Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.     

Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.     

A unique C-terminal repeat domain maintains the cytosolic localization of the placenta-specific tristetraprolin family member ZFP36L3

Members of the tristetraprolin family of CCCH tandem zinc finger proteins bind to AU-rich elements in certain cellular mRNAs, leading to their deadenylation and destabilization. Studies in knock-out mice demonstrated roles for three of the family members, tristetraprolin, ZFP36L1, and ZFP36L2, in inflammation, chorioallantoic fusion, and early embryonic development, respectively. However, little is known about a recently discovered placenta-specific tristetraprolin family member, ZFP36L3. Tristetraprolin, ZFP36L1, and ZFP36L2 have been shown to shuttle between the nucleus and cytoplasm, using typical hydrophobic amino acid-rich nuclear export sequences, and nuclear localization sequences located within the tandem zinc finger domain. In contrast, we previously showed that green fluorescent protein-labeled ZFP36L3, expressed in HEK 293 cells, remained cytosolic, even in the presence of the nuclear export blocker leptomycin B. We show here that the conserved tandem zinc finger domain contains an active nuclear localization signal. However, the sequence corresponding to the nuclear export signal in the other family members was nonfunctional, and thus did not contribute to the cytosolic localization. The unique C-terminal repeat domain could override the activity of the nuclear localization sequence, preventing the import of ZFP36L3 into the nucleus. Immunostaining of mouse placenta demonstrated that ZFP36L3 was located only in the cytoplasm of trophoblast cells. Thus, in contrast to the other mammalian members of this protein family, ZFP36L3 is a "full-time" cytosolic protein, rather than a nucleocytoplasmic shuttling protein. The significance of this difference in subcellular localization to the physiology of placental trophoblast cells, where ZFP36L3 is selectively expressed, remains to be determined.     

The active component of ginseng,ginsenoside Rb1, improves erythropoiesis in models of Diamond–Blackfan anemia by targeting Nemo-like kinase

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.     

Insulin increases tristetraprolin and decreases VEGF gene expression in mouse 3T3-L1 adipocytes

Objectives: Tristetraprolin (TTP) family proteins (TTP/ZFP36; ZFP36L1, ZFP36L2, ZFP36L3) destabilize adenylate uridylate‐rich element‐containing mRNAs encoding cytokines, such as tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Little is known about the expression and insulin regulation of TTP and related genes in adipocytes. We analyzed the relative abundance of TTP family mRNAs in 3T3‐L1 adipocytes compared to RAW264.7 macrophages and investigated insulin effects on the expression of 43 genes in 3T3‐L1 adipocytes. Methods and Procedures: Insulin was added to mouse 3T3‐L1 adipocytes. Relative abundance of mRNA levels was determined by quantitative real‐time PCR. TTP and ZFP36L1 proteins were detected by immunoblotting. Results: Zfp36l1 and Zfp36l2 genes were expressed at eight‐ to tenfold higher than Ttp in adipocytes. Zfp36l3 mRNA was detected at ～1% of Ttp mRNA levels in adipocytes and its low level expression was confirmed in RAW cells. Insulin at 10 and 100 nmol/l increased Ttp mRNA levels by five‐ to sevenfold, but decreased those of Zfp36l3 by 40% in adipocytes after a 30‐min treatment. Immunoblotting showed that insulin induced TTP but did not affect ZFP36L1 protein levels in adipocytes. Insulin decreased mRNA levels of Vegf and a number of other genes in adipocytes. Discussion: Insulin induced Ttp mRNA and protein expression and decreased Vegf mRNA levels in adipocytes. Zfp36l3 mRNA was detected, for the first time, in cells other than mouse placenta and extraembryonic tissues. This study established a basis for the investigation of TTP and VEGF genes in the regulation of obesity and suggested that Vegf mRNA may be a target of TTP in fat cells.     

A hematopoietic-specific transmembrane protein, Art-1, is possibly regulated by AML1

The functions of AML1 in hematopoietic differentiation are repressed by AML1-mutants including the AML1/ETO chimeric protein, which is seen in t(8;21) acute myeloid leukemia. Erythroid progenitors of the patients with t(8;21) AML expressed AML1/ETO. To investigate the effect of AML1/ETO in erythroid cells, we made a tetracycline-regulated AML1/ETO overexpression system in mouse erythroleukemic (MEL) cells. Enforced AML1/ETO repressed the terminal erythroid differentiation. Furthermore, we performed representational difference analysis using this MEL cell system to clone the downstream targets of AML1 in erythroid cell differentiation. We cloned a novel transmembrane protein, Art-1 (AML1-regulated transmembrane protein 1), which is a member of tetramembrane spanning superfamily. Art-1 expression was restricted in hematopoietic cells. It was upregulated by AML1 and downregulated by AML1/ETO in both erythroid and myeloid cells, and increased during erythroid cell differentiation. Art-1 may play an important role in the differentiation of erythroid cells, possibly as a direct downstream target of AML1.