Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization.

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.

Translational repression of pre-formed cytokine-encoding mRNA prevents chronic activation of memory T cells.

Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.

High-resolution sequencing and modeling identifies distinct dynamic RNA regulatory strategies.

Cells control dynamic transitions in transcript levels by regulating transcription, processing, and/or degradation through an integrated regulatory strategy. Here, we combine RNA metabolic labeling, rRNA-depleted RNA-seq, and DRiLL, a novel computational framework, to quantify the level; editing sites; and transcription, processing, and degradation rates of each transcript at a splice junction resolution during the LPS response of mouse dendritic cells. Four key regulatory strategies, dominated by RNA transcription changes, generate most temporal gene expression patterns. Noncanonical strategies that also employ dynamic posttranscriptional regulation control only a minority of genes, but provide unique signal processing features. We validate Tristetraprolin (TTP) as a major regulator of RNA degradation in one noncanonical strategy. Applying DRiLL to the regulation of noncoding RNAs and to zebrafish embryogenesis demonstrates its broad utility. Our study provides a new quantitative approach to discover transcriptional and posttranscriptional events that control dynamic changes in transcript levels using RNA sequencing data.

Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity.

Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD+ onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.

Tristetraprolin impairs myc-induced lymphoma and abolishes the malignant state.

Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis.

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

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.

Regulation of IFN-γ production by ZFP36L2 in T cells is time-dependent.

CD8+ T cells kill target cells by releasing cytotoxic molecules and proinflammatory cytokines, such as TNF and IFN-γ. The magnitude and duration of cytokine production are defined by posttranscriptional regulation, and critical regulator herein are RNA-binding proteins (RBPs). Although the functional importance of RBPs in regulating cytokine production is established, the kinetics and mode of action through which RBPs control cytokine production are not well understood. Previously, we showed that the RBP ZFP36L2 blocks the translation of preformed cytokine encoding mRNA in quiescent memory T cells. Here, we uncover that ZFP36L2 regulates cytokine production in a time-dependent manner. T cell-specific deletion of ZFP36L2 (CD4-cre) had no effect on T-cell development or cytokine production during early time points (2-6 h) of T-cell activation. In contrast, ZFP36L2 specifically dampened the production of IFN-γ during prolonged T-cell activation (20-48 h). ZFP36L2 deficiency also resulted in increased production of IFN-γ production in tumor-infiltrating T cells that are chronically exposed to antigens. Mechanistically, ZFP36L2 regulates IFN-γ production at late time points of activation by destabilizing Ifng mRNA in an AU-rich element-dependent manner. Together, our results reveal that ZFP36L2 employs different regulatory nodules in effector and memory T cells to regulate cytokine production.

Tristetraprolin regulates the skeletal phenotype and osteoclastogenic potential through monocytic myeloid-derived suppressor cells.

Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, in vitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype.

Hydroxychloroquine attenuates double-stranded RNA-stimulated hyper-phosphorylation of tristetraprolin/ZFP36 and AU-rich mRNA stabilization.

The human innate immune system recognizes dsRNA as a pathogen-associated molecular pattern that induces a potent inflammatory response. The primary source of pathogenic dsRNA is cells infected with replicating viruses, but can also be released from uninfected necrotic cells. Here, we show that the dsRNA poly(I:C) challenge in human macrophages activates the p38 MAPK-MK2 signalling pathway and subsequently the phosphorylation of tristetraprolin (TTP/ZFP36). The latter is an mRNA decay-promoting protein that controls the stability of AU-rich mRNAs (AREs) that code for many inflammatory mediators. Hydroxychloroquine (HCQ), a common anti-malaria drug, is used to treat inflammatory and autoimmune disorders and, controversially, during acute COVID-19 disease. We found that HCQ reduced the dsRNA-dependent phosphorylation of p38 MAPK and its downstream kinase MK2. Subsequently, HCQ reduced the abundance and protein stability of the inactive (phosphorylated) form of TTP. HCQ reduced the levels and the mRNA stability of poly (I:C)-induced cytokines and inflammatory mRNAs like TNF, IL-6, COX-2, and IL-8 in THP-1 and primary blood monocytes. Our results demonstrate a new mechanism of the anti-inflammatory role of HCQ at post-transcriptional level (TTP phosphorylation) in a model of dsRNA activation, which usually occurs in viral infections or RNA release from necrotic tissue.

