RESUMO
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.
Assuntos
Antígeno B7-H1/imunologia , Neoplasias Colorretais/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Tristetraprolina/imunologia , Evasão Tumoral , Animais , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Clivagem do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Tristetraprolina/genéticaRESUMO
Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. In eukaryotes, mRNAs containing specific AU-rich motifs are regulated by binding of tristetraprolin (TTP) family tandem zinc finger proteins, which promote mRNA deadenylation and decay, partly through interaction of a conserved C-terminal CNOT1 binding (CNB) domain with CCR4-NOT protein complexes. The social ameba Dictyostelium discoideum shared a common ancestor with humans more than a billion years ago, and expresses only one TTP family protein, TtpA, in contrast to three members expressed in humans. Evaluation of ttpA null-mutants identified six transcripts that were consistently upregulated compared to WT during growth and early development. The 3'-untranslated regions (3'-UTRs) of all six 'TtpA-target' mRNAs contained multiple TTP binding motifs (UUAUUUAUU), and one 3'-UTR conferred TtpA post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mutations in the core TTP-binding motifs. All six target transcripts were upregulated to similar extents in a C-terminal truncation mutant, in contrast to less severe effects of analogous mutants in mice. All six target transcripts encoded probable membrane proteins. In Dictyostelium, TtpA may control an 'RNA regulon', where a single RNA binding protein, TtpA, post-transcriptionally co-regulates expression of several functionally related proteins.
Assuntos
Dictyostelium/genética , Proteínas de Protozoários/metabolismo , Regulon , Tristetraprolina/metabolismo , Regiões 3' não Traduzidas , Dictyostelium/metabolismo , Mutação , Proteínas de Protozoários/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tristetraprolina/genéticaRESUMO
Decay of transcribed mRNA is a key determinant of steady state mRNA levels in cells. Global analysis of mRNA decay in cultured cells has revealed amazing heterogeneity in rates of decay under normal growth conditions, with calculated half-lives ranging from several minutes to many days. The factors that are responsible for this wide range of decay rates are largely unknown, although our knowledge of trans-acting RNA binding proteins and non-coding RNAs that can control decay rates is increasing. Many methods have been used to try to determine mRNA decay rates under various experimental conditions in cultured cells, and transcription inhibitors like actinomycin D have probably the longest history of any technique for this purpose. Despite this long history of use, the actinomycin D method has been criticized as prone to artifacts, and as ineffective for some promoters. With appropriate guidelines and controls, however, it can be a versatile, effective technique for measuring endogenous mRNA decay in cultured mammalian and insect cells, as well as the decay of exogenously-expressed transcripts. It can be used readily on a genome-wide level, and is remarkably cost-effective. In this short review, we will discuss our utilization of this approach in these cells; we hope that these methods will allow more investigators to apply this useful technique to study mRNA decay under the appropriate conditions.
Assuntos
Dactinomicina/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Análise de Sequência de RNA , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
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.
Assuntos
Inflamação/genética , RNA Mensageiro/genética , Tristetraprolina/genética , Aminoquinolinas/efeitos adversos , Animais , Artrite Experimental/genética , Células Cultivadas , Colágeno/imunologia , Dermatite/etiologia , Dermatite/genética , Encefalomielite Autoimune Experimental/genética , Imiquimode , Camundongos , Camundongos Transgênicos , Mutação , Tristetraprolina/metabolismoRESUMO
In most mammals, the Zfp36 gene family consists of three conserved members, with a fourth member, Zfp36l3, present only in rodents. The ZFP36 proteins regulate post-transcriptional gene expression at the level of mRNA stability in organisms from humans to yeasts, and appear to be expressed in all major groups of eukaryotes. In Mus musculus, Zfp36l3 expression is limited to the placenta and yolk sac, and is important for overall fecundity. We sequenced the Zfp36l3 gene from more than 20 representative species, from members of the Muridae, Cricetidae and Nesomyidae families. Zfp36l3 was not present in Dipodidae, or any families that branched earlier, indicating that this gene is exclusive to the Muroidea superfamily. We provide evidence that Zfp36l3 arose by retrotransposition of an mRNA encoded by a related gene, Zfp36l2 into an ancestral rodent X chromosome. Zfp36l3 has evolved rapidly since its origin, and numerous modifications have developed, including variations in start codon utilization, de novo intron formation by mechanisms including a nested retrotransposition, and the insertion of distinct repetitive regions. One of these repeat regions, a long alanine rich-sequence, is responsible for the full-time cytoplasmic localization of Mus musculus ZFP36L3. In contrast, this repeat sequence is lacking in Peromyscus maniculatus ZFP36L3, and this protein contains a novel nuclear export sequence that controls shuttling between the nucleus and cytosol. Zfp36l3 is an example of a recently acquired, rapidly evolving gene, and its various orthologues illustrate several different mechanisms by which new genes emerge and evolve.
