Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling

BACKGROUND: KSRP is a AU-rich element (ARE) binding protein that causes decay of select sets of transcripts in different cell types. We have recently described that phosphatidylinositol 3-kinase/AKT (PI3K-AKT) activation induces stabilization and accumulation of the labile β-catenin mRNA through an impairment of KSRP function. RESULTS: Aim of this study was to identify additional KSRP targets whose stability and steady-state levels are enhanced by PI3K-AKT activation. First, through microarray analyses of the AU-rich transcriptome in pituitary αT3-1 cells, we identified 34 ARE-containing transcripts upregulated in cells expressing a constitutively active form of AKT1. In parallel, by an affinity chromatography-based technique followed by microarray analyses, 12 mRNAs target of KSRP, additional to β-catenin, were identified. Among them, seven mRNAs were upregulated in cells expressing activated AKT1. Both steady-state levels and stability of these new KSRP targets were consistently increased by either KSRP knock-down or PI3K-AKT activation. CONCLUSION: Our study identified a set of transcripts that are targets of KSRP and whose expression is increased by PI3K-AKT activation. These mRNAs encode RNA binding proteins, signaling molecules and a replication-independent histone. The increased expression of these gene products upon PI3K-AKT activation could play a role in the cellular events initiated by this signaling pathway

RNase L Mediates Transient Control of The Interferon Response Through Modulation of The Double-stranded RNA-Dependent Protein Kinase PKR

The transient control of diverse biological responses that occurs in response to varied forms of stress is often a highly regulated process. During the interferon (IFN) response, translational repression due to phosphorylation of eukaryotic initiation factor 2α, eIF2α, by the double-stranded RNA-dependent protein kinase, PKR, constitutes a means of inhibiting viral replication. Here we show that the transient nature of the IFN response against acute viral infections is regulated, at least in part, by RNase L. During the IFN antiviral response in RNase L-null cells, PKR mRNA stability was enhanced, PKR induction was increased, and the phosphorylated form of eIF2α appeared with extended kinetics compared with similarly treated wild type cells. An enhanced IFN response in RNase L-null cells was also demonstrated by monitoring inhibition of viral protein synthesis. Furthermore, ectopic expression of RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses

RNase L Mediates Transient Control of The Interferon Response Through Modulation of The Double-stranded RNA-Dependent Protein Kinase PKR

The transient control of diverse biological responses that occurs in response to varied forms of stress is often a highly regulated process. During the interferon (IFN) response, translational repression due to phosphorylation of eukaryotic initiation factor 2α, eIF2α, by the double-stranded RNA-dependent protein kinase, PKR, constitutes a means of inhibiting viral replication. Here we show that the transient nature of the IFN response against acute viral infections is regulated, at least in part, by RNase L. During the IFN antiviral response in RNase L-null cells, PKR mRNA stability was enhanced, PKR induction was increased, and the phosphorylated form of eIF2α appeared with extended kinetics compared with similarly treated wild type cells. An enhanced IFN response in RNase L-null cells was also demonstrated by monitoring inhibition of viral protein synthesis. Furthermore, ectopic expression of RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses

Hurler disease (mucopolysaccharidosis type IH): clinical features and consanguinity in Tunisian population

Mucopolysaccharidosis type I (MPS I) was a group of rare autosomal recessive disorder caused by the deficiency of the lysosomal enzyme, alpha -L -iduronidase, and the resulting accumulation of undergraded dematan sulfate and heparan sulfate. MPS I patients have a wide range of clinical presentations, that makes it difficult to predict patient phenotype which is needed for genetic counseling and also impedes the selection and evaluation of patients undergoing therapy bone marrow transplantation

Alternative polyadenylation variants of the RNA binding protein, HuR: abundance, role of AU-rich elements and auto-Regulation

The RNA-binding protein, HuR, is involved in the stabilization of AU-rich element-containing mRNAs with products that are involved in cell-cycle progression, cell differentiation and inflammation. We show that there are multiple polyadenylation variants of HuR mRNA that differ in their abundance, using both bioinformatics and experimental approaches. A polyadenylation variant with distal poly(A) signal is a rare transcript that harbors functional AU-rich elements (ARE) in the 3′UTR. A minimal 60-nt region, but not a mutant form, fused to reporter-3′UTR constructs was able to downregulate the reporter activity. The most predominant and alternatively polyadenylated mature transcript does not contain the ARE. HuR itself binds HuR mRNA, and upregulated the activity of reporter from constructs fused with ARE-isoform and the HuR ARE. Wild-type tristetraprolin (TTP), but not the zinc finger mutant TTP, competes for HuR binding and upregulation of HuR mRNA. The study shows that the HuR gene codes for several polyadenylation variants differentially regulated by AU-rich elements, and demonstrates an auto-regulatory role of HuR

