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Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes.

Andreev DE, O'Connor PB, Zhdanov AV, Dmitriev RI, Shatsky IN, Papkovsky DB, Baranov PV - Genome Biol. (2015)

Bottom Line: While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes.Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames.These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry and Cell Biology, Western Gateway Building, University College Cork, Cork, Ireland. cycloheximide@yandex.ru.

ABSTRACT

Background: Oxygen and glucose metabolism play pivotal roles in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) during ischemia and stroke results in extensive tissue injury and cell death.

Results: Using time-resolved ribosome profiling, we assess gene expression levels in a neural cell line, PC12, during the first hour of OGD. The most substantial alterations are seen to occur within the first 20 minutes of OGD. While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes. This response involves reprogramming of initiation and elongation rates, as well as the stringency of start codon recognition. Genes involved in oxidative phosphorylation are most affected. Detailed analysis of ribosome profiles reveals salient alterations of ribosome densities on individual mRNAs. The mRNA-specific alterations include increased translation of upstream open reading frames, site-specific ribosome pauses, and production of alternative protein isoforms with amino-terminal extensions. Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames.

Conclusions: These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes.

No MeSH data available.


Related in: MedlinePlus

In vitro model of ischemia. (A) Analysis of cellular ATP during OGD. Reduction in ATP becomes significant after 2 h. In the presence of glucose, ATP levels remain unchanged. OD, oxygen deprivation. (B) Decreased intensities of TMRM and J-aggregated form of JC-1 (red) probes under OGD indicate ΔΨm depolarization. (C) Dissimilar to OD, only a minor transient elevation in Epas1 (also known as HIF-2α) levels is seen under OGD (arbitrary units (a.u.)). (D) Characteristic OGD-induced changes in phosphorylation of the proteins involved in regulation of translation. A minor increase in phosphorylated AMPKα under OD suggests a smaller effect of this condition on AMPK signaling. Scale bar is 50 μm. Asterisks demonstrate significant difference. Error bars show standard deviation (A,C,D) or a range in TMRM and JC-1 intensities (B), calculated for 40 to 50 randomly selected cells.
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Fig1: In vitro model of ischemia. (A) Analysis of cellular ATP during OGD. Reduction in ATP becomes significant after 2 h. In the presence of glucose, ATP levels remain unchanged. OD, oxygen deprivation. (B) Decreased intensities of TMRM and J-aggregated form of JC-1 (red) probes under OGD indicate ΔΨm depolarization. (C) Dissimilar to OD, only a minor transient elevation in Epas1 (also known as HIF-2α) levels is seen under OGD (arbitrary units (a.u.)). (D) Characteristic OGD-induced changes in phosphorylation of the proteins involved in regulation of translation. A minor increase in phosphorylated AMPKα under OD suggests a smaller effect of this condition on AMPK signaling. Scale bar is 50 μm. Asterisks demonstrate significant difference. Error bars show standard deviation (A,C,D) or a range in TMRM and JC-1 intensities (B), calculated for 40 to 50 randomly selected cells.

Mentions: After 1 h of OGD cellular ATP levels decreased to approximately 80% of control conditions and continued to decrease further almost linearly with time (Figure 1A). In line with this, the mitochondrial membrane potential (ΔΨm) probes TMRM (Tetramethylrhodamine, methyl ester) and JC-1 showed a progressive depolarization of mitochondria in cells exposed to OGD (Figure 1B; Figure A2A,B in Additional file 1). As expected, these effects were not observed when glucose was available: ATP and ΔΨm levels decreased only slightly due to the continued ATP generation via glycolysis and activity of F1Fo ATP synthase working in reverse mode to maintain mitochondrial polarization. Under energy stress imposed on the cells, only a minor transient elevation in Epas1 (also known as HIF-2α) was observed along with a substantial increase in AMPK phosphorylation (Thr172) during the course of OGD, contrasting with oxygen deprivation (OD) conditions (Figure 1C,D; Figure A2C in Additional file 1) [15].Figure 1


Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes.

