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At least 19 recordsLinked to original sources
PubMed2001

Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway.

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor alpha 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 alpha 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 cell-free 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 alpha.

3' Untranslated Regions↗
PubMed2004

The stability of tristetraprolin mRNA is regulated by mitogen-activated protein kinase p38 and by tristetraprolin itself.

Tristetraprolin (TTP) is an mRNA-destabilizing protein that negatively regulates the expression of proinflammatory mediators such as tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor, and cyclooxygenase 2. Here we investigate the regulation of TTP expression in the mouse macrophage cell line RAW264.7. We show that TTP mRNA is expressed in a biphasic manner following stimulation of cells with lipopolysaccharide and that the second phase of expression, like the first, is dependent on mitogen-activated protein kinase (MAPK) p38. MAPK p38 acts through a downstream kinase to stabilize TTP mRNA, and this stabilization is mediated by an adenosine/uridine-rich region at the 3'-end of the TTP 3'-untranslated region. Hence TTP is post-transcriptionally regulated in a similar manner to several proinflammatory genes. We also demonstrate that TTP is able to bind to its own 3'-untranslated region and negatively regulate its own expression, forming a feedback loop to limit expression levels.

3' Untranslated Regions↗
PubMed2005

Tristetraprolin regulates the expression of the human inducible nitric-oxide synthase gene.

The expression of human inducible NO synthase (iNOS) is regulated both by transcriptional and post-transcriptional mechanisms. Stabilization of mRNAs often depends on activation of p38 mitogen-activated protein kinase (p38 MAPK). In human DLD-1 cells, inhibition of p38 MAPK by the compound 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) or by overexpression of a dominant-negative p38 MAPKalpha protein resulted in a reduction of human iNOS mRNA and protein expression, whereas human iNOS promoter activity was not affected. An important RNA binding protein regulated by the p38 MAPK pathway and involved in the regulation of the stability of several mRNAs is tristetraprolin. RNase protection, quantitative real-time polymerase chain reaction, and Western blot experiments showed that cytokines used to induce iNOS expression in DLD-1 cells also enhanced tristetraprolin expression. SB203580 incubation reduced cytokine-mediated enhancement of tristetraprolin expression. Overexpression or down-regulation of tristetraprolin in stably transfected DLD-1- or A549/8 cells consistently resulted in enhanced or reduced iNOS expression by modulating iNOS-mRNA stability. In UV cross-linking experiments, recombinant tristetraprolin did not interact with the human iNOS mRNA. However, coimmunoprecipitation experiments showed interaction of tristetraprolin with the KH-type splicing regulatory protein (KSRP), which is known to recruit mRNAs containing AU-rich elements to the exosome for degradation. This tristetraprolin-KSRP interaction was enhanced by cytokines and reduced by SB203580 treatment. We conclude that tristetraprolin positively regulates human iNOS expression by enhancing the stability of human iNOS mRNA. Because tristetraprolin does not directly bind to the human iNOS mRNA but interacts with KSRP, tristetraprolin is likely to stabilize iNOS mRNA by capturing the KSRP-exosome complex.

Cell Line↗
PubMed2006

Tristetraprolin inhibits HIV-1 production by binding to genomic RNA.

HIV-1 genome has an AU-rich sequence and requires rapid nuclear export by Rev activity to prevent multiple splicing. HIV-1 infection occurs in activated CD4(+) T cells where the decay of mRNAs of cytokines and chemokines is regulated by the binding of AU-rich elements to the mRNA-destabilizing protein tristetraprolin. We here investigated the influence of tristetraprolin on the replication of HIV-1. Treatment of siRNA against tristetraprolin in a latently HIV-1 infected cell line increases HIV-1 production following stimulation. A chloramphenicol acetyltransferase and luciferase assay revealed that exogenous tristetraprolin reduced HIV-1 virion production and in contrast increased the multiply spliced products. Furthermore, quantitative RT-PCR analysis showed tristetraprolin increases the ratio of multiple-spliced RNAs to un-, single-spliced RNA. Moreover, an electrophoretic mobility shift assay showed that tristetraprolin binds to synthesized HIV-1 RNA with AU-rich sequence but not to RNA with less AU sequence. These results suggest that tristetraprolin is a regulator of HIV-1 replication and enhances splicing by direct binding to AU-rich sequence of HIV-1 RNAs.

