable for studying primary cultured cells and stable cell lines derived from transgenic mice and their respective controls, and provides for direct comparisons of mRNA decay rates in otherwise identical cells with and without the gene of interest.

Tristetraprolin (TTP) coordinately regulates primary and secondary cellular responses to proinflammatory stimuli.

TTP is an anti-inflammatory protein that acts by binding to AREs in its target mRNAs, such as Tnf mRNA, and promoting their deadenylation and decay. TNF released from inflammatory cells can then stimulate gene expression in tissue cells, such as fibroblasts. To determine whether TTP could affect the decay of TNF-induced transcripts in fibroblasts, we exposed primary embryonic fibroblasts and stable fibroblast cell lines, derived from WT and TTP KO mice, to TNF. The decay rates of transcripts encoded by several early-response genes, including Cxcl1, Cxcl2, Ier3, Ptgs2, and Lif, were significantly slowed in TTP-deficient fibroblasts after TNF stimulation. These changes were associated with TTP-dependent increases in CXCL1, CXCL2, and IER3 protein levels. The TTP-susceptible transcripts contained multiple, conserved, closely spaced, potential TTP binding sites in their 3'-UTRs. WT TTP, but not a nonbinding TTP zinc finger mutant, bound to RNA probes that were based on the mRNA sequences of Cxcl1, Cxcl2, Ptgs2, and Lif. TTP-promoted decay of transcripts encoding chemokines and other proinflammatory mediators is thus a critical post-transcriptional regulatory mechanism in the response of secondary cells, such as fibroblasts, to TNF released from primary immune cells.

Differential post-transcriptional regulation of IL-10 by TLR2 and TLR4-activated macrophages.

The activation of TLRs by microbial molecules triggers intracellular-signaling cascades and the expression of cytokines such as IL-10. Il10 expression is tightly controlled to ensure effective immune responses, while preventing pathology. Maximal TLR-induction of Il10 transcription in macrophages requires signaling through the MAPKs, ERK, and p38. Signals via p38 downstream of TLR4 activation also regulate IL-10 at the post-transcriptional level, but whether this mechanism operates downstream of other TLRs is not clear. We compared the regulation of IL-10 production in TLR2 and TLR4-stimulated BM-derived macrophages and found different stability profiles for the Il10 mRNA. TLR2 signals promoted a rapid induction and degradation of Il10 mRNA, whereas TLR4 signals protected Il10 mRNA from rapid degradation, due to the activation of Toll/IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) and enhanced p38 signaling. This differential post-transcriptional mechanism contributes to a stronger induction of IL-10 secretion via TLR4. Our study provides a molecular mechanism for the differential IL-10 production by TLR2- or TLR4-stimulated BMMs, showing that p38-induced stability is not common to all TLR-signaling pathways. This mechanism is also observed upon bacterial activation of TLR2 or TLR4 in BMMs, contributing to IL-10 modulation in these cells in an infection setting.

Mutational and structural analysis of the tandem zinc finger domain of tristetraprolin.

Tristetraprolin (TTP), the best known member of a class of tandem (R/K)YKTELCX8CX5CX3H zinc finger proteins, can destabilize target mRNAs by first binding to AU-rich elements (AREs) in their 3'-untranslated regions (UTRs) and subsequently promoting deadenylation and ultimate destruction of those mRNAs. This study sought to determine the roles of selected amino acids in the RNA binding domain, known as the tandem zinc finger (TZF) domain, in the ability of the full-length protein to bind to AREs within the tumor necrosis factor α (TNF) mRNA 3'-UTR. Within the CX8C region of the TZF domain, mutation of some of the residues specific to TTP, not found in other members of the TTP protein family, resulted in decreased binding to RNA as well as inhibited mRNA deadenylation and decay. Evaluation of simulation solution models revealed a distinct structure in the second zinc finger of TTP that was induced by the presence of these TTP-specific residues. In addition, mutations within the lead-in sequences preceding the first C of highly conserved residues within the CX5C or CX3H regions or within the linker region between the two fingers also perturbed both RNA binding and the simulation model of the TZF domain in complex with RNA. We conclude that, although the majority of conserved residues within the TZF domain of TTP are required for productive binding, not all residues at sequence-equivalent positions in the two zinc fingers of the TZF domain of TTP are functionally equivalent.

Regulation of suppressor of cytokine signaling 3 (SOCS3) mRNA stability by TNF-alpha involves activation of the MKK6/p38MAPK/MK2 cascade.

