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. 2015 Mar 31;43(6):3219-36.
doi: 10.1093/nar/gkv167. Epub 2015 Mar 8.

Hypoxia-induced Gene Expression Results From Selective mRNA Partitioning to the Endoplasmic Reticulum

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Free PMC article

Hypoxia-induced Gene Expression Results From Selective mRNA Partitioning to the Endoplasmic Reticulum

Jonas J Staudacher et al. Nucleic Acids Res. .
Free PMC article

Abstract

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. 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. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

Figures

Figure 1.
Figure 1.
Polysomal gradient analysis. HT1080 cells were incubated under control (21% oxygen) or hypoxic (1% oxygen) conditions for up to 36 h. (A) Schematic overview of polysomal gradient analysis, adopted from (39). (B) Typical ribosomal profiles after sucrose gradient ultra-centrifugation monitored at 254 nm absorbance from bottom (51% sucrose) to top (17% sucrose). (CG) Quantification of candidate transcripts in extracts of cells grown under normoxia or 36 h of hypoxia (1 % oxygen) following fractionation of sucrose gradients as indicated in (1B). n = 3.
Figure 2.
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. (AE) 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.
Figure 3.
Figure 3.. Dual fluorescence detection of transcript co-localization with the ER marker Calnexin by IF-FISH. HT1080 cells were cultured either under normoxic or hypoxic (1% oxygen) conditions for 36 h and hybridized with oligo(dT), VEGF, HIF-1α, β-Actin, P4HA1 and BLID mRNA probes (FITC, green) for in situ hybridization. Immunostaining of endogenous α-tubulin (Cy5, cyan) and Calnexin (Cy3, red) was carried out using specific antibodies. The fourth line shows the merged images for co-localization. In the fifth line, dotted squares indicate the magnified area represented in the enlargement. Cy3 and FITC fluorescence monitored along the dashed lines is shown as relative signal intensity in the bottom panel. Co-localization of the transcript with the ER marker Calnexin is indicated by a good match in peaks or troughs of both signals.
Figure 4.
Figure 4.
Summary of microarray data analysis. Groups of significantly up- (green) or down- (red) regulated candidates from total RNA (overall cellular expression) or ER-localized RNA (ER localization) were selected. The numbers of candidate genes are indicated in black. Intersections of candidates regulated by both expression level and localization were calculated. Gene ontology analysis was performed using the tools provided by WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/). For GO analysis only miRNAs and protein coding transcripts were used. **−P < 0.01, ***−P < 0.001. A detailed description of significantly enriched GOs and statistics is provided in Supplementary Tables S2–S7.
Figure 5.
Figure 5.
Sub-group specific UTR features. Transcripts of genes that were either up- or downregulated in their expression and/or ER localization as described in Figure 5 were used to test for different UTR features. (A and B) From all 5′UTR sequences we determined uAUG number (A) and UTR length. The AUG score (B) is the ratio of the number of uAUG and the length of the respective UTR. The gene specific AUG number/score was analysed with a non-parametric two factor ANOVA test with respect to location and expression level. The HIF target gene group consists of 78 verified candidates as described in (1) and is shown for comparison only. (C and D) Group-specific conservation scores of 5′-UTRs (C) or 3′UTRs (D). A hash sign (#) indicates a significantly higher conservation score compared to all other groups in the statistical post-hoc test. Asterisks (*) indicate a significantly lower conservation score compared to the other groups in the post-hoc test. The grey bars represent either all 5′- or all 3′-UTRs and were not included in the statistical analysis. They are shown for comparison only. A detailed post-hoc statistical analysis is presented in Supplementary Figure S14.
Figure 6.
Figure 6.
IF-FISH analysis of HT1080 cells transfected with Firefly-Luciferase (Luc) reporter plasmids by electroporation as indicated and cultured under normoxic or hypoxic conditions. Cells were hybridized with Luc mRNA probe (FITC, green). The native Luciferase mRNA is shown as control (ctrl.). Modified Luciferase transcripts contained insertions of 5′- or 3′-UTR cis-elements as shown in Table 1, with either the original sequence context (wt) or a mutated cis-Element (mut) as shown in Figure 7A. Nuclei were stained with DAPI (blue). Specific antibodies were used to co-stain endogenous a-tubulin (Cy5, cyan) and Calnexin (Cy3, red). Dotted squares indicate the magnified area represented in the enlargement. Cy3 and FITC fluorescence monitored along the dashed lines is shown as relative signal intensity in the bottom panel.
Figure 7.
Figure 7.
Verification of motif functionality by reporter gene assays. The pGL3-promoter vector (SV40 promoter) was used to insert 30–40-mers carrying selected motifs (boxes) in its center into the 5′- or 3′-UTR of luciferase mRNA. (A) Schematic illustration of cloning strategy and inserted sequences. Native motifs (wt) and mutated variants (mut) were tested. (BD) HT1080 cells were transfected and incubated for up to 40 h under control or hypoxic conditions. Relative fold changes as compared to control conditions are shown for the original pGL3-promoter construct as well as constructs carrying native or mutated cis-elements in the luciferase 5′UTR (5′UTR-cis1) or 3′UTR (3′UTR-cis1 and 3′UTR-cis22). n = 8. * indicates significantly changed luciferase activity of the wt-construct compared to the unmodified luciferase mRNA (pGL3p) and # indicates significance compared to the mutated variant (P < 0.05).

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