Assuntos
Evolução Molecular , Roedores/genética , Tristetraprolina/genética , Animais , Núcleo Celular/genética , Feminino , Humanos , Íntrons , Muridae/genética , Peromyscus/genética , Filogenia , Placenta/metabolismo , Gravidez , Proteínas/genética , RNA Mensageiro , Sequências Repetitivas de Ácido Nucleico , RetroelementosRESUMO
Tristetraprolin (TTP), the best known member of a class of tandem (R/K)YKTELCX8CX5CX3H zinc finger proteins, can destabilize target mRNAs by first binding to AU-rich elements (AREs) in their 3'-untranslated regions (UTRs) and subsequently promoting deadenylation and ultimate destruction of those mRNAs. This study sought to determine the roles of selected amino acids in the RNA binding domain, known as the tandem zinc finger (TZF) domain, in the ability of the full-length protein to bind to AREs within the tumor necrosis factor α (TNF) mRNA 3'-UTR. Within the CX8C region of the TZF domain, mutation of some of the residues specific to TTP, not found in other members of the TTP protein family, resulted in decreased binding to RNA as well as inhibited mRNA deadenylation and decay. Evaluation of simulation solution models revealed a distinct structure in the second zinc finger of TTP that was induced by the presence of these TTP-specific residues. In addition, mutations within the lead-in sequences preceding the first C of highly conserved residues within the CX5C or CX3H regions or within the linker region between the two fingers also perturbed both RNA binding and the simulation model of the TZF domain in complex with RNA. We conclude that, although the majority of conserved residues within the TZF domain of TTP are required for productive binding, not all residues at sequence-equivalent positions in the two zinc fingers of the TZF domain of TTP are functionally equivalent.
Assuntos
Modelos Moleculares , Mutação de Sentido Incorreto , Tristetraprolina/química , Dedos de Zinco/fisiologia , Regiões 3' não Traduzidas/fisiologia , Substituição de Aminoácidos , Humanos , Estrutura Terciária de Proteína , Estabilidade de RNA/fisiologia , Tristetraprolina/genética , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with "target" RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Elementos Ricos em Adenilato e Uridilato/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA/métodos , Homologia de Sequência de AminoácidosRESUMO
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-ß (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.
Assuntos
Interleucina-10/genética , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Processamento Pós-Transcricional do RNA , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Bactérias/imunologia , Ativação Enzimática , Feminino , Regulação da Expressão Gênica , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Tristetraprolina/deficiência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Removal of the poly(A) tail, or deadenylation, is a crucial step in destabilizing mRNAs in eukaryotes. In this chapter, we describe a cell-free deadenylation assay that uses cytoplasmic cell extracts from human HEK293 cells transiently transfected with DNA encoding RNA-binding proteins (RBP), and in vitro-transcribed, radiolabeled, RNA probes. We include methods to evaluate the effects of RBPs or deadenylases on various in vitro-transcribed probes, with or without poly(A) tails. Finally, we also demonstrate the adaptability of these assays to test purified protein components in our cell-free deadenylation assay. In our experience, these methods are well suited for the initial assessment of the effects of RBPs on the deadenylation of mRNAs.
Assuntos
Proteínas de Ligação a RNA , RNA , Animais , Humanos , Extratos Celulares , Células HEK293 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estabilidade de RNA , Poli A/metabolismo , Mamíferos/genéticaRESUMO
Members of the tristetraprolin (TTP) family of RNA-binding proteins can bind to and promote the decay of specific transcripts containing AU-rich motifs. ZFP36 (TTP) is best known for regulating pro-inflammatory cytokine expression in myeloid cells; however, its mammalian paralogues ZFP36L1 and ZFP36L2 have not been viewed as important in controlling inflammation. We knocked out these genes in myeloid cells in mice, singly and together. Single-gene myeloid-specific knockouts resulted in almost no spontaneous phenotypes. In contrast, mice with myeloid cell deficiency of all three genes developed severe inflammation, with a median survival of 8 wk. Macrophages from these mice expressed many more stabilized transcripts than cells from myeloid-specific TTP knockout mice; many of these encoded pro-inflammatory cytokines and chemokines. The failure of weight gain, arthritis, and early death could be prevented completely by two normal alleles of any of the three paralogues, and even one normal allele of Zfp36 or Zfp36l2 was enough to prevent the inflammatory phenotype. Our findings emphasize the importance of all three family members, acting in concert, in myeloid cell function.