The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-alpha. In contrast to IL-6, TNF-alpha increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38(MAPK)-cascade is required for TNF-alpha-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38(MAPK) cascade, basal SOCS3-expression is strongly reduced and TNF-alpha-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38(MAPK)-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3' untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-alpha. In summary, these data indicate that TNF-alpha regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38(MAPK) cascade and that the activation of MK2, a downstream target of p38(MAPK), is important for the regulation of SOCS3 expression.

Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts.

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-2beta. 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.

Influence of nonameric AU-rich tristetraprolin-binding sites on mRNA deadenylation and turnover.

Tristetraprolin (TTP), a member of the tandem CCCH zinc finger protein family, promotes deadenylation of tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to the AU-rich elements (ARE) in their 3'-untranslated regions. The high affinity TTP-ARE binding occurs between the tandem zinc finger domain and the preferred nonamer UUAUUUAUU. By mutating a well defined core sequence of 24 bases from the tumor necrosis factor-alpha ARE, we compared the influence of four possible nonameric TTP-binding sites in the wild-type ARE with that of a single binding site in the mutated probe on the binding of TTP to the RNA and the subsequent deadenylation of the poly(A) tail. By inserting this 24-base ARE into an otherwise stable transcript, we also attempted to determine the extent of the instability conferred by the presence of one or two TTP-binding sites. These sites were created or modified by mutating the As in the UUAUUUAUU nonamer or by changing the central U in the nonamer, in both cases to C residues. The results suggest that even a single nonamer TTP-binding site can confer at least partial sensitivity to the TTP-mediated mRNA turnover on an otherwise stable mRNA, but that two binding sites make the transcript much more unstable. Even though the central U of the nonamer binding site was predicted by structural studies possibly to permit base substitution, mutation of this U to C greatly inhibited the binding of TTP to the ARE, thus reducing the ability of the TTP to promote deadenylation and instability of the mRNA.

Zfp36l3, a rodent X chromosome gene encoding a placenta-specific member of the Tristetraprolin family of CCCH tandem zinc finger proteins.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger (TZF) proteins can bind directly to AU-rich elements (ARE) in mRNA, causing deadenylation and destabilization of the transcripts to which they bind. We describe here a novel fourth mammalian member of the TTP protein family, designated ZFP36L3, which could also bind directly to ARE-containing RNAs and could promote the deadenylation and degradation of ARE-containing target RNAs. Zfp36l3 transcript expression was detected only in placenta and extraembryonic tissues in the mouse. It was expressed throughout development in the placenta and was particularly highly expressed in the cells of the labyrinthine layer of the trophoblastic placenta. Unlike the other family members, the expression of a ZFP36L3-green fluorescent protein fusion protein was entirely cytoplasmic when expressed in 293 cells, even in the presence of the CRM1-dependent nuclear export inhibitor leptomycin B. Zfp36l3 was located on the mouse X chromosome; a similar predicted gene was present on the rat X chromosome, but there was no evidence for a similar gene in humans. ZFP36L3 may thus be a rodent-specific or even murine-specific member of the TTP protein family. Its presumed role in placental physiology may be unique to rodents or murine rodents, but this role may be subsumed by other family members in nonrodents.

The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis.

Tristetraprolin (TTP) is the best-studied member of a small family of three proteins in humans that is characterized by a tandem CCCH zinc finger (TZF) domain with highly conserved sequences and spacing. Although initially discovered as a gene that could be induced rapidly and transiently by the stimulation of fibroblasts with growth factors and mitogens, it is now known that TTP can bind to AU-rich elements in mRNA, leading to the removal of the poly(A) tail from that mRNA and increased rates of mRNA turnover. This activity was discovered after TTP-deficient mice were created and found to have a systemic inflammatory syndrome with severe polyarticular arthritis and autoimmunity, as well as medullary and extramedullary myeloid hyperplasia. The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine. The myeloid hyperplasia might be due in part to increased stability of granulocyte-macrophage colony-stimulating factor (GM-CSF). This review highlights briefly the characteristics of the TTP-deficiency syndrome in mice and its possible genetic modifiers, as well as recent data on the characteristics of the TTP-binding site in the TNF-alpha and GM-CSF mRNAs. Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex. We review the current knowledge of TTP sequence variants in humans and discuss the possible contributions of the TTP-related proteins in mouse physiology and in human monocytes. The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

Interaction of retroviral Tax oncoproteins with tristetraprolin and regulation of tumor necrosis factor-alpha expression.