Andreev DE, O'Connor PB, Zhdanov AV, Dmitriev RI, Shatsky IN, Papkovsky DB, Baranov PV - Genome Biol. (2015)

In vitro model of ischemia. (A) Analysis of cellular ATP during OGD. Reduction in ATP becomes significant after 2 h. In the presence of glucose, ATP levels remain unchanged. OD, oxygen deprivation. (B) Decreased intensities of TMRM and J-aggregated form of JC-1 (red) probes under OGD indicate ΔΨm depolarization. (C) Dissimilar to OD, only a minor transient elevation in Epas1 (also known as HIF-2α) levels is seen under OGD (arbitrary units (a.u.)). (D) Characteristic OGD-induced changes in phosphorylation of the proteins involved in regulation of translation. A minor increase in phosphorylated AMPKα under OD suggests a smaller effect of this condition on AMPK signaling. Scale bar is 50 μm. Asterisks demonstrate significant difference. Error bars show standard deviation (A,C,D) or a range in TMRM and JC-1 intensities (B), calculated for 40 to 50 randomly selected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419486&req=5

Fig1: In vitro model of ischemia. (A) Analysis of cellular ATP during OGD. Reduction in ATP becomes significant after 2 h. In the presence of glucose, ATP levels remain unchanged. OD, oxygen deprivation. (B) Decreased intensities of TMRM and J-aggregated form of JC-1 (red) probes under OGD indicate ΔΨm depolarization. (C) Dissimilar to OD, only a minor transient elevation in Epas1 (also known as HIF-2α) levels is seen under OGD (arbitrary units (a.u.)). (D) Characteristic OGD-induced changes in phosphorylation of the proteins involved in regulation of translation. A minor increase in phosphorylated AMPKα under OD suggests a smaller effect of this condition on AMPK signaling. Scale bar is 50 μm. Asterisks demonstrate significant difference. Error bars show standard deviation (A,C,D) or a range in TMRM and JC-1 intensities (B), calculated for 40 to 50 randomly selected cells.
Mentions: After 1 h of OGD cellular ATP levels decreased to approximately 80% of control conditions and continued to decrease further almost linearly with time (Figure 1A). In line with this, the mitochondrial membrane potential (ΔΨm) probes TMRM (Tetramethylrhodamine, methyl ester) and JC-1 showed a progressive depolarization of mitochondria in cells exposed to OGD (Figure 1B; Figure A2A,B in Additional file 1). As expected, these effects were not observed when glucose was available: ATP and ΔΨm levels decreased only slightly due to the continued ATP generation via glycolysis and activity of F1Fo ATP synthase working in reverse mode to maintain mitochondrial polarization. Under energy stress imposed on the cells, only a minor transient elevation in Epas1 (also known as HIF-2α) was observed along with a substantial increase in AMPK phosphorylation (Thr172) during the course of OGD, contrasting with oxygen deprivation (OD) conditions (Figure 1C,D; Figure A2C in Additional file 1) [15].Figure 1

Bottom Line: While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes.Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames.These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry and Cell Biology, Western Gateway Building, University College Cork, Cork, Ireland. cycloheximide@yandex.ru.

ABSTRACT

Background: Oxygen and glucose metabolism play pivotal roles in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) during ischemia and stroke results in extensive tissue injury and cell death.

Results: Using time-resolved ribosome profiling, we assess gene expression levels in a neural cell line, PC12, during the first hour of OGD. The most substantial alterations are seen to occur within the first 20 minutes of OGD. While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes. This response involves reprogramming of initiation and elongation rates, as well as the stringency of start codon recognition. Genes involved in oxidative phosphorylation are most affected. Detailed analysis of ribosome profiles reveals salient alterations of ribosome densities on individual mRNAs. The mRNA-specific alterations include increased translation of upstream open reading frames, site-specific ribosome pauses, and production of alternative protein isoforms with amino-terminal extensions. Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames.

Conclusions: These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes.

No MeSH data available.


Related in: MedlinePlus