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

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.

Figure 1

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.

Figure 2

Analysis of differential gene expression. (A) Reproducibility of ribo-seq experiment under control conditions. (B) Z-score transformation and false discovery rate (FDR) estimation. (C) Heat map diagram of differentially expressed (DE) genes. TE, translation efficiency. (D) DE gene enrichment over KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. B-H, Benjamini-Hochberg. (E) The magnitude of ribo-seq occupancy alteration during the first 20, 40 and 60 minutes of OGD for the genes detected as DE after 40 minutes. (F) Correlations across replicates of ribo-seq fold changes of genes with >32 reads per time point at different time intervals of OGD exposure. (G) Venn diagrams showing the lack of a relationship between DE genes, mTOR sensitivity and the presence of rHRE.

Figure 3

OGD-induced changes to initiation, elongation and termination. (A) Distribution of sequenced fragments across functional regions of mRNAs under different conditions. (B) Metagene profile of the ribosome density obtained under normal condition and after 1 h of OGD. Positions of footprints 5′ ends are shown relative to the coordinates of the start and stop codons of acORFs, which are set to 0. Over 5,000 mRNA profiles were aggregated to produce the profile. (C) Ribosomal density at Polr2l mRNA. The density in the 3′ UTR indicates leaky termination (readthrough). (D) Sequence logo of the nucleotide context surrounding stop codons in the 19 mRNAs exhibiting high levels of readthrough (top) and the change of stop codon readthrough efficiency during the time course of OGD. (E) Distribution of the Pearson’s correlations of ribosome densities for individual mRNA profiles between replicas and across conditions. (F) An example of mRNA (Mapk3) with changes in footprint density distribution between conditions and replicas.

Figure 4

The effect of 5′ leader translation on translation of acORFs and identification of regulatory uORFs. (A) Correlation between fold change of ribosome density in the 5′ leaders and in acORFs and the change in acORF translation in mRNAs with translated 5′ leaders (orange bars) relative to all mRNA (grey): absolute (left) and relative (right) frequencies of genes. (B) Change in the position of the centre of ribosome density for each mRNA during the course of OGD. mRNAs with upregulation of inhibitory uORFs are likely to be found in the left bottom corner. Ribosome profiles of corresponding mRNAs were chosen for manual examination. (C) acORF regions translated in more than one reading frame are expected to have altered triplet periodicity (high density of footprints with 5′ ends at the third subcodon position). This analysis was used to detect overlapping uORFs.

Figure 5

Ribosome profiles exhibiting mRNA-specific OGD-induced features. (A) Selected mRNAs whose ribosome densities increase at uORFs and decrease at acORFs during OGD. Ribosome profile for Eif1a mRNA is used as the legend. (B) Upregulation of parathymosin isoform with amino-terminal extension whose synthesis begins at a non-AUG codon. (C) Increased uORF density at Eif1b mRNA coincides with decreased density at the acORF and non-annotated downstream ORF. (D) Increased ribosome density downstream of Romo1 acORF that may occur as a result of translation of a long overlapping ORF. (E) Ribosome stalling or an abortive translation event at Srpr mRNA.