The human ZFP36 zinc finger protein family includes ZFP36 ZFP36L1 and

The human ZFP36 zinc finger protein family includes ZFP36 ZFP36L1 and ZFP36L2. Mouse monoclonal to FGB 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 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 Etofenamate knockdown in different cell types (BCL1 ACHN Ramos) resulted in increased levels of 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 construct made up of the adenine uridine rich element and removal of the adenine uridine rich core from the 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 mRNA as an important focus on by which ZFP36L1 mediates its pro-apoptotic results in malignant B-cells. Launch Etofenamate The individual ZFP36 proteins family members includes three widely-expressed people specifically ZFP36 (TIS11 TTP Nup475 GOS24) ZFP36L1 (Tis11b Berg36 ERF-1 BRF-1) and ZFP36L2 (Tis11d ERF-2 BRF-2) [1]-[3]. A 4th family member referred to in rodents Zfp36l3 shows placental-specific appearance but isn’t detectably expressed in virtually any individual tissues Etofenamate [4]. ZFP36-family proteins are also determined and characterised in a few various other species such as for example yeast and Xenopus [5]-[7]. These protein contain two tandemly repeated zinc finger motifs and function to modify gene expression at the post-transcriptional level by binding to adenine uridine (AU) rich elements (AREs) in the 3′ untranslated region (3′UTR) of units of mRNAs and mediating ARE-dependent mRNA decay [1]-[3]. In mammals ZFP36 family members have been shown to function in regulating development cell differentiation tumourigenesis the inflammatory response and apoptosis by targeting an extensive overlapping repertoire of mRNAs. These have been best characterised in the inflammatory/immune response in which all three ZFP36 family members elicit quick downregulation of important cytokines via destabilisation of their mRNAs (examined in [1]-[3]). Users of the ZFP36 family also target mRNAs encoding important transcription factors such as STAT5b in the regulation of erythropoiesis [8] and PRDM1/Blimp1 in terminal plasmacytoid differentiation of B cells [9]. We originally reported around the pro-apoptotic function of ZFP36L1 in Ramos Burkitt B lymphoma cells [10] and more recently in Rituximab-induced apoptosis of B-chronic lymphocytic leukaemia cells (BCLL) [11] from which the human gene was originally isolated as an early response gene cDNA [12]. Overexpression of ZFP36 Etofenamate family members has been shown by other laboratories to induce apoptosis in a variety of other mammalian cell lines including HeLa U20S SAOS2 and 3T3 [13] [14]. Induction of apoptosis by all three ZFP36 family members is completely abrogated in the presence of Bcl-2 or CrmA [13]. ZFP36 synergistically induces apoptosis with TNF-α in 3T3 cells and the zinc fingers and the N-terminal domain name of ZFP36 are completely required for this effect [14]. Mutant ZFP36 (TIS11) lacking the zinc finger motifs fails to induce apoptosis and is localised Etofenamate to the nucleus whereas the wild type ZFP36/TIS11 is usually localised in the cytoplasm [14]. Induction of apoptosis by ZFP36 family proteins therefore appears to require intactness of the zinc finger motifs and presumably mRNA binding. However the identities of mRNAs that are targeted by ZFP36 family members in mediating their pro-apoptotic effects are currently unknown. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1 we reverse-engineered a gene regulatory network for ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference [15] Of the final set of three inferred anti-apoptosis ZFP36L1 targets recognized by this analysis we focussed on experimental validation of mRNA.