AUF1 facilitates microRNA-mediated gene silencing.

Eukaryotic mRNA decay is tightly modulated by RNA-binding proteins (RBPs) and microRNAs (miRNAs). RBP AU-binding factor 1 (AUF1) has four isoforms resulting from alternative splicing and is critical for miRNA-mediated gene silencing with a distinct preference of target miRNAs. Previously, we have shown that AUF1 facilitates miRNA loading to Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex. Here, we further demonstrate that depletion of AUF1 abolishes the global interaction of miRNAs and AGO2. Single-molecule analysis revealed that AUF1 slowed down assembly of AGO2-let-7b-mRNA complex unexpectedly. However, target mRNAs recognized by both miRNA and AUF1 are less abundant upon AUF1 overexpression implying that AUF1 is a decay-promoting factor influencing multiple steps in AGO2-miRNA-mediated mRNA decay. Our findings indicate that AUF1 functions in promoting miRNA-mediated mRNA decay globally.

Accumulation of pre-let-7g and downregulation of mature let-7g with the depletion of EWS.

EWS functions in RNA splicing and transcription by encoding an RNA binding protein, which results in the chromosomal translocation t(11;22)(q24;q12) found in Ewing sarcoma. EWS interacts with the microprocessor complex involving Drosha and DGCR8, which play roles as the cofactors of primary microRNA processing. However, the role of EWS in microRNA biogenesis has not been investigated. Here, we show that endogenous EWS interacts with endogenous Drosha by IP-western blotting. In addition, EWS knockout mouse decreased the expression of Drosha. The depletion of EWS results in the accumulation of precursor let-7g but down-regulates mature let-7g in U2OS cells. Consistently, mature let 7g was suppressed in both Ewing sarcoma cell and primary Ewing sarcoma. Also, expression levels of Dicer and CCND1 (Cyclin D1), which are known target genes of the let-7 family were upregulated. Our findings suggest that EWS mediates generation of mature let-7g from pre-let-7g.

Tumor suppressor ras association domain family 5 (RASSF5/NORE1) mediates death receptor ligand-induced apoptosis.

Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-α and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-α-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-α-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-α- and TNF-related apoptosis-inducing ligand-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-α-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.

Redox-mediated modification of PLZF by SUMO-1 and ubiquitin.

Earlier, we reported that the transcriptional repressor promyelocytic leukemia zinc-finger protein (PLZF) is sumoylated at position K242, and the sumoylation regulated its biological function. Here, we show that the sumoylation site can be modified by ubiquitin. The stability and nuclear localization of PLZF were regulated by the antagonistic relationship between sumoylation and ubiquitination. We observed the antagonistic effects of ubiquitin and SUMO-1 on PLZF under oxidative stress induced by serum deprivation. Thus, the choice between modification of PLZF by SUMO or ubiquitin was determined by the intracellular level of ROS, which was generated by serum deprivation that inactivated the SUMO-conjugating enzymes Uba2 and Ubc9, and resulted in decrease of sumoylation. The ubiquitination was increased under these conditions. The expression of BID, a known transcriptional target protein of PLZF, was decreased, and the consequent apoptosis was induced by the ROS generated during serum starvation. On the basis of these results, we propose that PLZF post-translational modification is controlled by intracellular ROS, and the biological function of PLZF is regulated by sumoylation and ubiquitination.

Redox regulation of cytosolic glycerol-3-phosphate dehydrogenase: Cys(102) is the target of the redox control and essential for the catalytic activity.

Cytosolic glycerol-3-phosphate dehydrogenase (cG3PDH) occupies the branch point between the glycolytic pathway and triglyceride biosynthesis. However, the regulatory mechanism of the cG3PDH activity has remained obscure. Here we report that cG3PDH is efficiently inhibited by modification of the thiol group through a redox mechanism. In this study, we found that sodium selenite and nitric oxide (NO) donors such as S-nitroso-N-acetylpenicillamine and 3-morpholinosydnonimine inhibited cG3PDH activity, and that similar effects could be achieved with selenium metabolites such as selenocysteine and selenomethionine. Furthermore, we found that reducing agents, such as dithiothreitol and beta-mercaptoethanol, restored the cG3PDH activity suppressed by selenite and NO both in vitro and in cultured cells. Buthionine sulfoximine depleted levels of both reduced glutathione and the oxidized form but had no effect on the suppression of cG3PDH activity by selenite in cultured cells. Moreover, thiol-reactive agents, such as N-ethylmaleimide and o-iodosobenzoic acid, blocked the enzyme activity of cG3PDH through the modification of redox-sensitive cysteine residues in cG3PDH. The inhibitor of NO synthase, L-N(G)-nitro-arginine, restored the cG3PDH activity inhibited by NO in cultured cells, whereas the inhibitor of guanylyl cyclase, 1H-[1,2,4] oxadiazole[4,3-alpha] quinoxalin-1-one (ODQ), has no effect. NO directly inhibits cG3PDH activity not via a cGMP-dependent mechanism. Finally, using site-directed mutagenesis, we found that Cys(102) of cG3PDH was sensitive to both selenite and NO. From the results, we suggest that cG3PDH is a target of cellular redox regulation.

