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The insulin receptor cellular IRES confers resistance to eIF4A inhibition.

Olson CM, Donovan MR, Spellberg MJ, Marr MT - Elife (2013)

Bottom Line: Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition.In addition, the murine insulin receptor and insulin-like growth factor receptor 5' UTRs support cap-independent translation and have a similar resistance to hippuristanol.This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and the Rosenstiel Basic Medical Sciences Research Center , Brandeis University , Waltham , United States.

ABSTRACT
Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. We have shown previously that the 5' UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5' UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions. DOI:http://dx.doi.org/10.7554/eLife.00542.001.

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Foxo activates Pdcd4 in Drosophila cells.(A) Reanalysis of ChIP-chip data from Teleman et al. (2008). Genomic Browser view of Foxo binding to the Pdcd4 locus in starved larva. The data are plotted as the enrichment (log2) over mock precipitated samples. Primers used for ChIP and qPCR are indicated. (B) ChIP of Foxo at 4E-BP promoter and Pdcd4 locus in Drosophila S2 cells expressing constitutively active Foxo (FoxoCA). The data are plotted as fold enrichment over a background region 1 kb downstream of 4E-BP. Uninduced samples are plotted in white, induced samples in black (error bars indicate SD). (C) RT-qPCR of Pdcd4 mRNA and pre-mRNA in Drosophila S2 cells expressing FoxoCA. Data are plotted as fold-induction (error bars indicate SD). (D) Immunoblot of total protein from Drosophila S2 cells expressing FoxoCA. Positions of Pdcd4 and tubulin are indicated. (E) 4E-BP, GstD1, and Pdcd4 RNA levels in untreated and paraquat-treated animals. The levels of RNA were normalized to RP49 and are plotted as fold-induction relative to untreated animals (error bars indicate SEM).DOI:http://dx.doi.org/10.7554/eLife.00542.004
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fig2: Foxo activates Pdcd4 in Drosophila cells.(A) Reanalysis of ChIP-chip data from Teleman et al. (2008). Genomic Browser view of Foxo binding to the Pdcd4 locus in starved larva. The data are plotted as the enrichment (log2) over mock precipitated samples. Primers used for ChIP and qPCR are indicated. (B) ChIP of Foxo at 4E-BP promoter and Pdcd4 locus in Drosophila S2 cells expressing constitutively active Foxo (FoxoCA). The data are plotted as fold enrichment over a background region 1 kb downstream of 4E-BP. Uninduced samples are plotted in white, induced samples in black (error bars indicate SD). (C) RT-qPCR of Pdcd4 mRNA and pre-mRNA in Drosophila S2 cells expressing FoxoCA. Data are plotted as fold-induction (error bars indicate SD). (D) Immunoblot of total protein from Drosophila S2 cells expressing FoxoCA. Positions of Pdcd4 and tubulin are indicated. (E) 4E-BP, GstD1, and Pdcd4 RNA levels in untreated and paraquat-treated animals. The levels of RNA were normalized to RP49 and are plotted as fold-induction relative to untreated animals (error bars indicate SEM).DOI:http://dx.doi.org/10.7554/eLife.00542.004

Mentions: There are hints in the literature, based on microarray experiments, indicating this gene is induced in response to nutrient stress and might be controlled by Foxo (Gershman et al., 2007). In an effort to determine if Foxo binds to the Pdcd4 gene in nutrient stressed animals we reanalyzed the only publically available Foxo ChIP (Chromatin immunoprecipitation) dataset (Teleman et al., 2008). These experiments were performed on starved larva. We find Foxo binds the Pdcd4 gene in both the promoter and intronic regions with enrichment values as high as 16-fold over background (Figure 2A). To corroborate this finding, we performed ChIP on genomic DNA from a cell line with an inducible Foxo cDNA gene that has been modified so the Foxo protein produced is constitutively active because it is immune to the negative regulation by insulin signaling (FoxoCA) (Puig et al., 2003; Gershman et al., 2007). This allows us to induce Foxo under conditions of high nutrient signaling and remove possible crosstalk from upstream signaling pathways. We tested the enrichment of genomic sequences by qPCR using primers to the Pdcd4 promoter region (Figure 2A) compared to a region in the first intron of CG15414, a gene just downstream of 4E-BP. We find FoxoCA binds to the promoter region of Pdcd4 at levels comparable to a well-defined direct target, 4E-BP (Junger et al., 2003; Puig et al., 2003; Marr et al., 2007) (Figure 2B). To determine the effect on mRNA production under these conditions we performed quantitative RT-qPCR on induced cells. We find that the steady-state level of Pdcd4 mRNA is increased about threefold in cells expressing active Foxo (Figure 2C). To determine if the effect is due to mRNA stability changes or new transcription we assayed intron-containing pre-mRNAs by RT-qPCR. Since most splicing is co-transcriptional in Drosophila this is a good assay for new RNA synthesis (Khodor et al., 2011). We find that Pdcd4 pre-mRNA is increased, indicating an increase in transcription of the gene. The increased mRNA also leads to increased protein synthesis as determined by immuno-blot with antibodies directed against Drosophila Pdcd4 (Figure 2D). This is likely an underestimate of the effect since these experiments are all done under high serum and insulin conditions that should result in the rapid turnover of Pdcd4 protein (Dorrello et al., 2006).10.7554/eLife.00542.004Figure 2.Foxo activates Pdcd4 in Drosophila cells.


