Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of both acute and chronic inflammatory responses in many diseases. Tristetraprolin (TTP), the prototype of a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, inhibited TNF-alpha production from macrophages by destabilizing its messenger RNA. This effect appeared to result from direct TTP binding to the AU-rich element of the TNF-alpha messenger RNA. TTP is a cytosolic protein in these cells, and its biosynthesis was induced by the same agents that stimulate TNF-alpha production, including TNF-alpha itself. These findings identify TTP as a component of a negative feedback loop that interferes with TNF-alpha production by destabilizing its messenger RNA. This pathway represents a potential target for anti-TNF-alpha therapies.
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.
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.
SummaryThe immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3′ UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.
Tristetraprolin (TTP) is an RNA binding protein that controls the inflammatory response by limiting the expression of several proinflammatory cytokines. TTP post-transcriptionally represses gene expression by interacting with AU-rich elements (AREs) in 3′UTRs of target mRNAs and subsequently engenders their deadenylation and decay. TTP accomplishes these tasks, at least in part, by recruiting the multi subunit CCR4–NOT deadenylase complex to the mRNA. Here we identify an evolutionarily conserved C-terminal motif in human TTP that directly binds to a central domain of CNOT1, a core subunit of the CCR4–NOT complex. A high-resolution crystal structure of the TTP-CNOT1 complex was determined, providing the first structural insight into an ARE-binding protein bound to the CCR4–NOT complex. Mutations at the CNOT1-TTP interface impair TTP-mediated deadenylation, demonstrating the significance of this interaction in TTP-mediated gene silencing.
Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice and cells derived from them showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target mRNAs for TTP include tumor necrosis factor alpha, granulocytemacrophage colony-stimulating factor, and interleukin-2. Here we used microarray analysis of RNA from wild-type and TTP-deficient fibroblast cell lines to identify transcripts with different decay rates, after serum stimulation and actinomycin D treatment. Of 250 mRNAs apparently stabilized in the absence of TTP, 23 contained two or more conserved TTP binding sites; nine of these appeared to be stabilized on Northern blots. The most dramatically affected transcript encoded the protein Ier3, recently implicated in the physiological control of blood pressure. The Ier3 transcript contained several conserved TTP binding sites that could bind TTP directly and conferred TTP sensitivity to the mRNA in cell transfection studies. These studies have identified several new, physiologically relevant TTP target transcripts in fibroblasts; these target mRNAs encode proteins from a variety of functional classes.
Eukaryotic mRNA stability can be influenced by AU-rich elements (AREs) within mRNA primary sequences. Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that binds to ARE-containing transcripts and destabilizes them, apparently by first promoting the removal of their poly(A) tails. We developed a cell-free system in which TTP and its related proteins stimulated the deadenylation of ARE-containing, polyadenylated transcripts. Transcript deadenylation was not stimulated when a mutant TTP protein was used that was incapable of RNA binding, nor when a mutant ARE was present that did not bind TTP. The ability of TTP to promote transcript deadenylation required Mg 2؉ , but not ATP or prior capping of the RNA substrate. Cotransfection and additivity studies with the poly(A) RNase (PARN) demonstrated that TTP promoted the ability of this enzyme to deadenylate ARE-containing, polyadenylated transcripts, while having no effect on transcripts lacking an ARE. There was no effect of TTP to act synergistically with enzymatically inactive PARN mutants. We conclude that TTP can promote the deadenylation of ARE-containing, polyadenylated substrates by PARN. This interaction may be responsible for the ability of TTP and its family members to promote the deadenylation of such transcripts in intact cells.Steady-state levels of cellular mRNAs are determined by the balance between their biosynthesis and turnover. Different mRNAs can exhibit marked differences in turnover rates within the same cell, and the turnover rates of individual mRNAs can also vary significantly in response to changes in the cellular environment. In mammalian cells, the earliest step in mRNA turnover is thought to be removal of the poly(A) tail, or deadenylation, and this process in turn largely determines the overall decay rate of the mRNA (15,16,31,36).It has been appreciated for many years that cis-acting AUrich elements (AREs), often within the 3Ј-untranslated regions (3Ј-UTR) of the mRNA, can confer decreased stability on the mRNAs that contain them (32).
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