Interactions of CCCH Zinc Finger Proteins with mRNA

Abstract

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-␣ 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-␣ 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.The CCCH zinc finger motif has been found in proteins from organisms ranging from man to yeast (1-11). A small subset of these proteins contain two CCCH zinc fingers with the following characteristics. The fingers are 18 amino acids apart; they have the internal spacing CX 8 CX 5 CX 3 H, where X indicates variable amino acids; and both fingers have a highly conserved lead-in sequence, R(K)YKTEL. Three such proteins have been identified to date in mammals, with a fourth member recently discovered in Xenopus and fish (2, 12, 13). We will refer to this small group of proteins as the CCCH tandem zinc finger (TZF) 1 domain proteins.Our group has begun to elucidate a function for this group of proteins by analyzing the phenotype of mice deficient in tristetraprolin (TTP), the prototype of this group of proteins. These animals develop a complex phenotype consisting of cachexia, dermatitis, conjunctivitis, destructive arthritis, myeloid hyperplasia, and autoimmunity (14). Virtually all aspects of this phenotype can be prevented by the repeated injection of antibodies to tumor necrosis factor-␣ (TNF␣), implicating an effective excess of circulating TNF␣ in the pathogenesis of this condition (14). This was confirmed by studies showing that the phenotype can be largely prevented by interbreed...

Multiple tristetraprolin sequence domains required to induce apoptosis and modulate responses to TNFα through distinct pathways

“…Because we did not observe a strict correlation between induction of apoptosis and subcellular localization of TTP (Figures 2b and 3a), we conclude that the TTP Nand C-terminal regions are not required simply to localize the TTP zinc ®ngers, but are involved in interactions or signals that are important for TTP activity. Consistent with this view, expression of TTP zinc ®nger mutants that lack RNA binding activity stabilizes ARE-containing mRNAs, suggesting that these mutants may act as dominant negatives (Lai et al, 2002). In addition, phosphorylation of TTP within regions outside of the zinc ®ngers not only in¯uences TTP subcellular localization (Johnson et al, 2002), but also may inhibit its activity in the cell Zhu et al, 2001).…”

RNA-destabilizing Factor Tristetraprolin Negatively Regulates NF-κB Signaling

“…We observed that the first zinc finger mutant of TTP also significantly suppressed NF-B activation with less potency compared with the wild-type TTP protein. Because any zinc finger mutation of TTP protein completely loses its RNA-binding activity (29), our data suggest that the effect of TTP on NF-B signaling may be independent of its RNA-destabilizing activity but that its two zinc fingers may be required for transcription repression.…”

Characterization of zfs1 as an mRNA-binding and -destabilizing Protein in Schizosaccharomyces pombe

“…The solution structure of the TZF domain of ZFP36L2 (TIS11D) bound to the UUAUUUAUU nonamer has been solved (13), providing insight into the interaction between the TZF domain and a target ARE. A core UAUUUAU heptamer unit appears to be necessary for optimal binding affinity of the TZF domain for an ARE (10,14).…”

“…Interactions between the TZF domain and type II AU-rich elements (AREs) found in the 3Ј-untranslated regions (UTRs) of target mRNAs have been the focus of multiple investigations (9 -14). These have included the use of a synthetic TTP TZF domain peptide to examine specificity of ARE target variants (9,11,12), the evaluation of target mRNA decay as it is influenced by mutated TZF domains expressed in co-transfected cells (14), cell-free deadenylation of ARE-containing mRNAs (10,15), and the use of engineered stable transcripts to assess ARE specificity, as measured through mRNA decay (10). The solution structure of the TZF domain of ZFP36L2 (TIS11D) bound to the UUAUUUAUU nonamer has been solved (13), providing insight into the interaction between the TZF domain and a target ARE.…”