Variants in NLRP2 and ZFP36L2, non-core components of the human subcortical maternal complex, cause female infertility with embryonic development arrest.

The subcortical maternal complex (SCMC), which is vital in oocyte maturation and embryogenesis, consists of core proteins (NLRP5, TLE6, OOEP), non-core proteins (PADI6, KHDC3L, NLRP2, NLRP7), and other unknown proteins that are encoded by maternal effect genes. Some variants of SCMC genes have been linked to female infertility characterized by embryonic development arrest. However, so far, the candidate non-core SCMC components associated with embryonic development need further exploration and the pathogenic variants that have been identified are still limited. In this study, we discovered two novel variants [p.(Ala131Val) and p.(Met326Val)] of NLRP2 in patients with primary infertility displaying embryonic development arrest from large families. In vitro studies using 293T cells and mouse oocytes, respectively, showed that these variants significantly decreased protein expression and caused the phenotype of embryonic development arrest. Additionally, we combined the 'DevOmics' database with the whole exome sequence data of our cohort and screened out a new candidate non-core SCMC gene ZFP36L2. Its variants [p.(Ala241Pro) and p.(Pro291dup)] were found to be responsible for embryonic development arrest. Co-immunoprecipitation experiments in 293T cells, used to demonstrate the interaction between proteins, verified that ZFP36L2 is one of the human SCMC components, and microinjection of ZFP36L2 complementary RNA variants into mouse oocytes affected embryonic development. Furthermore, the ZFP36L2 variants were associated with disrupted stability of its target mRNAs, which resulted in aberrant H3K4me3 and H3K9me3 levels. These disruptions decreased oocyte quality and further developmental potential. Overall, this is the first report of ZFP36L2 as a non-core component of the human SCMC and we found four novel pathogenic variants in the NLRP2 and ZFP36L2 genes in 4 of 161 patients that caused human embryonic development arrest. These findings contribute to the genetic diagnosis of female infertility and provide new insights into the physiological function of SCMC in female reproduction.

ZFP36 family expression is suppressed by Th2 cells in asthma, leading to enhanced synthesis of inflammatory cytokines and cell surface molecules.

Asthma is a chronic inflammatory airway disease, in which inflammatory cytokines play a pivotal role. The zinc finger binding protein 36 (ZFP36) family includes ZFP36, ZFP36L1, and ZFP36L2 and is among the RNA-binding proteins (RBPs) reported to cause inflammation. The present study aimed to clarify the roles of the ZFP36 family in asthma, particularly highlighting the relationship between the ZFP36 family and Th2 cells, which are key players in type 2 inflammation in asthma. Real-time PCR analysis revealed the preferential expression of ZFP36 family mRNAs in human white blood cells. Gene expression analysis using public datasets from the GEO database (https://www.ncbi.nlm.nih.gov/gds) showed significantly suppressed expression of ZFP36 family mRNAs in patients with asthma compared to that in healthy controls. Using multiple cytokine assays, Th2 cell transfection with ZFP36 family siRNAs enhanced the expression of inflammatory cytokines IL-8, IFN-γ, CCL3/MIP-1α, CCL4/MIP-1ß, and TNF-α and cell surface molecules CCR4 (CD194) and PSGL-1 (CD162). Treatment with IL-2, 4, and 15 significantly suppressed, and corticosteroid significantly enhanced the expressions of ZFP36 family mRNAs by Th2 cells. In conclusion, the ZFP36 family expressed by Th2 cells was suppressed in patients with asthma, leading to the enhanced expression of cytokines and cell surface molecules. Suppressed ZFP36 expression in asthma may be involved in the enhancement of airway inflammation, and the ZFP36 family may be a therapeutic target for inflammatory diseases, including asthma.

Post-transcriptional regulation of BIRC5/survivin expression and induction of apoptosis in breast cancer cells by tristetraprolin.

Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3' UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.