BACKGROUND: The Tax oncoproteins are transcriptional regulators of viral expression involved in pathogenesis induced by complex leukemogenic retroviruses (or delta-retroviruses, i.e., primate T-cell leukemia viruses and bovine leukemia virus). To better understand the molecular pathways leading to cell transformation, we aimed to identify cellular proteins interacting with Tax. METHODS: We used a yeast two-hybrid system to identify interacting cellular proteins. Interactions between Tax and candidate interacting cellular proteins were confirmed by glutathione S-transferase (GST) pulldown assays, co-immunoprecipitation, and confocal microscopy. Functional interactions between Tax and one interacting protein, tristetraprolin (TTP), were assessed by analyzing the expression of tumor necrosis factor-alpha (TNF-alpha), which is regulated by TTP, in mammalian cells (HeLa, D17, HEK 293, and RAW 264.7) transiently transfected with combinations of intact and mutant Tax and TTP. RESULTS: We obtained seven interacting cellular proteins, of which one, TTP, was further characterized. Tax and TTP were found to interact specifically through their respective carboxyl-terminal domains. The proteins colocalized in the cytoplasm in a region surrounding the nucleus of HeLa cells. Furthermore, coexpression of Tax was associated with nuclear accumulation of TTP. TTP is an immediate-early protein that inhibits expression of TNF-alpha at the post-transcriptional level. Expression of Tax reverted this inhibition, both in transient transfection experiments and in stably transfected macrophage cell lines. CONCLUSION: Tax, through its interactions with the TTP repressor, indirectly increases TNF-alpha expression. This observation is of importance for the cell transformation process induced by leukemogenic retroviruses, because TNF-alpha overexpression plays a central role in pathogenesis.

Characteristics of the interaction of a synthetic human tristetraprolin tandem zinc finger peptide with AU-rich element-containing RNA substrates.

Tristetraprolin (TTP) and its two known mammalian family members are tandem CCCH zinc finger proteins that can bind to AU-rich elements (AREs) in cellular mRNAs and destabilize those transcripts, apparently by initiating their deadenylation. Previous studies have shown that the approximately 70-amino acid tandem zinc finger domain of TTP is required and sufficient for RNA binding, and that the integrity of both zinc fingers is also required. However, little is known about the kinetics or structure of the peptide-RNA interaction, in part because of difficulties in obtaining soluble recombinant protein or peptides. We characterized the binding of a synthetic 73-amino acid peptide from human TTP to the tumor necrosis factor (TNF) ARE by gel mobility shift analyses and fluorescence anisotropy experiments. Both types of studies yielded a peptide-RNA dissociation constant of approximately 10 nM. Surprisingly, we found that the "footprint" from the TNF ARE required for peptide binding was only approximately 9 bases and that two molecules of peptide could bind to probes containing as little as 19 bases. An identical recombinant peptide exhibited gel shift characteristics similar to those of the synthetic peptide. NMR analysis of the 15N-labeled recombinant peptide suggested that its first zinc finger was structured in solution but that the second was not. The titration of oligonucleotides representing 17, 13, and even 9 bases of the TNF ARE caused an essentially identical, dramatic shift of existing resonances, and the appearance of new resonances in the peptide spectra, so that all amino acids could be assigned. These data suggest that this TTP peptide-RNA complex is structured in solution and might be amenable to NMR structure determination.

Interactions of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs.

Tristetraprolin (TTP), the prototype of a small family of CCCH tandem zinc finger (TZF) domain proteins, is a physiological stimulator of instability of the mRNAs encoding tumor necrosis factor-alpha and granulocyte/macrophage colony-stimulating factor in certain cell types. TTP stimulates mRNA turnover after binding to class II AU-rich elements (AREs) within the 3'-untranslated regions of both mRNAs. In turn, this binding is dependent upon the key CCCH residues in the TZF domain. To evaluate other primary sequence requirements for ARE binding in this novel mRNA-binding domain, we mutated many of the conserved residues within the TZF domain of human TTP and evaluated the effects of these mutations on RNA binding in a cell-free system and TTP-induced mRNA instability in cell transfection experiments. These mutations revealed a number of conserved amino acids that were required for binding and begin to define the primary protein sequence requirements for this novel mRNA-binding motif. Unexpectedly, all of the point mutations that prevented TTP binding to RNA also caused an increase in steady-state levels of ARE-containing mRNAs in cell transfection experiments. Actinomycin D experiments suggested that this effect was due to inhibition of mRNA turnover. Although expression of the mutant form of TTP could also inhibit the destruction of tumor necrosis factor-alpha mRNA by wild-type TTP, the primary mechanism did not involve heterodimerization with wild-type TTP because the 293 cells used in these studies express no detectable endogenous TTP. These data suggest that TTP may act, at least in part, by physically interacting with an enzyme activity or protein complex and functionally stimulating its ability to deadenylate class II ARE-containing mRNAs.