Cell Line↗
PubMed2001

HuA and tristetraprolin are induced following T cell activation and display distinct but overlapping RNA binding specificities.

AU-rich elements found in the 3'-untranslated regions of cytokine and proto-oncogene transcripts regulate mRNA degradation and function as binding sites for the mRNA-stabilizing protein HuA and the mRNA-destabilizing protein tristetraprolin. Experiments were performed to evaluate the expression of HuA and tristetraprolin in purified human T lymphocytes and to evaluate the ability of these proteins to recognize specific AU-rich sequences. HuA is a predominantly nuclear protein that can also be found in the cytoplasm of resting T lymphocytes. Within 1 h after stimulation of T lymphocytes with anti-T cell receptor antibodies or a combination of a phorbol myristate acetate and ionomycin, an increase in cytoplasmic HuA RNA-binding activity was observed. Although absent in resting cells, cytoplasmic tristetraprolin protein was detected 3-6 h following activation. HuA recognized specific AU-rich sequences found in c-jun or c-myc mRNA that were poorly recognized by tristetraprolin. In contrast, tristetraprolin recognized an AU-rich sequence in interleukin-2 mRNA that was poorly recognized by HuA. Both HuA and tristetraprolin, however, recognized AU-rich sequences from c-fos, interleukin-3, tumor necrosis factor-alpha, and granulocyte/macrophage colony-stimulating factor mRNA. HuA may transiently stabilize a subset of AU-rich element-containing transcripts following T lymphocyte activation, and tristetraprolin may subsequently mediate their degradation.

3' Untranslated Regions↗
PubMed2004

Herpes simplex virus 1 induces cytoplasmic accumulation of TIA-1/TIAR and both synthesis and cytoplasmic accumulation of tristetraprolin, two cellular proteins that bind and destabilize AU-rich RNAs.

Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the U(L)41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3'-to-5' degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking U(L)41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking U(L)41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

Cell Line, Tumor↗
PubMed2003

Tristetraprolin binds to the 3'-untranslated region of cyclooxygenase-2 mRNA. A polyadenylation variant in a cancer cell line lacks the binding site.

In human colorectal adenocarcinoma cell lines, we found two major transcripts of cyclooxygenase-2, the full-length mRNA and a short polyadenylation variant (2577 kb) lacking the distal segment of the 3'-untranslated region. Tristetraprolin, an mRNA-binding protein that promotes message instability, was shown to bind the cyclooxygenase-2 mRNA in the region of the 3'-untranslated region between nucleotides 3125 and 3432 and to reduce levels of the full-length mRNA. During cell growth and confluence, the expression of tristetraprolin mRNA was inversely correlated with that of the full-length cyclooxygenase-2 transcript, and transfection of tristetraprolin into HCA-7 cells reduced the level of full-length cyclooxygenase-2 mRNA. However, the truncated transcript escaped tristetraprolin binding and downregulation.

3' Untranslated Regions↗
PubMed2006

Expression of TNF-alpha, tristetraprolin, T-cell intracellular antigen-1 and Hu antigen R genes in synovium of patients with rheumatoid arthritis.

Post-transcriptional regulation through the AU-rich element (ARE) by ARE binding proteins (ARBPs) has an important role in controlling the production of cytokines, including tumor necrosis factor (TNF)-alpha. Therefore, expression of ARBPs may influence, or may be influenced, by the severity of rheumatoid arthritis (RA). We measured the gene expression of ARBPs, including tristetraprolin, T-cell intracellular antigen (TIA)-1 and Hu antigen R (HuR), in synovial tissues from RA and osteoarthritis patients. cDNA was constructed from synovial tissues obtained from 21 patients with RA, and those from 12 patients with osteoarthritis. Gene expression was measured using the TaqMan PCR real-time quantification method. No significant differences were observed in the expression of tristetraprolin, TIA-1 or HuR genes between RA and osteo-arthritis synovium samples. No significant relationships between expression of tristetraprolin, TIA-1 or HuR genes and TNF-alpha gene expression serum CRP levels in samples from RA patients were observed. A significant positive relationship was observed between gene expression levels of TIA-1 and HuR. While HuR stabilizes TNF-alpha mRNA and enhances TNF-alpha production, TIA-1 acts as a post-transcriptional silencer, and suppresses the production of the TNF-alpha protein. The clear positive relationship between the expression of these two ARBPs may imply that the expression of either gene affects the expression of the other, or the mechanisms that control the expression of these genes have some factors in common.