The insulin receptor cellular IRES confers resistance to eIF4A inhibition.

Olson CM, Donovan MR, Spellberg MJ, Marr MT - Elife (2013)

Foxo activates Pdcd4 in Drosophila cells.(A) Reanalysis of ChIP-chip data from Teleman et al. (2008). Genomic Browser view of Foxo binding to the Pdcd4 locus in starved larva. The data are plotted as the enrichment (log2) over mock precipitated samples. Primers used for ChIP and qPCR are indicated. (B) ChIP of Foxo at 4E-BP promoter and Pdcd4 locus in Drosophila S2 cells expressing constitutively active Foxo (FoxoCA). The data are plotted as fold enrichment over a background region 1 kb downstream of 4E-BP. Uninduced samples are plotted in white, induced samples in black (error bars indicate SD). (C) RT-qPCR of Pdcd4 mRNA and pre-mRNA in Drosophila S2 cells expressing FoxoCA. Data are plotted as fold-induction (error bars indicate SD). (D) Immunoblot of total protein from Drosophila S2 cells expressing FoxoCA. Positions of Pdcd4 and tubulin are indicated. (E) 4E-BP, GstD1, and Pdcd4 RNA levels in untreated and paraquat-treated animals. The levels of RNA were normalized to RP49 and are plotted as fold-induction relative to untreated animals (error bars indicate SEM).DOI:http://dx.doi.org/10.7554/eLife.00542.004
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Related In: Results  -  Collection

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fig2: Foxo activates Pdcd4 in Drosophila cells.(A) Reanalysis of ChIP-chip data from Teleman et al. (2008). Genomic Browser view of Foxo binding to the Pdcd4 locus in starved larva. The data are plotted as the enrichment (log2) over mock precipitated samples. Primers used for ChIP and qPCR are indicated. (B) ChIP of Foxo at 4E-BP promoter and Pdcd4 locus in Drosophila S2 cells expressing constitutively active Foxo (FoxoCA). The data are plotted as fold enrichment over a background region 1 kb downstream of 4E-BP. Uninduced samples are plotted in white, induced samples in black (error bars indicate SD). (C) RT-qPCR of Pdcd4 mRNA and pre-mRNA in Drosophila S2 cells expressing FoxoCA. Data are plotted as fold-induction (error bars indicate SD). (D) Immunoblot of total protein from Drosophila S2 cells expressing FoxoCA. Positions of Pdcd4 and tubulin are indicated. (E) 4E-BP, GstD1, and Pdcd4 RNA levels in untreated and paraquat-treated animals. The levels of RNA were normalized to RP49 and are plotted as fold-induction relative to untreated animals (error bars indicate SEM).DOI:http://dx.doi.org/10.7554/eLife.00542.004
Mentions: There are hints in the literature, based on microarray experiments, indicating this gene is induced in response to nutrient stress and might be controlled by Foxo (Gershman et al., 2007). In an effort to determine if Foxo binds to the Pdcd4 gene in nutrient stressed animals we reanalyzed the only publically available Foxo ChIP (Chromatin immunoprecipitation) dataset (Teleman et al., 2008). These experiments were performed on starved larva. We find Foxo binds the Pdcd4 gene in both the promoter and intronic regions with enrichment values as high as 16-fold over background (Figure 2A). To corroborate this finding, we performed ChIP on genomic DNA from a cell line with an inducible Foxo cDNA gene that has been modified so the Foxo protein produced is constitutively active because it is immune to the negative regulation by insulin signaling (FoxoCA) (Puig et al., 2003; Gershman et al., 2007). This allows us to induce Foxo under conditions of high nutrient signaling and remove possible crosstalk from upstream signaling pathways. We tested the enrichment of genomic sequences by qPCR using primers to the Pdcd4 promoter region (Figure 2A) compared to a region in the first intron of CG15414, a gene just downstream of 4E-BP. We find FoxoCA binds to the promoter region of Pdcd4 at levels comparable to a well-defined direct target, 4E-BP (Junger et al., 2003; Puig et al., 2003; Marr et al., 2007) (Figure 2B). To determine the effect on mRNA production under these conditions we performed quantitative RT-qPCR on induced cells. We find that the steady-state level of Pdcd4 mRNA is increased about threefold in cells expressing active Foxo (Figure 2C). To determine if the effect is due to mRNA stability changes or new transcription we assayed intron-containing pre-mRNAs by RT-qPCR. Since most splicing is co-transcriptional in Drosophila this is a good assay for new RNA synthesis (Khodor et al., 2011). We find that Pdcd4 pre-mRNA is increased, indicating an increase in transcription of the gene. The increased mRNA also leads to increased protein synthesis as determined by immuno-blot with antibodies directed against Drosophila Pdcd4 (Figure 2D). This is likely an underestimate of the effect since these experiments are all done under high serum and insulin conditions that should result in the rapid turnover of Pdcd4 protein (Dorrello et al., 2006).10.7554/eLife.00542.004Figure 2.Foxo activates Pdcd4 in Drosophila cells.

Bottom Line: Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition.In addition, the murine insulin receptor and insulin-like growth factor receptor 5' UTRs support cap-independent translation and have a similar resistance to hippuristanol.This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and the Rosenstiel Basic Medical Sciences Research Center , Brandeis University , Waltham , United States.

ABSTRACT
Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. We have shown previously that the 5' UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5' UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions. DOI:http://dx.doi.org/10.7554/eLife.00542.001.

Show MeSH