Selection of AU-rich transiently expressed sequences: Reversal of cDNA abundance

Study of early and transient response gene expression is important for understanding the mechanisms of response to growth stimuli and exogenous agents such as microbes, stress, and radiation. Many of the cytokines, proto-oncogenes, and other transiently expressed gene products are encoded by mRNAs that contain AU-rich elements (AREs) in their 3′ untranslated regions (UTRs). In this article, we describe an approach to selectively synthesize ARE-containing cDNA (ARE-cDNA) using an innovative combination of culture treatment, thermostabilization of reverse transcriptase (RT) by the disaccharide trehalose, and use of optimized ARE-specific oligomers. The monocytic cell line, THP-1, was treated with cycloheximide and endotoxin to enrich for ARE-mediated gene expression followed by the RT procedure. Selection of ARE-cDNA with simultaneous suppression of abundant cDNA was made possible using the procedure as monitored by the preferential expression of IL-8, an ARE-cDNA molecule, over the abundant housekeeping cDNA, β-actin. The use of trehalose dramatically reversed cDNA abundance, resulting in almost complete suppression of housekeeping cDNA. Finally, construction of specialized ARE-cDNA libraries confirmed the selectivity of ARE-cDNAs and the presence of rare genes. The ability to reverse the abundance of housekeeping and other highly expressed genes toward ARE genes facilitates the discovery and study of rare early response and transiently expressed genes

Transcriptional regulation of the proto-oncogene Zfp521 by SPI1 (PU.1) and HOXC13

The mouse zinc‐finger gene Zfp521 (also known as ecotropic viral insertion site 3; Evi3; and ZNF521 in humans) has been identified as a B‐cell proto‐oncogene, causing leukemia in mice following retroviral insertions in its promoter region that drive Zfp521 over‐expression. Furthermore, ZNF521 is expressed in human hematopoietic cells, and translocations between ZNF521 and PAX5 are associated with pediatric acute lymphoblastic leukemia. However, the regulatory factors that control Zfp521 expression directly have not been characterized. Here we demonstrate that the transcription factors SPI1 (PU.1) and HOXC13 synergistically regulate Zfp521 expression, and identify the regions of the Zfp521 promoter required for this transcriptional activity. We also show that SPI1 and HOXC13 activate Zfp521 in a dose‐dependent manner. Our data support a role for this regulatory mechanism in vivo, as transgenic mice over‐expressing Hoxc13 in the fetal liver show a strong correlation between Hoxc13 expression levels and Zfp521 expression. Overall these experiments provide insights into the regulation of Zfp521 expression in a nononcogenic context. The identification of transcription factors capable of activating Zfp521 provides a foundation for further investigation of the regulatory mechanisms involved in ZFP521‐driven cell differentiation processes and diseases linked to Zfp521 mis‐expression

PI3K-AKT signaling stabilizes a set of KSRP-interacting mRNAs and increases their expression

<p><b>Copyright information:</b></p><p>Taken from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"</p><p>http://www.biomedcentral.com/1471-2199/8/28</p><p>BMC Molecular Biology 2007;8():28-28.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858702.</p><p></p> (A) Either mock-αT3-1 or αT3-1-myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of KSRP-interacting mRNAs and β2-MG (control transcript), monitored by RT-PCR, in either mock-αT3-1 or αT3-1-myrAKT1 cells. (C) Semi quantitative RT-PCR analysis of both KSRP-interacting mRNAs and β2-MG (control transcript) in either mock-αT3-1 (red lines) or αT3-1-myrAKT1 (blue lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file

KSRP associates with AUF1p45 and hnRNPA1 in cytoplasmic extracts of aT3-1 cells

<p><b>Copyright information:</b></p><p>Taken from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"</p><p>http://www.biomedcentral.com/1471-2199/8/28</p><p>BMC Molecular Biology 2007;8():28-28.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858702.</p><p></p> (A) S100 extracts from αT3-1 cells were subjected to gel filtration chromatography on a Superose 6 column. Aliquots of the eluted fractions were analyzed by Western blotting using the indicated antibodies. (B) RNase A-treated S100 extracts from αT3-1 cells were immunoprecipitated with preimmune (lane 2) or anti-KSRP (lane 3) sera and analyzed by immunoblotting with either anti-AUF1 (top) or anti-HnRNPA1 (bottom) antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1, while the asterisk marks a anti-AUF1 cross-reacting band. (C) GST-pulldown of either endogenous AUF1p45 (top) or endogenous hnRNPA1 (bottom) from S100 extracts of αT3-1 cells using either control GST or GST-KSRP. Proteins were analyzed by immunoblotting using the indicated antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1