LncRNA PCBP1-AS1 suppresses cell growth in oral squamous cell carcinoma by targeting miR-34c-5p/ZFP36 axis.

Oral squamous cell carcinoma (OSCC) is the most frequently diagnosed oral malignancy and poses a great threat to public health. According to bioinformatics analysis, long noncoding RNA PCBP1-AS1 is downregulated in OSCC. In this work, the functions and mechanism of PCBP1-AS1 in OSCC were further investigated. PCBP1-AS1 expression in OSCC cells was measured by quantitative polymerase chain reaction. Cell viability and proliferation were detected using CCK-8 assays and colony-forming assays. TUNEL assays as well as flow cytometry analyses were carried out to detect OSCC cell apoptosis. Binding relationship between PCBP1-AS1 and miR-34c-5p or that between miR-34c-5p and ZFP36 in OSCC cells was identified using RNA immunoprecipitation assays, RNA pulldown assays, and luciferase reporter assays. Experimental results revealed that PCBP1-AS1 was downregulated in OSCC cells. PCBP1-AS1 overexpression hampered cell proliferation and enhanced cell apoptosis in OSCC. PCBP1-AS1 interacted with miR-34c-5p in OSCC and negatively regulated miR-34c-5p. ZFP36 3'untranslated region was targeted by miR-34c-5p. PCBP1-AS1 positively regulated ZFP36 expression. ZFP36 silencing abrogated the suppressive impact of PCBP1-AS1 on OSCC cell growth. In summary, PCBP1-AS1 suppresses cell growth in OSCC by upregulating ZFP36 through interaction with miR-34c-5p.

Intrinsically disordered regions of tristetraprolin and DCP2 directly interact to mediate decay of ARE-mRNA.

The RNA-binding protein tristetraprolin (TTP) is a potent activator of mRNA decay, specifically for transcripts bearing AU-rich elements (AREs) in their 3'-untranslated regions. TTP functions as a mediator for mRNA decay by interacting with the decay machinery and recruiting it to the target ARE-mRNA. In this study, we report a weak, but direct interaction between TTP and the human decapping enzyme DCP2, which impacts the stability of ARE transcripts. The TTP-DCP2 interaction is unusual as it involves intrinsically disordered regions (IDRs) of both binding partners. We show that the IDR of DCP2 has a propensity for oligomerization and liquid-liquid phase separation in vitro. Binding of TTP to DCP2 leads to its partitioning into phase-separated droplets formed by DCP2, suggesting that molecular crowding might facilitate the weak interaction between the two proteins and enable assembly of a decapping-competent mRNA-protein complex on TTP-bound transcripts in cells. Our studies underline the role of weak interactions in the cellular interaction network and their contribution towards cellular functionality.

Sequence and tissue targeting specificity of ZFP36L2 reveals Elavl2 as a novel target with co-regulation potential.

Zinc finger protein 36 like 2 (ZFP36L2) is an RNA-binding protein that destabilizes transcripts containing adenine-uridine rich elements (AREs). The overlap between ZFP36L2 targets in different tissues is minimal, suggesting that ZFP36L2-targeting is highly tissue specific. We developed a novel Zfp36l2-lacking mouse model (L2-fKO) to identify factors governing this tissue specificity. We found 549 upregulated genes in the L2-fKO spleen by RNA-seq. These upregulated genes were enriched in ARE motifs in the 3'UTRs, which suggests that they are ZFP36L2 targets, however the precise sequence requirement for targeting was not evident from motif analysis alone. We therefore used gel-shift mobility assays on 12 novel putative targets and established that ZFP36L2 requires a 7-mer (UAUUUAU) motif to bind. We observed a statistically significant enrichment of 7-mer ARE motifs in upregulated genes and determined that ZFP36L2 targets are enriched for multiple 7-mer motifs. Elavl2 mRNA, which has three 7-mer (UAUUUAU) motifs, was also upregulated in L2-fKO spleens. Overexpression of ZFP36L2, but not a ZFP36L2(C176S) mutant, reduced Elavl2 mRNA expression, suggesting a direct negative effect. Additionally, a reporter assay demonstrated that the ZFP36L2 effect on Elavl2 decay is dependent on the Elavl2-3'UTR and requires the 7-mer AREs. Our data indicate that Elavl2 mRNA is a novel target of ZFP36L2, specific to the spleen. Likely, ZFP36L2 combined with other RNA binding proteins, such as ELAVL2, governs tissue specificity.

