Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

Abstract

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...

Tristetraprolin Regulates CXCL1 (KC) mRNA Stability

“…The ARE of TNF-␣ contains multiple overlapping AUUUA pentamers, suggesting that such sequences might be specific targets for TTP recognition and function. This has been supported by studies directed at defining the sequence recognition specificity of TTP, in which the highest affinity recognition sites were demonstrated to contain three to four uridine residues flanked by at least one adenine, with the highest affinity being identified for a nonameric sequence element (UUAUUUAUU) (40,47,48). Our deletion analysis of KC 3Ј-UTR demonstrates that the clustered as well as isolated pentamercontaining sequences exhibit sensitivity to TTP.…”

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

“…However, they are probably minor phosphorylation sites since truncated TTP fragments are not significantly phosphorylated in transfected cells by in vivo radioactive labeling [55]. The zinc finger domains synthesized chemically or expressed in E. coli [2] can bind to the same ARE as the recombinant full-length TTP with similar binding affinity by the electrophoretic mobility shift assay [3]. However, it is impossible to rule out minor effects of phosphorylation at these sites since the binding assay is a semiquantitative method.…”

“…TTP binds to AU-rich elements (AREs) with high affinity for UUAUUUAUU nucleotides within mRNA sequences [2][3][4][5][6][7]. The specific binding of TTP to AREs causes destabilization of mRNA molecules encoding proteins such as tumor necrosis factor-alpha (TNFα) [3,[8][9][10], granulocyte-macrophage colony-stimulating factor (GM-CSF) [11,12], cyclooxygenase 2 (COX2) [13,14], interleukin 2 (IL2) [15] and transcription factor E47 [16].…”

The Protein Zfand5 Binds and Stabilizes mRNAs with AU-rich Elements in Their 3′-Untranslated Regions

“…TTP binds directly to the class II AREs and accelerates breakdown of its target mRNAs (43)(44)(45). We hypothesized that Zfand5 may stabilize Class II ARE-containing mRNA by competing with TTP-ARE binding to reduce the destabilizing effect of TTP on its targets.…”