Project description:Tino is an A+U-Rich Element (ARE) binding protein first identified through its ability to bind to bcl-2 mRNA and to contribute to its degradation. It has recently been recognized as a shorter form of the human Mex-3D protein (hMex-3D), one of the four members of the family of Mex-3 RNA-binding phosphoproteins. In C. elegans, ceMex-3 is a translational regulator that plays a key role in early embryonic development and in the maintenance of worm germ line totipotency. To examine the potential functional conservation between ceMex-3 and hMex3, we have used complementary microarray-based approaches to identify mRNAs directly bound to Tino/hMex-3D. Computational analysis of these target mRNAs resulted in the identification of an U-rich, 34- to 39-nucleotide long, consensus, forming loops of variable sizes. Remarkably, more than half of Tino/hMex-3D targets also contain the consensus for Quaking, which is the human ortholog of GLD-1, a regulator of nematode gametogenesis. All together, our results suggest that Tino/hMex-3D belongs to a regulatory circuit of mRNA trans-acting factors involved in cell fate and differentiation. Keywords: RIP-chip and (recombinant)RIP-Chip analysis of Tino/hMEX-3D mRNPs To identify the transcripts directly bound in vivo by Tino/hMex-3D, we have used two complementary strategies. First, using Tino-his transfected HEK293 cells, we have analysed the RNAs immunoprecipitated by Tino/hMex-3D (RNA-IP complexes). Second, we have developed an in vitro nitrocellulose RNA-protein binding assay using a truncated form of Tino, TinoΔRING-his. First for the in vivo approach, we have transiently transfected HEK293 cells with a His-tagged Tino expressing construct (see Materials) and isolated Tino target mRNAs by immunoprecipitation (IP) assays carried out under conditions preserving mRNA-protein complex integrity. Second, for the in vitro approach, cRNAs were prepared from a human placenta cDNA library and incubated with TinoΔRING-his. This deleted form of Tino-his protein conserves its mRNA binding activity. mRNAs directly bound to TinoΔRING-his protein were subsequently isolated using a nitrocellulose RNA-protein binding assay. A supplementary file of ordered genes based on z-ratios, derived from experimental and negative control samples for each Affymetrix probe, is appended below.

Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. This SuperSeries is composed of the following subset Series:; GSE11496: Expression data from Gcn2 wild-type and knockout mouse liver perfused with or without methionine; GSE11684: Expression data from Perk wild-type and knockout mouse liver perfused without or with 2,5-di-tert-butylhydroquinone Experiment Overall Design: Refer to individual Series

Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Gcn2 wild-type or knockout mouse liver were perfused with complete amino acids media or media lacking methionine for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Gcn2+/+, +Met or -Met; total and polysome fraction of Gcn2-/-, +Met or -Met. Each array analysis was done in duplicate.

Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Perk wild-type or knockout mouse liver were perfused without or with 2,4-di-tert-butylhydroquinone (tBuHQ) for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Perk+/+, -tBuHQ or +tBuHQ; total and polysome fraction of Perk-/-, -tBuHQ or +tBuHQ. Each array analysis was done in duplicate.

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Microarray analysis of normal and tumor-like bone cells from neonatal-onset multisystem inflammatory disease (NOMID)

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