The mRNA-destabilizing protein Tristetraprolin targets "meiosis arrester" Nppc mRNA in mammalian preovulatory follicles.

C-natriuretic peptide (CNP) and its receptor guanylyl cyclase, natriuretic peptide receptor 2 (NPR2), are key regulators of cyclic guanosine monophosphate (cGMP) homeostasis. The CNP-NPR2-cGMP signaling cascade plays an important role in the progression of oocyte meiosis, which is essential for fertility in female mammals. In preovulatory ovarian follicles, the luteinizing hormone (LH)-induced decrease in CNP and its encoding messenger RNA (mRNA) natriuretic peptide precursor C (Nppc) are a prerequisite for oocyte meiotic resumption. However, it has never been determined how LH decreases CNP/Nppc In the present study, we identified that tristetraprolin (TTP), also known as zinc finger protein 36 (ZFP36), a ubiquitously expressed mRNA-destabilizing protein, is the critical mechanism that underlies the LH-induced decrease in Nppc mRNA. Zfp36 mRNA was transiently up-regulated in mural granulosa cells (MGCs) in response to the LH surge. Loss- and gain-of-function analyses indicated that TTP is required for Nppc mRNA degradation in preovulatory MGCs by targeting the rare noncanonical AU-rich element harbored in the Nppc 3' UTR. Moreover, MGC-specific knockout of Zfp36, as well as lentivirus-mediated knockdown in vivo, impaired the LH/hCG-induced Nppc mRNA decline and oocyte meiotic resumption. Furthermore, we found that LH/hCG activates Zfp36/TTP expression through the EGFR-ERK1/2-dependent pathway. Our findings reveal a functional role of TTP-induced mRNA degradation, a global posttranscriptional regulation mechanism, in orchestrating the progression of oocyte meiosis. We also provided a mechanism for understanding CNP-dependent cGMP homeostasis in diverse cellular processes.

Identification of novel compound heterozygous ZFP36L2 variants implicated in oocyte maturation defects and female infertility.

PURPOSE: To explore the pathogenesis of oocyte maturation defects. METHODS: Whole exome sequencing was conducted to identify potential variants, which were then confirmed within the pedigree through Sanger sequencing. The functional characterization of the identified variants responsible for the disease, including their subcellular localization, protein levels, and interactions with other proteins, was verified through transient transfection in HeLa cells in vitro. Additionally, we employed real-time RT-PCR and single-cell RNA sequencing to examine the impact of ZFP36L2 pathogenic variants on mRNA metabolism in both HeLa cells and mouse or human oocytes. RESULTS: A novel compound heterozygous variant in ZFP36L2 (c.186T > G, p.His62Gln and c.869 C > T, p.Pro290Leu) was discovered in a patient with oocyte maturation defects. Our findings indicate that these variants lead to compromised binding capacity of the ZFP36L2-CONT6L complex and impaired mRNA degradation in HeLa cells and mouse oocytes. Furthermore, we characterized the changes in the human oocyte transcriptome associated with ZFP36L2 variants, with a particular emphasis on cell division, mitochondrial function, and ribosome metabolism. CONCLUSIONS: This study broadens the mutation spectrum of ZFP36L2 and constitutes the first report of human oocyte transcriptome alterations linked to ZFP36L2 variants. In conjunction with existing knowledge of ZFP36L2, our research lays the groundwork for genetic counseling aimed at addressing female infertility.

Activation of the MKK3-p38-MK2-ZFP36 Axis by Coronavirus Infection Restricts the Upregulation of AU-Rich Element-Containing Transcripts in Proinflammatory Responses.