Assuntos
Inflamação , Tristetraprolina , Camundongos , Animais , Tristetraprolina/genética , Tristetraprolina/metabolismo , Inflamação/genética , Inflamação/metabolismo , Células Mieloides/metabolismo , Macrófagos/metabolismo , Camundongos Knockout , Citocinas/metabolismo , Mamíferos/metabolismoRESUMO
Both innate and adaptive immunity in birds are different from their mammalian counterparts. Understanding bird immunity is important because of the enormous potential impact of avian infectious diseases, both in their role as food animals and as potential carriers of zoonotic diseases in man. The anti-inflammatory protein tristetraprolin (TTP) is an important component of the mammalian innate immune response, in that it binds to and destabilizes key cytokine mRNAs. TTP knockout mice exhibit a severe systemic inflammatory syndrome, and they are abnormally sensitive to innate immune stimuli such as LPS. TTP orthologs have been found in most vertebrates studied, including frogs. Here, we attempted to identify TTP orthologs in chicken and other birds, using database searches and deep mRNA sequencing. Although sequences encoding the two other widely expressed TTP family members, ZFP36L1 and ZFP36L2, were identified, we did not find sequences corresponding to TTP in any bird species. Sequences corresponding to TTP were identified in both lizards and alligators, close evolutionary relatives of birds. The induction kinetics of Zfp36l1 and Zfp36l2 mRNAs in LPS-stimulated chicken macrophages or serum-stimulated chick embryo fibroblasts did not resemble the normal mammalian TTP response to these stimuli, suggesting that the other two family members might not compensate for the TTP deficiency in regulating rapidly induced mRNA targets. Several mammalian TTP target transcripts have chicken counterparts that contain one or more potential TTP binding sites, raising the possibility that birds express other proteins that subsume TTP's function as a rapidly inducible regulator of AU-rich element (ARE)-dependent mRNA turnover.
Assuntos
Proteínas Aviárias/deficiência , Galinhas/metabolismo , Imunidade Inata , Inflamação/prevenção & controle , Tristetraprolina/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/genética , Sequência de Bases , Bovinos , Linhagem Celular , Galinhas/genética , Galinhas/imunologia , Bases de Dados Genéticas , Regulação da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Cinética , Lipopolissacarídeos/farmacologia , Camundongos , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/metabolismo , Répteis/genética , Répteis/imunologia , Répteis/metabolismo , Proteínas de Répteis/genética , Proteínas de Répteis/metabolismo , Análise de Sequência de DNA , Fatores de Tempo , Transfecção , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
BACKGROUND/PURPOSE: Adipose tissue contains progenitor cells that contribute to beneficial tissue expansion when needed by de novo adipocyte formation (classical white or beige fat cells with thermogenic potential). However, in chronic obesity, they can exhibit an activated pro-fibrotic, extracellular matrix (ECM)-depositing phenotype that highly aggravates obesity-related adipose tissue dysfunction. METHODS: Given that progenitors' fibrotic activation and fat cell browning appear to be antagonistic cell fates, we have examined the anti-fibrotic potential of pro-browning agents in an obesogenic condition. RESULTS: In obese mice fed a high fat diet, thermoneutral housing, which induces brown fat cell dormancy, increases the expression of ECM gene programs compared to conventionally raised animals, indicating aggravation of obesity-related tissue fibrosis at thermoneutrality. In a model of primary cultured murine adipose progenitors, we found that exposure to ß-hydroxybutyrate selectively reduced Tgfß-dependent profibrotic responses of ECM genes like Ctgf, Loxl2 and Fn1. This effect is observed in both subcutaneous and visceral-derived adipose progenitors, as well as in 3T3-L1 fibroblasts. In 30 patients with obesity eligible for bariatric surgery, those with higher circulating ß-hydroxybutyrate levels have lower subcutaneous adipose tissue fibrotic scores. Mechanistically, ß-hydroxybutyrate limits Tgfß-dependent collagen accumulation and reduces Smad2-3 protein expression and phosphorylation in visceral progenitors. Moreover, ß-hydroxybutyrate induces the expression of the ZFP36 gene, encoding a post-transcriptional regulator that promotes the degradation of mRNA by binding to AU-rich sites within 3'UTRs. Importantly, complete ZFP36 deficiency in a mouse embryonic fibroblast line from null mice, or siRNA knock-down in primary progenitors, indicate that ZFP36 is required for ß-hydroxybutyrate anti-fibrotic effects. CONCLUSION: These data unravel the potential of ß-hydroxybutyrate to limit adipose tissue matrix deposition, a finding that might exploited in an obesogenic context.
