Characteristics of the Intron Involvement in the Mitogen-induced Expression of Zfp-36

Abstract

Zfp-36, the gene encoding the putative zinc finger protein tristetraprolin (TTP), is rapidly induced in fibroblasts by a variety of growth factors. Recent gene knockout experiments have shown that TTP-deficient mice developed arthritis, cachexia, and autoimmunity, all apparently mediated by an excess of tumor necrosis factor ␣. We recently showed that full serum inducibility of Zfp-36 requires elements in the promoter; in addition, removal of the single intron strikingly inhibited seruminduced TTP expression. We show here that replacement of the intron with unrelated sequences, or removal of 95% of the intron but retention of the splice sites, each resulted in the maintenance of approximately 45 and 19%, respectively, of full serum-induced expression. In addition, deletion of intron sequences base pairs 601-655 decreased the serum-induced expression of TTP by 65%. Sequence base pairs 618 -626 bound specifically to the transcription factor Sp1; mutation of this binding motif decreased TTP expression by 70%, suggesting that Sp1 binding to this motif contributes to serum induction of Zfp-36. We conclude that full serum-induced expression of Zfp-36 depends on the activation of conventional promoter elements as well as elements in the single intron, and that the presence per se of the intron in its natural location also contributes significantly to the regulated expression of this gene. Tristetraprolin (TTP)1 is a widely expressed protein containing two putative zinc fingers of the unusual CCCH class (1-5). It is the prototype of an enlarging group of proteins containing similar zinc fingers (6 -9). Although predominantly nuclear in quiescent fibroblasts, TTP is rapidly translocated from the nucleus to the cytoplasm by serum and other mitogens (10), an event that occurs concomitantly with stimulated serine phosphorylation (11). Although TTP has no proven function, disruption of its gene, Zfp-36, in mice leads to a complex syndrome that includes erosive arthritis, conjunctivitis, myeloid hyperplasia, cachexia, and autoimmunity (12). All aspects of the syndrome were prevented by pretreating the animals with monoclonal antibodies to tumor necrosis factor ␣ (TNF␣) (12). These data suggest that a potential function of TTP is to regulate the production of TNF␣ by certain cell types. They also indicate the possibility that defects in Zfp-36 might be involved in the pathogenesis of certain human conditions in which TNF␣ excess plays a role, such as rheumatoid arthritis and systemic lupus erythematosus.The transcription of Zfp-36 is rapidly and dramatically stimulated by a variety of growth factors and mitogens, but not by agents acting solely through increases in cAMP levels (1, 3, 4). Several transcription factor-binding sites have been identified in the Zfp-36 promoter that are each partially responsible for activating transcription in response to serum or insulin (13). In addition, we showed that the single intron of Zfp-36 also participates in the regulation of its transcription, since deletion of the intron results in a...

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

“…11G and H) in which the hTTP-GFP fusion protein expression was driven by the native hTTP promoter and intron. Both the promoter and the single intron of TTP play important roles in its expression (23,24). Similar predominantly cytosolic distribution was seen in HeLa cells transfected with the same constructs (data not shown).…”

“…supplemented with 10% fetal bovine serum, 100 U of penicillin per ml, and 100 g of streptomycin per ml. Transient transfection of 2 ϫ 10 6 cells with CMV.mTNF-␣ or other constructs in calcium-phosphate precipitates was performed as described previously (23,24), except that the transfection mixture was allowed to stay on the cells for 16 to 20 h and the glycerol shock step was omitted. In some experiments, pXGH5 (Nichols Institute Diagnostics) was also cotransfected to monitor transfection efficiency.…”

“…In some experiments, pXGH5 (Nichols Institute Diagnostics) was also cotransfected to monitor transfection efficiency. Assays of released HGH were performed as described previously (23,24).…”

Interactions of CCCH Zinc Finger Proteins with mRNA

“…The TTP zinc finger mutants of expression plasmid CMV.hTTP.tag were made by the PCR primer-overlapping mutagenesis technique (21). Correct sequences of all intended mutations were confirmed by dRhodamine Terminator Cycle sequencing (PerkinElmer Life Sciences).…”

Evaluating the Control of mRNA Decay in Fission Yeast

“…TTP is a protein encoded by Zfp36 in mice whose absence results in a severe autoimmune and inflammatory syndrome Taylor et al, 1996). TTP deficiency is associated with tumor necrosis factor-alpha (TNFα) overexpression (Blackshear, 2002;Carballo et al, 1998;Lai et al, 1998), and TTP has been shown to promote deadenylation and destruction of TNFα mRNA (Carballo et al, 1998;Lai et al, 1999). TTP may regulate overlapping aspects of immune cell function, inflammation and autoimmunity by targeting, in addition to TNFα, other essential mRNAs (Carballo et al, 1998;Carballo et al, 2000;Lai et al, 2006;Ogilvie et al, 2005).…”