Figure 3. Stress-induced remodeling of translatomes.

Identification of translation classes of mRNA. A) Analysis of the translation change after stress using the classical metabolic labeling of newly-made proteins with [³⁵S]-Met. NIH3T3 cells were treated with thapsigargin for the indicated times, labeled for 30′ and proteins were analyzed by SDS-PAGE and autoradiography. Note that [³⁵S]-Met incorporation was inhibited by 90% at 1h and by 70% at 3 h after stress treatment. The phosphorylation state of eIF2α was analyzed by western-blot in parallel (lower panel). B) The synthesis of proteins that account for most of cellular translation was preferentially inhibited after stress. Based on 2D PAGE analysis and mass spectroscopy (MS) data extracted from the literature, we built a list with the 46 most abundant proteins found in NIH3T3 and Jurkat cells (see Table S1). The mean ±SD of log₂P/FM values for this mRNA subset under control and stress conditions are shown and compared with values obtained for all mRNA in both cell types. C) Plots showing the change in translation efficiencies (log₂P/FM stress-log₂P/FM control) after thapsigargin treatment (3 h for NIH3T3, 1h and 3 h for Jurkat). In parentheses are the number of mRNA used in the analysis. Quadrants were set to identify the translation classes according to values in log₂P/FM change upon stress. The sensitive (S) class comprises mRNA whose translation decreased≥0.8 log₂ (40–50% of mRNAs in both NIH3T3 and Jurkat). A representative member of this group is the ACTB mRNA. Resistant (R) class includes those mRNA that continue to translate at moderate to high rates during stress. These mRNA show a log₂P/FM≥0.8 in both control and stressed cells, and comprises about 3–4% of total mRNA in NIH3T3 and up to 13% in Jurkat cells. A representative member of this group is the HSPA5 (the BiP chaperone) mRNA. Translation inducible class (I) comprises mRNA with low translation efficiencies under control conditions (log₂ P/FM≤0) that increased upon stress (log₂ change≥1). This group comprises about 8% of mRNA in NIH3T3 cells and 1.5% in Jurkat cells. A representative member of this group is the transcription factor ATF4.