Assuntos
Tecido Adiposo Marrom , Tecido Adiposo Branco , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacologia , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Fibroblastos/metabolismo , Fibrose , Humanos , Camundongos , Obesidade/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Tristetraprolina/metabolismoRESUMO
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-2beta. 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.
Assuntos
Fibroblastos/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Tristetraprolina/deficiência , Tristetraprolina/metabolismo , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Soro/fisiologia , Transcrição Gênica , Tristetraprolina/genéticaRESUMO
Tristetraprolin (TTP), the prototype member of the protein family of the same name, was originally discovered as the product of a rapidly inducible gene in mouse cells. Development of a knockout (KO) mouse established that absence of the protein led to a severe inflammatory syndrome, due in part to elevated levels of tumor necrosis factor (TNF). TTP was found to bind directly and with high affinity to specific AU-rich sequences in the 3'-untranslated region of the TNF mRNA. This initial binding led to promotion of TNF mRNA decay and inhibition of its translation. Many additional TTP target mRNAs have since been identified, some of which are cytokines and chemokines involved in the inflammatory response. There are three other proteins in the mouse with similar activities and domain structures, but whose KO phenotypes are remarkably different. Moreover, proteins with similar domain structures and activities have been found throughout eukaryotes, demonstrating that this protein family arose from an ancient ancestor. The defining characteristic of this protein family is the tandem zinc finger (TZF) domain, a 64 amino acid sequence with many conserved residues that is responsible for the direct RNA binding. We discuss here many aspects of this protein domain that have been elucidated since the original discovery of TTP, including its sequence conservation throughout eukarya; its apparent continued evolution in some lineages; its functional dependence on many key conserved residues; its "interchangeability" among evolutionarily distant species; and the evidence that RNA binding is required for the physiological functions of the proteins. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
Assuntos
Células Eucarióticas/enzimologia , Células Eucarióticas/metabolismo , Estabilidade de RNA , Motivos de Ligação ao RNA , Tristetraprolina/química , Tristetraprolina/metabolismo , Dedos de Zinco , Regulação da Expressão Gênica , Tristetraprolina/genéticaRESUMO
Tristetraprolin (TTP) is an anti-inflammatory protein that modulates the stability of certain cytokine/chemokine mRNAs. After initial high-affinity binding to AU-rich elements in 3' untranslated regions of target mRNAs, mediated through its tandem zinc finger (TZF) domain, TTP promotes the deadenylation and ultimate decay of target transcripts. These transcripts and their encoded proteins accumulate abnormally in TTP knockout (KO) mice, leading to a severe inflammatory syndrome. To assess the importance of the highly conserved C-terminal CNOT1 binding domain (CNBD) of TTP to the TTP deficiency phenotype in mice, we created a mouse model in which TTP lacked its CNBD. CNBD deletion mice exhibited a less severe phenotype than the complete TTP KO mice. In macrophages, the stabilization of target transcripts seen in KO mice was partially normalized in the CNBD deletion mice. In cell-free experiments, recombinant TTP lacking its CNBD could still activate target mRNA deadenylation by purified recombinant Schizosaccharomyces pombe CCR4/NOT complexes, although to a lesser extent than full-length TTP. Thus, TTP lacking its CNBD can still act to promote target mRNA instability in vitro and in vivo These data have implications for TTP family members throughout the eukarya, since species from all four kingdoms contain proteins with linked TZF and CNOT1 binding domains.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Deleção de Sequência , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Fenótipo , Estabilidade de RNA , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that can bind to AU-rich element-containing mRNAs and promote their decay. TTP knockout mice develop a severe inflammatory syndrome, largely due to excess tumor necrosis factor (TNF), whose mRNA is a direct target of TTP binding and destabilization. TTP's RNA binding activity and its ability to promote mRNA decay are lost when one of the zinc-coordinating residues of either zinc finger is mutated. To address several long-standing questions about TTP activity in intact animals, we developed a knock-in mouse with a cysteine-to-arginine mutation within the first zinc finger. Homozygous knock-in mice developed a severe inflammatory syndrome that was essentially identical to that of complete TTP deficiency, suggesting that TTP's critical anti-inflammatory role in mammalian physiology is secondary to its ability to bind RNA. In addition, there was no evidence for a "dominant-negative" effect of the mutant allele in heterozygotes, as suggested by previous experiments. Finally, mRNA decay experiments in mutant macrophages demonstrated that TTP can regulate the stability of its own mRNA, albeit to a minor extent. These studies suggest that RNA binding is an essential first step in the physiological activities of members of this protein family.