EWS-WT1 oncoprotein activates neuronal reprogramming factor ASCL1 and promotes neural differentiation.

The oncogenic fusion gene EWS-WT1 is the defining chromosomal translocation in desmoplastic small round-cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS-WT1 functions as an aberrant transcription factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS-WT1 under the native murine Ews promoter. EWS-WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural reprogramming factor ASCL1. Mechanistic analyses demonstrated that EWS-WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS-WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS-WT1. Taken together, our findings reveal that EWS-WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT.

A multifunctional protein, EWS, is essential for early brown fat lineage determination.

The recent surge in obesity has provided an impetus to better understand the mechanisms of adipogenesis, particularly in brown adipose tissue (BAT) because of its potential utilization for antiobesity therapy. Postnatal brown adipocytes arise from early muscle progenitors, but how brown fat lineage is determined is not completely understood. Here, we show that a multifunctional protein, Ewing Sarcoma (EWS), is essential for determining brown fat lineage during development. BATs from Ews null embryos and newborns are developmentally arrested. Ews mutant brown preadipocytes fail to differentiate due to loss of Bmp7 expression, a critical early brown adipogenic factor. We demonstrate that EWS, along with its binding partner Y-box binding protein 1 (YBX1), activates Bmp7 transcription. Depletion of either Ews or Ybx1 leads to loss of Bmp7 expression and brown adipogenesis. Remarkably, Ews null BATs and brown preadipocytes ectopically express myogenic genes. These results demonstrate that EWS is essential for early brown fat lineage determination.

Antioxidative role of selenoprotein W in oxidant-induced mouse embryonic neuronal cell death.

It has been reported that selenoprotein W (SelW) mRNA is highly expressed in the developing central nerve system of rats, and its expression is maintained until the early postnatal stage. We here found that SelW protein significantly increased in mouse brains of postnatal day 8 and 20 relative to embryonic day 15. This was accompanied by increased expression of SOD1 and SOD2. When the expression of SelW in primary cultured cells derived from embryonic cerebral cortex was knocked down with small interfering RNAs (siRNAs), SelW siRNA-transfected neuronal cells were more sensitive to the oxidative stress induced by treatment of H2O2 than control cells. TUNEL assays revealed that H2O2-induced apoptotic cell death occurred at a higher frequency in the siRNA-transfected cells than in the control cells. Taken together, our findings suggest that SelW plays an important role in protection of neurons from oxidative stress during neuronal development.

Modification of promyelocytic leukemia zinc finger protein (PLZF) by SUMO-1 conjugation regulates its transcriptional repressor activity.

Promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding protein that represses the transcriptional activity of target genes such as those for cyclin A and the interleukin-3 receptor alpha chain. The PLZF gene becomes fused to the retinoic acid receptor alpha gene as a result of the t(11, 17)(q23;q21) chromosomal translocation that is associated with acute promyelocytic leukemia. We now show that endogenous PLZF in human promyelocytic leukemia HL-60 cells is modified by conjugation with SUMO-1 (small ubiquitin-related modifier-1) and that PLZF colocalizes with SUMO-1 in the nucleus of transfected human embryonic kidney 293T cells. Site-directed mutagenesis identified lysine 242 in the RD2 domain of human PLZF as the sumoylation site. A luciferase reporter gene assay suggested that SUMO-1 modification of this residue is required for transcriptional repression by PLZF, and an electrophoretic mobility shift assay showed that this modification increases the DNA binding activity of PLZF. PLZF-mediated regulation of the cell cycle and transcriptional repression of the cyclin A2 gene were also dependent on sumoylation of PLZF on lysine 242. These results demonstrate that PLZF is modified by SUMO-1 conjugation and that this modification regulates the biological functions of PLZF.