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

Abstract

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.

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications,

“…Nuclear localization of TTP in normal and stimulated cells is very minimal, with a high abundance in dividing cells (3). The localization of TTP primarily in the cytosol is in agreement with previous studies using THP-1 myelomonocytic cells (5,38), mouse tissues and cells (3), and GFP-tagged TTP in transfected cells (7). It is known that in response to mitogen stimulation, TTP is able to shuttle rapidly from the nuclei to the cytosol in NIH/ 3T3 mouse fibroblasts (39), and that the extent of cytoplasmic localization of TTP is also increased by association with 14-3-3 protein (25).…”

“…Full-length TTP was expressed as a His-tagged protein (His-hTTP) in HEK293 cells. This cell line does not have detectable endogenous TTP mRNA (7). Cells were transiently transfected with pHis-hTTP plasmids encoding the wildtype, S186A, and T271A proteins, using the calcium phosphate precipitation method as described previously (7).…”

“…TTP and its related proteins can bind to the 3′-untranslated AREs of certain clinically important mRNAs, such as those encoding tumor necrosis factor α (TNF) (5,7,9), granulocyte-macrophage colony-stimulating factor (GM-CSF) (6), inter-leukin 3 (8), cyclooxygenase 2 (12,13), and plasminogen activator inhibitor type 2 (14). This specific binding of TTP to the AREs results in the destabilization of the mRNAs in transfected human embryonic kidney (HEK) 293 cells (5,7). Mice deficient in TTP develop a severe inflammatory syndrome, including polyarticular arthritis, myeloid hyperplasia, autoimmunity, and cachexia (15,16).…”

The Role of Human Antigen R, an RNA-binding Protein, in Mediating the Stabilization of Toll-Like Receptor 4 mRNA Induced by Endotoxin

“…18 TTP is critically implicated in inflammation and is a member of zinc finger proteins that bind to AREs and destabilize mRNAs of tumor necrosis factor (TNF-␣). 19 However, TTP knockout causes a severe inflammatory syndrome in vivo, 20 and it is possible that the effects of TTP are countered by HuR. 21 Our previous data showed that LPS-enhanced TLR4 expression in human aortic smooth muscle cells (HASMCs) is mediated by mRNA stabilization, although we have not yet clarified whether RNA-binding proteins are involved in this process.…”

Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA

“…The CCCH-type zinc-finger motifs have been shown to be required for ZAP to bind the target RNA (Guo et al, 2004). Similar zinc-finger motifs also exist in tristetraproline (TTP) (DuBois et al, 1990), which specifically targets the AU-rich element (ARE) often found in the 3' untranslated regions of cytokines, growth factors and proto-oncogenes (Lai et al, 1999(Lai et al, , 2000Blackshear, 2002;Carrick et al, 2004;Barreau et al, 2005), and characterized by the AUUUA motif. These results suggest that sequence-specific interaction between ZAP and RNA is important for ZAP to bind the RNA.…”