Ruggiero, T. et al. Identification of a set of KSRP target transcripts up-regulated by PI3K-AKT signaling. BMC Mol. Biol. 8, 28-43

Istituto Nazionale per la Ricerca sul Cancro (IST), 16132 Genova, Italy. <>
BMC Molecular Biology (Impact Factor: 2.19). 02/2007; 8(1):28. DOI: 10.1186/1471-2199-8-28
Source: PubMed

ABSTRACT 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 beta-catenin mRNA through an impairment of KSRP function.
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 alphaT3-1 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 beta-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.
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.

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    • "HuR, an RNA binding protein, is the only known factor able to stabilize b-catenin mRNA and to enhance b-catenin protein production, and it contributes to b-catenin accumulation in colon cancer (Lopez de Silanes et al, 2003). On the other hand, factors that destabilize b-catenin mRNA or reduce b-catenin protein production have also been identified, including KSRP, eIF6 and Quaking (Bikkavilli & Malbon, 2010; Gherzi et al, 2006; Ji et al, 2008; Ruggiero et al, 2007; Yang et al, 2010). Compared with our understanding of b-catenin degradation, the regulation of b-catenin protein production is far less clear and therefore requires further investigation. "
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    • "Coimmunoprecipitation experiments have demonstrated that various ARE-BPs can directly interact with one another including: HuR/AUF1 [10]; HuR/TIA-1 [11,12]; KSRP/AUF1 [13] and KSRP/TIA-1 [14]. What cannot be determined by immunoprecipitation, however, is where within the cell these interactions are taking place. "
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    ABSTRACT: A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts. All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1. Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and proteins and RNA is not always indicative of interaction. To this point, using FRET and immuno-FRET, we have demonstrated that RNA-BPs can visually colocalize without producing a FRET signal. In contrast, proteins that appear to be delimited to one or another intracellular compartment can be shown to interact when those compartments are juxtaposed.
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