Aged↗
PubMed2002

Analysis of the function, expression, and subcellular distribution of human tristetraprolin.

OBJECTIVE: The zinc-finger protein tristetraprolin (TTP) has been demonstrated to regulate tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) instability in murine macrophages. We sought to develop a model system to characterize the effects of human TTP (hTTP) on TNFalpha 3'-untranslated region (3'-UTR)-mediated expression. We also generated a specific polyclonal antibody against hTTP that enabled the examination of the subcellular distribution of hTTP and its RNA binding in vivo. METHODS: Transfection of reporter gene constructs were used to functionally characterize the role of hTTP in regulating TNFalpha expression in a 3'-UTR-dependent manner. An immunoprecipitation reverse transcription-polymerase chain reaction technique, immunoblotting, immunocytochemistry, and sucrose density fractionation were used to identify and localize hTTP. RESULTS: We found that hTTP interacted with human TNFalpha mRNA in the cytoplasm. The presence of the TNFalpha 3'-UTR was sufficient to confer binding by TTP in vivo. This interaction resulted in reduced luciferase reporter gene activity in a TNFalpha 3'-UTR adenine-uridine-rich element (ARE)-dependent manner. Immunoblotting and immunocytochemistry indicated that endogenous and transfected hTTP localized to the cytoplasm. Results of sucrose density fractionation studies were consistent with a polysomal location of hTTP. In rheumatoid synovium, hTTP expression was restricted to cells in the synovial lining layers. CONCLUSION: Through the development of an antiserum specific for hTTP, we have been able to demonstrate that hTTP binds specifically to the TNFalpha 3'-UTR and reduces reporter gene expression in an ARE-specific manner. These studies establish that hTTP is likely to function in a similar, if not identical manner, in the posttranscriptional regulation of TNFalpha. Understanding the posttranscriptional regulation of TNFalpha biosynthesis is important for the development of novel treatment strategies in rheumatoid arthritis.

3' Untranslated Regions↗
PubMed2007

Tristetraprolin recruits functional mRNA decay complexes to ARE sequences.

AU-rich elements (AREs) in the 3' untranslated region (UTR) of numerous mammalian transcripts function as instability elements that promote rapid mRNA degradation. Tristetraprolin (TTP) is an ARE-binding protein that promotes rapid mRNA decay through mechanisms that are poorly understood. A 31 nucleotide ARE sequences from the TNF-alpha 3' UTR promoted TTP-dependent mRNA decay when it was inserted into the 3' UTR of a beta-globin reporter transcript, indicating that this short sequence was sufficient for TTP function. We used a gel shift assay to identify a TTP-containing complex in cytoplasmic extracts from TTP-transfected HeLa cells that bound specifically to short ARE sequences. This TTP-containing complex also contained the 5'-3' exonuclease Xrn1 and the exosome component PM-scl75 because it was super-shifted with anti-Xrn1 or anti-PMscl75 antibodies. RNA affinity purification verified that these proteins associated specifically with ARE sequences in a TTP-dependent manner. Using a competition binding assay, we found that the TTP-containing complex bound with high affinity to short ARE sequences from GM-CSF, IL-3, TNF-alpha, IL-2, and c-fos, but did not bind to a U-rich sequence from c-myc, a 22 nucleotide poly U sequence or a mutated GM-CSF control sequence. High affinity binding by the TTP-containing complex correlated with TTP-dependent deadenylation and decay of capped, polyadenylated transcripts in a cell-free mRNA decay assay, suggesting that the TTP-containing complex was functional. These data support a model whereby TTP functions to enhance mRNA decay by recruiting components of the cellular mRNA decay machinery to the transcript.

3' Untranslated Regions↗
PubMed2004

Direct association of tristetraprolin with the nucleoporin CAN/Nup214.