Coronavirus infections induce the expression of multiple proinflammatory cytokines and chemokines. We have previously shown that in cells infected with gammacoronavirus infectious bronchitis virus (IBV), interleukin 6 (IL-6), and IL-8 were drastically upregulated, and the MAP kinase p38 and the integrated stress response pathways were implicated in this process. In this study, we report that coronavirus infection activates a negative regulatory loop that restricts the upregulation of a number of proinflammatory genes. As revealed by the initial transcriptomic and subsequent validation analyses, the anti-inflammatory adenine-uridine (AU)-rich element (ARE)-binding protein, zinc finger protein 36 (ZFP36), and its related family members were upregulated in cells infected with IBV and three other coronaviruses, alphacoronaviruses porcine epidemic diarrhea virus (PEDV), human coronavirus 229E (HCoV-229E), and betacoronavirus HCoV-OC43, respectively. Characterization of the functional roles of ZFP36 during IBV infection demonstrated that ZFP36 promoted the degradation of transcripts coding for IL-6, IL-8, dual-specificity phosphatase 1 (DUSP1), prostaglandin-endoperoxide synthase 2 (PTGS2) and TNF-α-induced protein 3 (TNFAIP3), through binding to AREs in these transcripts. Consistently, knockdown and inhibition of JNK and p38 kinase activities reduced the expression of ZFP36, as well as the expression of IL-6 and IL-8. On the contrary, overexpression of mitogen-activated protein kinase kinase 3 (MKK3) and MAPKAP kinase-2 (MK2), the upstream and downstream kinases of p38, respectively, increased the expression of ZFP36 and decreased the expression of IL-8. Taken together, this study reveals an important regulatory role of the MKK3-p38-MK2-ZFP36 axis in coronavirus infection-induced proinflammatory response. IMPORTANCE Excessive and uncontrolled induction and release of proinflammatory cytokines and chemokines, the so-called cytokine release syndrome (CRS), would cause life-threatening complications and multiple organ failure in severe coronavirus infections, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and COVID-19. This study reveals that coronavirus infection also induces the expression of ZFP36, an anti-inflammatory ARE-binding protein, promoting the degradation of ARE-containing transcripts coding for IL-6 and IL-8 as well as a number of other proteins related to inflammatory response. Furthermore, the p38 MAP kinase, its upstream kinase MKK3 and downstream kinase MK2 were shown to play a regulatory role in upregulation of ZFP36 during coronavirus infection cycles. This MKK3-p38-MK2-ZFP36 axis would constitute a potential therapeutic target for severe coronavirus infections.

Pb(II) coordination to the nonclassical zinc finger tristetraprolin: retained function with an altered fold.

Tristetraprolin (TTP) is a nonclassical CCCH zinc finger (ZF) that plays a crucial role in regulating inflammation. TTP regulates cytokine mRNAs by specific binding of its two conserved ZF domains (CysX8CysX5CysX3His) to adenylate-uridylate-rich sequences (AREs) at the 3'-untranslated region, leading to degradation of the RNA. Dysregulation of TTP in animal models has demonstrated several cytokine-related syndromes, including chronic inflammation and autoimmune disorders. Exposure to Pb(II), a prevalent environmental toxin, is known to contribute to similar pathologies, in part by disruption of and/or competition with cysteine-rich metalloproteins. TTP's role during stress as a ubiquitous translational regulator of cell signaling (and dysfunction), which may underpin various phenotypes of Pb(II) toxicity, highlights the importance of understanding the interaction between TTP and Pb(II). The impact of Pb(II) binding on TTP's fold and RNA-binding function was analyzed via UV-Vis spectroscopy, circular dichroism, X-ray absorption spectroscopy, nuclear magnetic resonance spectroscopy, and fluorescence anisotropy. A construct containing the two ZF domains of TTP (TTP-2D) bound to Pb(II) with nanomolar affinity and exhibited a different geometry and fold in comparison to Zn2-TTP-2D. Despite the altered secondary structure, Pb(II)-substituted TTP-2D bound a canonical ARE sequence more selectively than Zn2-TTP-2D. Taken together, these data suggest that Pb(II) may interfere with proper TTP regulation and hinder the cell's ability to respond to inflammation.

A post-transcriptional regulon controlled by TtpA, the single tristetraprolin family member expressed in Dictyostelium discoideum.

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.