Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus

Abstract

Protein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, and not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4F(H). O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, given that they occur during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus.

Keywords: HIF; RNA sequencing; SILAC; cancer; eIF4E; eIF4E2; eIF4F; hypoxia; oxygen; translation.

Figure 1. O₂-dependent remodeling of the cellular translatome

(A) RNA-Seq was performed on MO and P fractions of normoxic and hypoxic U87MG. MO/P demarcation was selected based on the induction pattern of hypoxic translation of several mRNAs (Uniacke et al., 2012; Uniacke et al., 2014). (B) Plots of R_ss in hypoxic (R_ss^H) versus normoxic (R_ss^N) (left panel) U87MG (left panel) and transcripts with R_ss^H/R_ss^N (R_ss^H/N) ratios between <2x and >0.5x (right panel, black). Transcripts with R_ss <1 were excluded from analysis. (C) Schematic of pSILAC workflow. (D) Normalized heavy intensities of newly synthesized proteins determined by pSILAC in hypoxic (^pS^H) and normoxic (^pS^N) U87MG (left panel) and from transcripts with R_ss^H/N ratios between <2x and >0.5x (right panel). Proteins that were only detected in normoxic (red) and hypoxic (blue) U87MG were given the maximum fold change observed. Canonical hypoxia-inducible genes are highlighted in green. (E) Plots of T_e in hypoxic (T_e^H) versus normoxic (T_e^N) U87MG (left panel), and transcripts with R_ss^H/N ratios between <2x and >0.5x (right panel). (F) Concordance analysis between R_ss^H/N, ^pS^H/N and T_e^H/N.

Figure 2. The eIF4F and eIF4F^H translation machineries govern O₂-dependent translatome remodeling

(A) Global translation rates of U87MG transiently transfected with siRNA against the indicated proteins and non-silencing (NS) control siRNA were measured by puromycin incorporation. Loading was performed on an equal cell basis. %puro inc., percent puromycin incorporation. Immunoblots of silenced proteins are shown. β-actin was used as a loading control. (B) Schematic of eIF4F^H and eIF4F. (C) Immunoblots of eIF4E, eIF4E2, and HIF-2α endogenous IPs in normoxic and hypoxic U87MG. WCL, 5% whole cell lysate. (D) Polysome profiles and (E) pSILAC analysis of hypoxic U87MG stably expressing eIF4E2-specific or non-silencing (NS) control shRNA.

Figure 3. Global reorganization of translation efficiency during O₂ deprivation

(A) Classification of all transcripts and (B) transcripts with R_ss^H/N ratios between <2x and >0.5x into three major classes according to T_e^H/T_e^N ratios. Class I transcripts (T_e^H/T_e^N ≤0.5-fold, red); Class II transcripts (purple); Class III transcripts (T_e^H/T_e^N ≥1-fold, blue). Low abundance transcripts (R_ss <10) were excluded from the analysis. (C) Immunoblots of representative proteins in normoxic and hypoxic U87MG from each class. (D) Global translation rates in normoxic and hypoxic 786-O transiently transfected with eIF4E-specific, eIF4E2-specific, or NS control siRNA were measured using puromycin incorporation. Immunoblots of silenced proteins are shown. (E) Immunoblots of representative proteins in normoxic and hypoxic 786-O as measured in (C).

Figure 4. Translation efficiency determines protein output in response to hypoxia

(A) RNA-Seq analysis identified 1079 different proteins derived from Class III mRNAs with R_ss^H/N ratios between <2x and >0.5x (blue), and 50 proteins derived from HIF target mRNAs with R_ss^H/N ratios ≥2 (green) (see Figure 1B). (B) Plot of change in R_ss against change in T_e for 50 HIF target mRNAs in hypoxic versus normoxic U87MG (see Figure 1B). Blue dots; representative Class III candidates with minimal change in R_ss levels. (C) Immunoblots of HIF target proteins in U87MG transiently transfected with HIF-1β-specific or NS siRNA, and subjected to a hypoxic time course. (D) Corresponding 24 hr hypoxia/normoxia steady-state mRNA levels of proteins measured in (C). * denotes statistical significance (p<0.05) compared to 0 hr hypoxia. (E) Immunoblots of hypoxia-inducible proteins in U87MG treated with Act. D or DMSO for 20 min, followed by 6 hr of hypoxic or normoxic treatment. (F) Corresponding hypoxia/normoxia steady-state mRNA levels of proteins measured in (E). * denotes p<0.05 compared to the corresponding normoxia control. (G) Plot of change in mRNA levels against change in T_e for 5 representative HIF target mRNAs in transcriptionally silent versus active U87MG under hypoxic versus normoxic conditions. (H) Immunoblots of HIF target proteins in U87MG stably expressing shRNA targeting eIF4E2 (inactive eIF4F^H) or NS shRNA (active eIF4F^H), and treated with Act. D as in (E). (I) Corresponding hypoxic induction of steady-state mRNA levels of proteins measured in (H). * denotes p≤0.05 compared to the corresponding normoxia control. (J) Immunoblots of HIF target proteins in normoxic and hypoxic 786-O transiently transfected with eIF4E-specific, eIF4E2-specific, or NS siRNA. (K) Global translation rates in normoxic and hypoxic U87MG transiently transfected with eIF4E2-specific or NS siRNA, and treated with Act. D as in (E), were measured using puromycin incorporation. Immunoblots of silenced proteins are shown.