Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome mediated by excess tumor necrosis factor alpha (TNF-␣). Macrophages derived from these mice oversecrete TNF-␣, by a mechanism that involves stabilization of TNF-␣ mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-␣ mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-␣ ARE is dependent upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely attenuated the binding of TTP to the TNF-␣ ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-␣ mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-␣ mRNA size persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller size was due to TTP-promoted deadenylation of TNF-␣ mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-␣ mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.

Eukaryotic mRNA stability can be influenced by AU-rich elements (AREs) within mRNA primary sequences. Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that binds to ARE-containing transcripts and destabilizes them, apparently by first promoting the removal of their poly(A) tails. We developed a cell-free system in which TTP and its related proteins stimulated the deadenylation of ARE-containing, polyadenylated transcripts. Transcript deadenylation was not stimulated when a mutant TTP protein was used that was incapable of RNA binding, nor when a mutant ARE was present that did not bind TTP. The ability of TTP to promote transcript deadenylation required Mg 2؉ , but not ATP or prior capping of the RNA substrate. Cotransfection and additivity studies with the poly(A) RNase (PARN) demonstrated that TTP promoted the ability of this enzyme to deadenylate ARE-containing, polyadenylated transcripts, while having no effect on transcripts lacking an ARE. There was no effect of TTP to act synergistically with enzymatically inactive PARN mutants. We conclude that TTP can promote the deadenylation of ARE-containing, polyadenylated substrates by PARN. This interaction may be responsible for the ability of TTP and its family members to promote the deadenylation of such transcripts in intact cells.Steady-state levels of cellular mRNAs are determined by the balance between their biosynthesis and turnover. Different mRNAs can exhibit marked differences in turnover rates within the same cell, and the turnover rates of individual mRNAs can also vary significantly in response to changes in the cellular environment. In mammalian cells, the earliest step in mRNA turnover is thought to be removal of the poly(A) tail, or deadenylation, and this process in turn largely determines the overall decay rate of the mRNA (15,16,31,36).It has been appreciated for many years that cis-acting AUrich elements (AREs), often within the 3Ј-untranslated regions (3Ј-UTR) of the mRNA, can confer decreased stability on the mRNAs that contain them (32).

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor ␣ secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor ␣ and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3-untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cellfree assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor ␣. Lipolysaccharide (LPS)1 -induced production of tumor necrosis factor ␣ (TNF␣) by monocyte/macrophages is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of TNF␣ synthesis occurs in part by modulation of its mRNA stability. This in turn is dependent upon a so-called class II AU-rich element (ARE) found in the 3Ј-untranslated region of TNF␣ transcripts (1). This ARE has been implicated in the regulation of both TNF␣ mRNA stability and its translation (2, 3). Targeted deletion of the TNF␣ mRNA ARE in mice (⌬ARE mice) results in the overproduction of TNF␣ and the development of a systemic inflammatory syndrome (4). A role for the protein serine/threonine kinase p38 has been suggested in ARE-mediated TNF␣ mRNA processing by numerous studies (5-7), and it was found recently that macrophages from the ⌬ARE mice were relatively insensitive to the p38 inhibitor, SB203580 (4). Conflicting studies suggest that these p38 inhibitors can regulate TNF␣ synthesis at either the mRNA stability or protein translation level (8 -10). Mice lacking the p38 substrate MAPKAPK-2 have been reported to have defective TNF␣ synthesis following an LPS challenge (11). In this case, the regulation appears not to be due to a decrease in either TNF␣ mRNA levels or stability but rather to inhibition of translation, suggesting that the effects of the p38 pathway on mRNA stability and translation may be independent and uncoupled.These and other studies have indicated a role for the p38 signaling pathway in the post-transcriptional regulation of TNF␣ synthesis through a mechanism invol...

BackgroundZFP36, also known as tristetraprolin or TTP, and ELAVL1, also known as HuR, are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embryonic kidney cells, and examined the combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1.ResultsTargets bound and negatively regulated by ZFP36 include transcripts encoding proteins necessary for immune function and cancer, and transcripts encoding other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance upon ZFP36 overexpression independent of effects from confounding features. Genes with increased mRNA half-lives in ZFP36 knockout versus wild-type mouse cells were significantly enriched for our human ZFP36 targets. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes, and found that ZFP36 degrades transcripts through specific AU-rich sequences, representing a subset of the U-rich sequences ELAVL1 interacts with to stabilize transcripts.ConclusionsZFP36-RNA target specificities in vivo are quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to unravel in vivo combinatorial regulation by RNA-binding proteins.

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor ␣ (TNF␣) release as a consequence of increased stability of TNF␣ mRNA. TTP was then shown to destabilize TNF␣ mRNA after binding directly to the AU-rich region (ARE) of the 3-untranslated region of the TNF␣ mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNF␣ ARE in a cell-free system and, in most cases, promote the breakdown of TNF␣ mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino-and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNF␣ ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNF␣ mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.

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 ϳ70-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 ϳ10 nM. Surprisingly, we found that the "footprint" from the TNF ARE required for peptide binding was only ϳ9 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 15 N-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. Tristetraprolin (TTP)1 is the prototype of a small family of eukaryotic CCCH tandem zinc finger proteins (see Ref. 1 for recent review). In mammals, there are three known members of this protein family, whereas fish and frogs express a fourth member that seems to consist of a group of closely related proteins (2-4). The characteristic tandem zinc finger domain of these proteins consists of two zinc fingers with 18 amino acids between fingers, strict intrafinger spacing of Cx8Cx5Cx3H, and a characteristic sequence motif leading into each finger of R(K)YKTEL or a closely related variant.Studies in TTP knockout mice and cells derived from them revealed that TTP acts in normal physiology to regulate the synthesis and secretion of two clinically important cytokines, tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (5-9). The increased secretion of these cytokines in the TTP knockout mice was secondary to increased steady state levels of their mRNAs, which in turn was caused by excessive stabilization of the transcripts. TTP was found to confer instability on these two transcripts by binding to the so-called class II AREs contained within the 3Ј-untranslated regions of these mRNAs (7, 10 -13). The tandem zinc finger domain was found to be the RNA binding domain, and the invariant cys...

The CCCH tandem zinc finger protein, Zfp36l2, like its better-known relative tristetraprolin (TTP), can decrease the stability of AU-rich element-containing transcripts in cell transfection studies; however, its physiological importance is unknown. We disrupted Zfp36l2 in mice, resulting in decreased expression of a truncated protein in which the N-terminal 29 amino acids had been deleted (DeltaN-Zfp36l2). Mice derived from different clones of ES cells exhibited complete female infertility, despite evidence from embryo and ovary transplantation experiments that they could gestate and rear wild-type young. DeltaN-Zfp36l2 females apparently cycled and ovulated normally, and their ova could be fertilized; however, the embryos did not progress beyond the two-cell stage of development. These mice represent a specific model of disruption of the earliest stages of embryogenesis, implicating Zfp36l2, a probable mRNA-binding and destabilizing protein, in the physiological control of female fertility at the level of early embryonic development. This newly identified biological role for Zfp36l2 may have implications for maternal mRNA turnover in normal embryogenesis, and conceivably could be involved in some cases of unexplained human female infertility.

Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 were identified by MS analyses using MALDI (matrix-assisted laser-desorption-ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)-tandem MS and multidimensional protein identification technology. Mutations of Ser197, Ser218 and Ser228 to alanine in the human protein significantly increased TTP's gel mobility (likely to be stoichiometric), whereas mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labelling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) respectively. The present study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.