Wi S. Lai

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-α). Macrophages derived from these mice oversecrete TNF-α, by a mechanism that involves stabilization of TNF-α mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-α mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001–1005, 1998). We show here that TTP binding to the TNF-α 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-α ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-α mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-α 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-α mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-α mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.

Novel mRNA Targets for Tristetraprolin (TTP) Identified by Global Analysis of Stabilized Transcripts in TTP-Deficient Fibroblasts†

ABSTRACT 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, granulocyte-macrophage colony-stimulating factor, and interleukin-2β. 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.

Zfp36l3, a Rodent X Chromosome Gene Encoding a Placenta-Specific Member of the Tristetraprolin Family of CCCH Tandem Zinc Finger Proteins

Abstract Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger (TZF) proteins can bind directly to AU-rich elements (ARE) in mRNA, causing deadenylation and destabilization of the transcripts to which they bind. We describe here a novel fourth mammalian member of the TTP protein family, designated ZFP36L3, which could also bind directly to ARE-containing RNAs and could promote the deadenylation and degradation of ARE-containing target RNAs. Zfp36l3 transcript expression was detected only in placenta and extraembryonic tissues in the mouse. It was expressed throughout development in the placenta and was particularly highly expressed in the cells of the labyrinthine layer of the trophoblastic placenta. Unlike the other family members, the expression of a ZFP36L3-green fluorescent protein fusion protein was entirely cytoplasmic when expressed in 293 cells, even in the presence of the CRM1-dependent nuclear export inhibitor leptomycin B. Zfp36l3 was located on the mouse X chromosome; a similar predicted gene was present on the rat X chromosome, but there was no evidence for a similar gene in humans. ZFP36L3 may thus be a rodent-specific or even murine-specific member of the TTP protein family. Its presumed role in placental physiology may be unique to rodents or murine rodents, but this role may be subsumed by other family members in nonrodents.

Identification of four CCCH zinc finger proteins in Xenopus, including a novel vertebrate protein with four zinc fingers and severely restricted expression

Tristetraprolin (TTP), the prototype of a class of CCCH zinc finger proteins, is a phosphoprotein that is rapidly and transiently induced by growth factors and serum in fibroblasts. Recent evidence suggests that a physiological function of TTP is to inhibit tumor necrosis factor alpha secretion from macrophages by binding to and destabilizing its mRNA (Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Science, 281, 1001-1005). To investigate possible functions of CCCH proteins in early development of Xenopus, we isolated four Xenopus cDNAs encoding members of this class. Based on 49% overall amino acid identity and 84% amino acid identity within the double zinc finger domain, one of the Xenopus proteins (XC3H-1) appears to be the homologue of TTP. By similar analyses, XC3H-2 and XC3H-3 are homologues of ERF-1 (cMG1, TIS11B) and ERF-2 (TIS11D). A fourth protein, XC3H-4, is a previously unidentified member of the CCCH class of vertebrate zinc finger proteins; it contains four Cx8Cx5Cx3H repeats, two of which are YKTEL Cx8Cx5Cx3H repeats that are closely related to sequences found in the other CCCH proteins. Whereas XC3H-1, XC3H-2, and XC3H-3 were widely expressed in adult tissues, XC3H-4 mRNA was not detected in any of the adult tissues studied except for the ovary. Its expression appeared to be limited to the ovary, oocyte, egg and the early embryonic stages leading up to the mid-blastula transition. Its mRNA was highly expressed in oocytes of all ages, and was enriched in the animal pole cytosol of mature oocytes. Maternal expression was also seen with the other three messages, suggesting the possibility that these proteins are involved in regulating mRNA stability in oocyte maturation and/or early embryogenesis.

Inflammation: cytokines and RNA-based regulation

The outcome of an inflammatory response depends upon the coordinated regulation of a variety of both pro‐inflammatory and anti‐inflammatory cytokines and other proteins. Regulation of these inflammation mediators can occur at multiple levels, including transcription, mRNA translation, post‐translational modifications, and mRNA degradation. Post‐transcriptional regulation has been shown to play an important role in controlling the expression of these mediators, allowing for normal initiation and resolution of the inflammatory response. Many inflammatory mediators have unstable mRNAs due, in part, to the presence of AU‐rich elements in their 3′‐untranslated regions. Increasing numbers of RNA‐binding proteins have been identified that can bind to these AU‐rich elements and then regulate the stability and/or translation of the mRNA. This review summarizes current knowledge about the role of several RNA‐binding proteins that act through AU‐rich elements to post‐transcriptionally regulate the biosynthesis of proteins involved in inflammation. Copyright

Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies

Significance Inflammation is involved in the pathogenesis of many chronic diseases. Many deleterious effects of inflammation are mediated through increased production of proinflammatory mediators, known as cytokines and chemokines. Many current therapies for these diseases involve blocking single proinflammatory mediators, such as TNF, using parenteral administration of recombinant binding proteins. We demonstrate here that a genetic modification in the mouse that increases the expression of an endogenous antiinflammatory protein, tristetraprolin (TTP), results in protection against mouse models for several human inflammatory diseases, including rheumatoid arthritis, psoriasis, and multiple sclerosis, presumably by decreasing the production of proinflammatory cytokines. Our results suggest that increasing TTP expression may be an effective therapeutic strategy in the treatment of certain inflammatory diseases. Tristetraprolin (TTP) is an inducible, tandem zinc-finger mRNA binding protein that binds to adenylate-uridylate–rich elements (AREs) in the 3′-untranslated regions (3′UTRs) of specific mRNAs, such as that encoding TNF, and increases their rates of deadenylation and turnover. Stabilization of Tnf mRNA and other cytokine transcripts in TTP-deficient mice results in the development of a profound, chronic inflammatory syndrome characterized by polyarticular arthritis, dermatitis, myeloid hyperplasia, and autoimmunity. To address the hypothesis that increasing endogenous levels of TTP in an intact animal might be beneficial in the treatment of inflammatory diseases, we generated a mouse model (TTPΔARE) in which a 136-base instability motif in the 3′UTR of TTP mRNA was deleted in the endogenous genetic locus. These mice appeared normal, but cultured fibroblasts and macrophages derived from them exhibited increased stability of the otherwise highly labile TTP mRNA. This resulted in increased TTP protein expression in LPS-stimulated macrophages and increased levels of TTP protein in mouse tissues. TTPΔARE mice were protected from collagen antibody-induced arthritis, exhibited significantly reduced inflammation in imiquimod-induced dermatitis, and were resistant to induction of experimental autoimmune encephalomyelitis, presumably by dampening the excessive production of proinflammatory mediators in all cases. These data suggest that increased systemic levels of TTP, secondary to increased stability of its mRNA throughout the body, can be protective against inflammatory disease in certain models and might be viewed as an attractive therapeutic target for the treatment of human inflammatory diseases.

Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA

Summary The 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. Graphical Abstract Figure. No caption available. HighlightsOncogenic RAS signaling via MEK increases PD‐L1 expressionRAS regulates PD‐L1 through AU‐rich elements (AREs) in the 3′ UTR of PD‐L1 mRNAThe ARE‐binding protein tristetraprolin (TTP) negatively regulates PD‐L1 expressionRestoration of tumor cell TTP activity enhances anti‐tumor immunity &NA; Coelho et al. demonstrate a post‐transcriptional mechanism whereby oncogenic RAS signaling increases PD‐L1 expression. Mechanistically, PD‐L1 mRNA is targeted by TTP through AU‐rich elements in the 3′ UTR, making it unstable. Oncogenic RAS signaling reduces TTP activity and stabilizes the PD‐L1 transcript. Restoring TTP activity reduces PD‐L1 expression and enhances anti‐tumor immunity.

The NIEHS Xenopus maternal EST project: interim analysis of the first 13,879 ESTs from unfertilized eggs

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 contigs comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.

Differential post-transcriptional regulation of IL-10 by TLR2 and TLR4-activated macrophages

The activation of TLRs by microbial molecules triggers intracellular‐signaling cascades and the expression of cytokines such as IL‐10. Il10 expression is tightly controlled to ensure effective immune responses, while preventing pathology. Maximal TLR‐induction of Il10 transcription in macrophages requires signaling through the MAPKs, ERK, and p38. Signals via p38 downstream of TLR4 activation also regulate IL‐10 at the post‐transcriptional level, but whether this mechanism operates downstream of other TLRs is not clear. We compared the regulation of IL‐10 production in TLR2 and TLR4‐stimulated BM‐derived macrophages and found different stability profiles for the Il10 mRNA. TLR2 signals promoted a rapid induction and degradation of Il10 mRNA, whereas TLR4 signals protected Il10 mRNA from rapid degradation, due to the activation of Toll/IL‐1 receptor domain‐containing adaptor inducing IFN‐β (TRIF) and enhanced p38 signaling. This differential post‐transcriptional mechanism contributes to a stronger induction of IL‐10 secretion via TLR4. Our study provides a molecular mechanism for the differential IL‐10 production by TLR2‐ or TLR4‐stimulated BMMs, showing that p38‐induced stability is not common to all TLR‐signaling pathways. This mechanism is also observed upon bacterial activation of TLR2 or TLR4 in BMMs, contributing to IL‐10 modulation in these cells in an infection setting.

Tandem CCCH Zinc Finger Proteins in mRNA Binding

A small family of mammalian zinc finger proteins containing an unusual putative tandem zinc finger motif was identified approximately 13 years ago. The tandem zinc finger domain was characterized by two hypothetical fingers with identical Cx8Cx5Cx3H spacing, with exactly 18 amino acids between the carboxyl terminal H of the first zinc finger and the amino terminal C of the second zinc finger. The two fingers also shared a characteristic amino-terminal lead-in sequence of RYKTEL or a close variant. Although first thought to be transcription factors, these proteins are becoming better understood as the result of experiments with knockout mice for tristetraprolin (TTP), currently the best-studied member of the family. These mice developed a systemic inflammatory syndrome found to be secondary to elevations of tumor necrosis factor alpha (TNF) and possibly granulocyte-macrophage colony-stimulating factor (GM-CSF). These elevations were found to be due to stabilized mRNAs for these cytokines, and subsequent work found that TTP could bind to the AU-rich elements within the 3′-untranslated region of these mRNAs and destabilize them, apparently by initiating a process of 3′–5′ deadenylation. This chapter will summarize some of our current thinking about this small but interesting protein family, including binding site and binding domain characterization, and recent developments in mutagenesis and structure determination.