Evidence that Tristetraprolin Binds to AU-Rich Elements and Promotes the Deadenylation and Destabilization of Tumor Necrosis Factor Alpha mRNA

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-alpha). Macrophages derived from these mice oversecrete TNF-alpha, by a mechanism that involves stabilization of TNF-alpha mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-alpha mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-alpha 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-alpha ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-alpha mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-alpha 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-alpha mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-alpha mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.     

Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins

Tristetraprolin (TTP) is the prototype of a family of CCCH tandem zinc finger proteins that can bind to AU-rich elements in mRNAs and promote their decay. TTP binds to mRNA through its central tandem zinc finger domain; it then promotes mRNA deadenylation, considered to be the rate-limiting step in eukaryotic mRNA decay. We found that TTP and its related family members could bind to certain isoforms of another AU-rich element-binding protein, HNRNPD/AUF1, as well as a related protein, laAUF1. The interaction domain within AUF1p45 appeared to be a C-terminal "GY" region, and the interaction domain within TTP was the tandem zinc finger domain. Surprisingly, binding of AUF1p45 to TTP occurred even with TTP mutants that lacked RNA binding activity. In cell extracts, binding of AUF1p45 to TTP potentiated TTP binding to ARE-containing RNA probes, as determined by RNA gel shift assays; AUF1p45 did not bind to the RNA probes under these conditions. Using purified, recombinant proteins and a synthetic RNA target in FRET assays, we demonstrated that AUF1p45, but not AUF1p37, increased TTP binding affinity for RNA ～5-fold. These data suggest that certain isoforms of AUF1 can serve as "co-activators" of TTP family protein binding to RNA. The results raise interesting questions about the ability of AUF1 isoforms to regulate the mRNA binding and decay-promoting activities of TTP and its family members as well as the ability of AUF1 proteins to serve as possible physical links between TTP and other mRNA decay proteins and structures.     

Recruitment of the 4EHP-GYF2 cap-binding complex to tetraproline motifs of tristetraprolin promotes repression and degradation of mRNAs with AU-rich elements

The zinc finger protein tristetraprolin (TTP) promotes translation repression and degradation of mRNAs containing AU-rich elements (AREs). Although much attention has been directed toward understanding the decay process and machinery involved, the translation repression role of TTP has remained poorly understood. Here we identify the cap-binding translation repression 4EHP-GYF2 complex as a cofactor of TTP. Immunoprecipitation and in vitro pull-down assays demonstrate that TTP associates with the 4EHP-GYF2 complex via direct interaction with GYF2, and mutational analyses show that this interaction occurs via conserved tetraproline motifs of TTP. Mutant TTP with diminished 4EHP-GYF2 binding is impaired in its ability to repress a luciferase reporter ARE-mRNA. 4EHP knockout mouse embryonic fibroblasts (MEFs) display increased induction and slower turnover of TTP-target mRNAs as compared to wild-type MEFs. Our work highlights the function of the conserved tetraproline motifs of TTP and identifies 4EHP-GYF2 as a cofactor in translational repression and mRNA decay by TTP.     

Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha 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 TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha 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 TNFalpha 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 TNFalpha 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.     

Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.     

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.     

Rapid Proteasomal Degradation of Posttranscriptional Regulators of the TIS11/Tristetraprolin Family Is Induced by an Intrinsically Unstructured Region Independently of Ubiquitination

The TIS11/tristetraprolin (TTP) CCCH tandem zinc finger proteins are major effectors in the destabilization of mRNAs bearing AU-rich elements (ARE) in their 3′ untranslated regions. In this report, we demonstrate that the Drosophila melanogaster dTIS11 protein is short-lived due to its rapid ubiquitin-independent degradation by the proteasome. Our data indicate that this mechanism is tightly associated with the intrinsically unstructured, disordered N- and C-terminal domains of the protein. Furthermore, we show that TTP, the mammalian TIS11/TTP protein prototype, shares the same three-dimensional characteristics and is degraded by the same proteolytic pathway as dTIS11, thereby indicating that this mechanism has been conserved across evolution. Finally, we observed a phosphorylation-dependent inhibition of dTIS11 and TTP degradation by the proteasome in vitro, raising the possibility that such modifications directly affect proteasomal recognition for these proteins. As a group, RNA-binding proteins (RNA-BPs) have been described as enriched in intrinsically disordered regions, thus raising the possibility that the mechanism that we uncovered for TIS11/TTP turnover is widespread among other RNA-BPs.     

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.     

Identification of porcine glycogen synthase kinase 3α (GSK-3α) gene and its association with carcass traits

GSK-3 plays an important role on numerous cellular processes involved in the regulation of embryonic development, protein synthesis, glycogen metabolism, inflammatory, mitosis and apoptosis. In this study, we obtained the cDNA and promoter sequences of the porcine GSK-3α gene, analyzed its genomic organization and mapped it to SSC6q12 through comparative mapping method. Moreover, the qRT-PCR analysis revealed that porcine GSK-3α gene was widely expressed in many tissues, and a high expression level was observed in the brain and spleen. In addition, seven single-nucleotide polymorphisms were detected in the promoter region of porcine GSK-3α gene. Association analysis revealed that the GSK-3α Hin1I and MspI polymorphisms both had significant associations (p < 0.05) with loin muscle area, average backfat thickness, thorax–waist fat thickness, and buttock fat thickness. These results provide useful information for further investigation on the function of porcine GSK-3α gene.     

Functional analysis of KSRP interaction with the AU-rich element of interleukin-8 and identification of inflammatory mRNA targets

mRNA stability is a major determinant of inflammatory gene expression. Rapid degradation of interleukin-8 (IL-8) mRNA is imposed by a bipartite AU-rich element (ARE) in the 3′ untranslated region (R. Winzen et al., Mol. Cell. Biol. 24:4835-4847, 2004). Small interfering RNA-mediated knockdown of the ARE-binding protein KSRP resulted in stabilization of IL-8 mRNA or of a β-globin reporter mRNA containing the IL-8 ARE. Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.