Tristetraprolin (TTP) is a widely expressed, zinc finger-containing protein that has been implicated in the regulation of TNFalpha production in mice. Stimulus-dependent cytoplasmic translocation of TTP has been demonstrated in several cells. In this report we used the yeast two-hybrid screen to identify proteins able to interact with full length, human TTP. One of the isolated TTP-interacting clones encoded the FG repeat region of the nuclear pore protein Nup214. Full length Nup214 co-precipitated with TTP from resting and LPS-stimulated THP-1 cells, indicating that this interaction occurred in intact cells. The ability of TTP to associate with Nup214 was dependent on two intact zinc fingers within TTP. In contrast to wild type TTP that localized primarily in the cytosol, a mutant unable to associate with Nup214 localized throughout the cell, suggesting that the interaction with Nup214 regulates TTP localization.

Active Transport, Cell Nucleus↗
PubMed2005

Inhibition of tristetraprolin expression by dexamethasone in activated macrophages.

Tristetraprolin (TTP) is a factor that regulates mRNA stability and the expression of certain inflammatory genes. In the present study, we found that TTP expression was increased in macrophages exposed to bacterial lipopolysaccharide (LPS). Dexamethasone and dissociated steroid RU24858 inhibited LPS-induced TTP protein and mRNA expression and the inhibitory effect was reversed by a glucocorticoid receptor antagonist mifepristone. Histone deacetylase inhibitors trichostatin A (TSA) and apicidin reduced the inhibitory effect of dexamethasone and RU24858 on TTP expression, but the glucocorticoids did not alter TTP mRNA half-life. These results suggest that anti-inflammatory steroids reduce TTP expression in activated macrophages by a glucocorticoid response element (GRE)-independent mechanism, possibly through histone deacetylation and transcriptional silencing.

Animals↗
PubMed2003

Expression and purification of recombinant tristetraprolin that can bind to tumor necrosis factor-alpha mRNA and serve as a substrate for mitogen-activated protein kinases.

Tristetraprolin (TTP) is an mRNA-binding protein, but studies of this interaction have been difficult due to problems with the purification of recombinant TTP. In the present study, we expressed human and mouse TTP as glutathione S-transferase and maltose-binding protein (MBP) fusion proteins in Escherichia coli, and purified them by affinity resins and Mono Q chromatography. TTP cleaved from the fusion protein was identified by immunoblotting, MALDI-MS, and protein sequencing, and was further purified to homogeneity by continuous-elution SDS-gel electrophoresis. Purified recombinant TTP bound to the AU-rich element of tumor necrosis factor-alpha (TNFalpha) mRNA and this binding was dependent on Zn(2+). Results from sizing columns suggested that the active species might be in the form of an oligomer of MBP-TTP. Recombinant TTP was phosphorylated by three members of the mitogen-activated protein (MAP) kinase family, p42, p38, and JNK, with half-maximal phosphorylation occurring at approximately 0.5, 0.25, and 0.25 microM protein, respectively. Phosphorylation by these kinases did not appear to affect the ability of TTP to bind to TNFalpha mRNA under the assay conditions. This study describes a procedure for purifying nonfusion protein TTP to homogeneity, demonstrates that TTP's RNA-binding activity is zinc dependent, and that TTP can be phosphorylated by JNK as well as by the other members of the greater MAP kinase family.

Animals↗
PubMed2003

Polymorphisms in the genes encoding members of the tristetraprolin family of human tandem CCCH zinc finger proteins.

The three known mammalian CCCH tandem zinc finger proteins of the tristetraprolin (TTP) class have recently been demonstrated to be mRNA-binding proteins. The prototype, TTP, functions in normal physiology to promote the instability of the tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor mRNAs. Conversely, these mRNAs are stabilized in TTP-deficient mice, leading to an inflammatory phenotype characterized by overproduction of these cytokines. To explore sequence variations in TTP and its two related proteins, we sequenced genomic DNA encoding the TTP protein (ZFP36) and those of its two known mammalian relatives, ZFP36L1 and ZFP36L2, from 72 to 92 anonymous human subjects from various geographical and ethnic backgrounds. We also sequenced ZFP36 in genomic DNA from 92 subjects exhibiting evidence of excessive TNFalpha action. The resequencing strategy identified 13 polymorphisms in the protein-coding regions of these three genes, of which six would result in amino acid changes; other putative polymorphisms were identified by EST searches. One mutation in ZFP36L1 was a dinucleotide substitution that would prevent splicing of the single intron. This mutation was identified in only one allele of the original 144 sequenced from an adult female Aka Pygmy from the Central African Republic; a second individual with the same variant allele was found by genotyping 58 additional Aka DNA samples. Analysis of mRNA from one of these subject's lymphoblasts confirmed that ZFP36L1 mRNA levels were approximately 50% of those in a comparable sample without the mutation. The functional significance of this and the other polymorphisms identified remains to be determined by both biochemical and population linkage studies.

