Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress

Abstract

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.

Keywords: ATF4; ISR; hypertonic; mTOR; mitochondria; neMito mRNAs; ribosome stalling; stress; translation.

Figure 1.. Inhibition of mRNA translation with increasing hyperosmotic stress intensity

MEFs treated with the indicated hyperosmotic stress conditions were analyzed by (A) cell counting, (B) protein synthesis assays, (C) Western blotting, and (D) polysome profiling. Means ± SEM of triplicate determinations are shown. *p < 0.01

Figure 2.. Adaptation to increasing stress intensity involves differential enrichment of ribosome-associated mRNAs

(A) Scatter plot comparing fold changes of ribosomal footprints (y-axis) and cytoplasmic RNA levels (x-axis) in MEFs exposed to the indicated hyperosmotic stress intensities. (B) Distribution of select mRNAs according to changes in their ribo^ocp (ribosome footprints normalized to corresponding mRNA levels). The number of mRNAs in each group is indicated on the x-axis. (C) Hierarchical clustering of q-values associated with enriched GO terms of the ribo^ocp-up groups. (D) Scatterplot comparing changes in ribosome footprints and cytoplasmic RNA levels of mRNAs encoding mitochondrial respiration complexes. (E) Ribosome footprints across select mRNA sequences. Footprints in the coding region (CDS) are highlighted in red, and in the untranslated regions (UTRs) in gray.

Figure 3.. Sustained inhibition of protein synthesis during severe stress is accompanied by ribosome pausing at translation initiation codons of select mRNAs

MEFs exposed to the indicated stress conditions were analyzed for (A) mean density of ribosomal footprints relative to the translation initiation codon (top row) and translation termination codon (bottom row). (B) Heat map depicting ribosome occupancies on the different codons (left) based on their relative location on the mRNAs (x-axis). (C) Scatter plots comparing ribosome occupancies of different codons (codon occupancy) in ORFs, excluding authentic translation initiation codons. (D) Percentage of paused and non-paused mRNAs in each group after 700 mOsm stress for 2 h. (E) Distribution of footprints on Sat1 mRNA. (F) Distribution of individual mRNAs on polysome profiles. Data are representative of three biological replicates. (G) Western blot analysis and quantification for indicated proteins in sucrose gradient fractions. Data are representative and quantification is inclusive of three independent experiments. *p < 0.01

Figure 4.. Pausing at translation initiation codon is stress duration-dependent and reversed by the return to isoosmolar media

MEFs treated with hyperosmolar media for the indicated times were analyzed for (A) cumulative distribution (y-axis) as a function of the fold change of ribo^ocp values (x-axis) for mRNAs that were either paused (red) or non-paused (cyan) after 2 h of 700 mOsm stress. Ribo^ocp values were calculated as ribosome footprints normalized to corresponding mRNA levels. (B) Relative changes of the ribo^ocp values of neMito mRNAs. (C) Scatter plot comparing fold changes of ribosomal footprints (y-axis) and cytoplasmic RNA levels (x-axis). Comparisons were made against values from 700 mOsm stress (2 h). (D) Protein synthesis levels. Means ± SEM for triplicate determinations are shown. *p < 0.05. (E) Heat map depicting ribosome occupancies on different codons (y-axis) based on their relative location on mRNAs (x-axis).

Figure 5.. Re-shaping of the translatome/transcriptome in recovery from severe stress is determined by stress duration

MEFs treated for the indicated times were analyzed by (A) scatter plot comparing fold changes of ribosomal footprints (y-axis) and cytoplasmic RNA levels (x-axis) compared to control (isoosmotic) conditions. (B) KEGG enrichment pathway analysis. (C) Representative confocal microscopy images of mitochondria (TOM20, green) and nuclei (Hoechst, blue). Treatment for 2 h with CCCP served as a positive control for mitochondrial fragmentation. Scale bar: 10 μm. (D) Quantification of cells with fragmented mitochondria. (E) Mitochondrial membrane potential. Means ± SEM of triplicate determinations are shown. *p < 0.01

Figure 6.. ISR is a hallmark of the recovery from adaptive pausing in response to severe stress

MEFs treated with the indicated stress conditions were analyzed by (A) protein synthesis assays, (B) Western blotting, (C) eIF2B activity assays, (D) polysome profiles, (E) cell survival assays, (F) mitochondria fragmentation assays, and (G) flow cytometry of propidium iodide-stained cells in the presence or absence of GADD34/PP1 inhibitor sephin1. Percentage of cells in each cell cycle phase are indicated. (H) Temporal cellular responses to severe hyperosmotic stress. The diagonal arrow indicates the development of APR (graded red color) as cells transition through different states (represented by quadrants). The processes that associate with APR development are listed at the right. Means ± SEM for triplicate determinations are shown. *p < 0.01

Figure 7.. Restored mitochondria morphology during recovery from severe stress requires reversal of mRNA translation inhibition caused by the signaling of eIF2α phosphorylation

(A) Representative confocal microscopy images of mitochondria (TOM20, green) and nuclei (Hoechst, blue) in MEFs treated as indicated. Scale bar: 10 μm. (B) Quantification of cells with fragmented mitochondria after indicated treatments. Means ± SEM of triplicate determinations are shown. *p < 0.01. Protein synthesis was inhibited during recovery from 700 mOsm stress via the use of chemicals inhibiting translation initiation (Hippuristanol and Harringtonine) or eIF2B activity (Salubrinal). (C) Western blot analysis of cells treated with 700 mOsm for 2 h and allowed to recover in isoosmolar media with or without Torin 1.