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Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Staudacher JJ, Naarmann-de Vries IS, Ujvari SJ, Klinger B, Kasim M, Benko E, Ostareck-Lederer A, Ostareck DH, Bondke Persson A, Lorenzen S, Meier JC, Blüthgen N, Persson PB, Henrion-Caude A, Mrowka R, Fähling M - Nucleic Acids Res. (2015)

Bottom Line: Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism.We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions.We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

View Article: PubMed Central - PubMed

Affiliation: Charité - Universitätsmedizin Berlin, Institut für Vegetative Physiologie, Charitéplatz 1, D-10117 Berlin, Germany.

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Estimation of gene expression rate and mRNA distribution in hypoxia. HT1080 cells were incubated under control and hypoxic conditions for 36 h. Gene expression levels (total RNA & total protein, left panel), mRNA localization in the cytoplasm (polysomal versus non-polysomal fractions, middle panel) and at the ER (right panel) are shown. (A–E) Total mRNA levels were determined by qPCR. Corresponding protein levels were estimated by western blotting as shown in Supplementary Figure S4. Levels for mRNAs in polysomal and non-polysomal fractions were determined following ultra-centrifugation of cytoplasmic extracts at 100 000 x g. ER-RNA was isolated using a commercially available ER-isolation kit. n = 5. *−P < 0.05, **−P < 0.01, ***−P < 0.001.
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Figure 2: Estimation of gene expression rate and mRNA distribution in hypoxia. HT1080 cells were incubated under control and hypoxic conditions for 36 h. Gene expression levels (total RNA & total protein, left panel), mRNA localization in the cytoplasm (polysomal versus non-polysomal fractions, middle panel) and at the ER (right panel) are shown. (A–E) Total mRNA levels were determined by qPCR. Corresponding protein levels were estimated by western blotting as shown in Supplementary Figure S4. Levels for mRNAs in polysomal and non-polysomal fractions were determined following ultra-centrifugation of cytoplasmic extracts at 100 000 x g. ER-RNA was isolated using a commercially available ER-isolation kit. n = 5. *−P < 0.05, **−P < 0.01, ***−P < 0.001.

Mentions: To assess the impact of mRNA partitioning on protein synthesis, we measured gene expression levels as determined by total RNA and protein levels as well as transcript levels of selected candidates in different subcellular compartments relevant for mRNA translation, i.e. cytoplasmic ribosomes or ER associated ribosomes. The cytoplasm was separated into translationally active sediment (polysomes) and translationally inactive supernatant (monosomes and RNP) fractions by ultra-centrifugation at 100 000 x g. A pure ER fraction was obtained by biochemical purification using a commercially available ER-isolation kit. Purity of the ER fraction was ensured by assessing the localization of cytoplasmic markers, i.e. GAPDH and β-Actin, and ER-specific markers i.e. aromatase and protein disulfide isomerase (PDI) (Supplementary Figure S3). Thus, cytoplasmic fractions generated by ultracentrifugation and biochemically purified ER were used for further analysis. A summary of the data is presented in Figure 2. Original western blotting results are presented in Supplementary Figure S4.


Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Staudacher JJ, Naarmann-de Vries IS, Ujvari SJ, Klinger B, Kasim M, Benko E, Ostareck-Lederer A, Ostareck DH, Bondke Persson A, Lorenzen S, Meier JC, Blüthgen N, Persson PB, Henrion-Caude A, Mrowka R, Fähling M - Nucleic Acids Res. (2015)

Estimation of gene expression rate and mRNA distribution in hypoxia. HT1080 cells were incubated under control and hypoxic conditions for 36 h. Gene expression levels (total RNA & total protein, left panel), mRNA localization in the cytoplasm (polysomal versus non-polysomal fractions, middle panel) and at the ER (right panel) are shown. (A–E) Total mRNA levels were determined by qPCR. Corresponding protein levels were estimated by western blotting as shown in Supplementary Figure S4. Levels for mRNAs in polysomal and non-polysomal fractions were determined following ultra-centrifugation of cytoplasmic extracts at 100 000 x g. ER-RNA was isolated using a commercially available ER-isolation kit. n = 5. *−P < 0.05, **−P < 0.01, ***−P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4381074&req=5

Figure 2: Estimation of gene expression rate and mRNA distribution in hypoxia. HT1080 cells were incubated under control and hypoxic conditions for 36 h. Gene expression levels (total RNA & total protein, left panel), mRNA localization in the cytoplasm (polysomal versus non-polysomal fractions, middle panel) and at the ER (right panel) are shown. (A–E) Total mRNA levels were determined by qPCR. Corresponding protein levels were estimated by western blotting as shown in Supplementary Figure S4. Levels for mRNAs in polysomal and non-polysomal fractions were determined following ultra-centrifugation of cytoplasmic extracts at 100 000 x g. ER-RNA was isolated using a commercially available ER-isolation kit. n = 5. *−P < 0.05, **−P < 0.01, ***−P < 0.001.
Mentions: To assess the impact of mRNA partitioning on protein synthesis, we measured gene expression levels as determined by total RNA and protein levels as well as transcript levels of selected candidates in different subcellular compartments relevant for mRNA translation, i.e. cytoplasmic ribosomes or ER associated ribosomes. The cytoplasm was separated into translationally active sediment (polysomes) and translationally inactive supernatant (monosomes and RNP) fractions by ultra-centrifugation at 100 000 x g. A pure ER fraction was obtained by biochemical purification using a commercially available ER-isolation kit. Purity of the ER fraction was ensured by assessing the localization of cytoplasmic markers, i.e. GAPDH and β-Actin, and ER-specific markers i.e. aromatase and protein disulfide isomerase (PDI) (Supplementary Figure S3). Thus, cytoplasmic fractions generated by ultracentrifugation and biochemically purified ER were used for further analysis. A summary of the data is presented in Figure 2. Original western blotting results are presented in Supplementary Figure S4.

Bottom Line: Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism.We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions.We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

View Article: PubMed Central - PubMed

Affiliation: Charité - Universitätsmedizin Berlin, Institut für Vegetative Physiologie, Charitéplatz 1, D-10117 Berlin, Germany.

Show MeSH
Related in: MedlinePlus