Amino Acid Sequence↗
PubMed1996

A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency.

Tristetraprolin (TTP) is a widely expressed potential transcription factor that contains two unusual CCCH zinc fingers and is encoded by the immediate-early response gene, Zfp-36. Mice made deficient in TTP by gene targeting appeared normal at birth, but soon manifested marked medullary and extramedullary myeloid hyperplasia associated with cachexia, erosive arthritis, dermatitis, conjunctivitis, glomerular mesangial thickening, and high titers of anti-DNA and antinuclear antibodies. Myeloid progenitors from these mice showed no increase in sensitivity to growth factors. Treatment of young TTP-deficient mice with antibodies to tumor necrosis factor alpha (TNF alpha) prevented the development of essentially all aspects of the phenotype. These results indicate a role for TTP in regulating TNF alpha synthesis, secretion, turnover, or action. TTP-deficient mice may serve as useful models of the autoimmune inflammatory state resulting from chronic effective TNF alpha excess.

Animals↗
PubMed2003

Selective RNA binding by a single CCCH zinc-binding domain from Nup475 (Tristetraprolin).

Regulation of gene expression takes place at several different levels and involves specific domains involved in specific protein-nucleic acid interactions. The protein Nup475 (also known as Tristetraprolin and TS11) binds to AU-rich sequence elements in certain mRNA molecules and favors the degradation of these mRNAs. The nucleic acid binding domain of Nup475 consists of two CCCH zinc-binding domains. A 36-amino acid peptide corresponding to the first of these CCCH domains has been synthesized and characterized. This peptide binds metal ions such as zinc(II) and cobalt(II) with affinities comparable to those of other authenticated zinc-binding domains. The zinc(II) complex of this peptide binds the RNA oligonucleotide UUUAUUU labeled with fluorescein on the 3'-end with an affinity of approximately 5 microM and discriminates against other sequences lacking the central A or the flanking U residues. These results demonstrate for the first time that a single CCCH domain is capable of binding single-stranded RNA with considerable affinity and selectivity. The combination of this well-behaved domain and the fluorescence-based binding assay sets the stage for more detailed structure-activity studies.

Anisotropy↗
PubMed2004

Expression, purification, and biochemical characterization of the antiinflammatory tristetraprolin: a zinc-dependent mRNA binding protein affected by posttranslational modifications.

Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor alpha mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 microM Zn(2+). The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zinc-dependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP-ARE complex.

Animals↗
PubMed2006

Functional characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475-2D): RNA binding affinity and selectivity.

The protein tristetraprolin (TTP, also known as NUP475 and TIS11) is a nonclassical zinc finger protein that is involved in regulating the inflammatory response. Specifically, TTP binds to AU-rich sequence elements located at the 3'-untranslated region of cytokine mRNAs forming a complex that is degraded by the exosome. The nucleic acid binding region of TTP is comprised of two CysX(8)CysX(5)CysX(3)His domains that are activated in the presence of zinc. A two-domain construct of TTP (TTP-2D) has been cloned and overexpressed in E. coli. TTP-2D picks up visible red coloration from the expression media, unless it is expressed under iron-restricted conditions. The iron-binding properties of TTP-2D and the effect of iron substitution on RNA recognition have been investigated. Both Fe(II) and Fe(III) bind to TTP-2D and a full titration of Fe(III) with TTP-2D revealed that this metal ion binds with micromolar affinity. Upon reconstitution of TTP-2D with either Fe(II) or Fe(III), the protein recognizes a canonical RNA-binding sequence, UUUAUUUAUUU, with nanomolar affinity. Substitution of a single adenine or both adenines results in a decreased affinity of TTP-2D for the RNA molecule, demonstrating that both Fe(II)-TTP-2D and Fe(III)-TTP-2D selectively recognize a physiologically relevant RNA sequence. The relative affinities of Fe(II)-TTP-2D and Fe(III)-TTP-2D for the series of RNA sequences mirror those observed for Zn(II)-TTP-2D and suggest that iron is a viable substitute for zinc in this protein.

Animals↗