Assuntos
Tristetraprolina/genética , Elementos Ricos em Adenilato e Uridilato , Sequência de Aminoácidos , Animais , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Mutação Puntual , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tristetraprolina/metabolismo , Dedos de ZincoRESUMO
BACKGROUND: Histone deacetylase inhibitors (HDACi) suppress cytokine production in immune and stromal cells of patients with rheumatoid arthritis (RA). Here, we investigated the effects of the HDACi givinostat (ITF2357) on the transcriptional and post-transcriptional regulation of inflammatory markers in RA fibroblast-like synoviocytes (FLS). METHODS: The effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-1ß-inducible genes in FLS were analyzed using array-based qPCR and Luminex. The expression of primary and mature cytokine transcripts, the mRNA levels of tristetraprolin (TTP, or ZFP36) and other AU-rich element binding proteins (ARE-BP) and the cytokine profile of fibroblasts derived from ZFP36+/+ and ZFP36-/- mice was measured by qPCR. ARE-BP silencing was performed by small interfering RNA (siRNA)-mediated knockdown, and TTP post-translational modifications were analyzed by immunoblotting. RESULTS: ITF2357 reduced the expression of 85% of the analyzed IL-1ß-inducible transcripts, including cytokines (IL6, IL8), chemokines (CXCL2, CXCL5, CXCL6, CXCL10), matrix-degrading enzymes (MMP1, ADAMTS1) and other inflammatory mediators. Analyses of mRNA stability demonstrated that ITF2357 accelerates IL6, IL8, PTGS2 and CXCL2 mRNA degradation, a phenomenon associated with the enhanced transcription of TTP, but not other ARE-BP, and the altered post-translational status of TTP protein. TTP knockdown potentiated cytokine production in RA FLS and murine fibroblasts, which in the latter case was insensitive to inhibition by ITF2357 treatment. CONCLUSIONS: Our study identifies that regulation of cytokine mRNA stability is a predominant mechanism underlying ITF2357 anti-inflammatory properties, occurring via regulation of TTP. These results highlight the therapeutic potential of ITF2357 in the treatment of RA.
Assuntos
Artrite Reumatoide/metabolismo , Citocinas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Sinoviócitos/efeitos dos fármacos , Animais , Artrite Reumatoide/imunologia , Células Cultivadas , Citocinas/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Sinoviócitos/metabolismo , Tristetraprolina/biossínteseRESUMO
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.
Assuntos
Adenina/metabolismo , Proteínas de Ligação a DNA , Exorribonucleases/metabolismo , Proteínas Imediatamente Precoces/metabolismo , RNA Mensageiro/metabolismo , Uracila/metabolismo , Composição de Bases , Linhagem Celular , Sistema Livre de Células , Exorribonucleases/genética , Genes Reporter , Humanos , Proteínas Imediatamente Precoces/genética , Mutação , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Tristetraprolina , Dedos de ZincoRESUMO
mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real time RT-PCR. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in the former case. This protocol is suitable for studying primary cultured cells and stable cell lines derived from transgenic mice and their respective controls, and provides for direct comparisons of mRNA decay rates in otherwise identical cells with and without the gene of interest.
RESUMO
Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity. Here, we describe procedures for mouse embryonic fibroblast cell isolation, primary culture, and stimulation. Specifically, we have optimized a step of serum starvation prior to stimulation. This step is necessary to maintain the quiescent status of these cells before they are exposed to pro-inflammatory stimuli for optimal responses. As shown in our previous studies, these mouse fibroblasts do not express Tnf, Csf2, or Il2 mRNAs at levels readily detectable by routine northern blotting techniques (Lai WS et al., 2006).