Nutrient deprivation elicits a transcriptional and translational inflammatory response coupled to decreased protein synthesis. Nutrient deprivation elicits a transcriptional and translational inflammatory response coupled to decreased protein synthesis

Purpose: The goal of this study is to examine gene expression regulation at the transcriptional and translational levels in response to various forms of nutrient deprivation, and whether there are differences between isogenic transformed and non-transformed cells. Mehods: We apply high-throughput sequencing of ribosome footprints (ribosome profiling) and poly(A) RNA (RNA sequencing) in an isogenic pair of transformed (tamoxifen-treated) and non-transformed (ethanol control) MCF10A-ER-Src cells subjected to the metabolic stresses that differentially affect global protein synthesis (Figure 1): deprivation of glutamine (for 30min and 4 hours), glucose (4hours), cysteine/cystine (4hours) or leucine/isoleucine/valine (brach-chain aminoacids - BCAA) (4hours). The same experiments were also performed in transformed ER-Src MCF10A treated with torin1 (500nM) for 4 hours. Results: Genome-wide translational profiling of glutamine deprived ER-Src MCF10A cells (for 30 minutes) shows increased translation of uORFs-containing mRNAs and down-regulation of ribosomal protein mRNAs, which is followed by increased translation and transcription of cytokine and inflammatory mRNAs (after 4 hours of glutamine deprivation). The transcription and translation of inflammatory and cytokine mRNAs is also stimulated in response to 4 hours deprivation of glucose, cysteine/cystine and BCAA, with the extent of stimulation correlating with the i) decrease in global protein synthesis and ii) down-regulation of all translationally-repressed mRNAs or ribosomal protein mRNAs. Conclusions: Pro-inflammatory gene expression is associated with translational repression in response to short-term nutrient deprivation. Overall design: Ribosome occupancy and mRNAs levels in transformed and non-transformed cells cultured for i) 30 minutes under complete (Ctrl) or glutamine-free (NoQ) DMEM medium, and ii) 4 hours under complete (Ctrl), glucose-free (NoG), glutamine-free (NoQ), cysteine/cystine-free (NoCys) or leucine, isoleucine, valine (NoBCAA)-free DMEM medium. The translation efficiency (TE) was calculated as the log2 ratio of RPKM measured by ribosome profiling over RPKM measured by RNA-seq. Differences in ribosome occupancy (RO) and TE between the metabolic stress condition and the reference sample (either TAM or EtOH cells grown in complete DMEM) were used to examine the transcriptional and translational changes, respectively. The ribosome profiling and RNA sequencing experiments were performed in duplicate with the exception of RNA-seq at 30 minutes (single experiments), ribosome profiling in cysteine/cystine-deprived EtOH cells (triplicate experiments), and the Torin1 ribosome profiling and RNA-seq (single experiment). The complete dataset comprises the raw (.fastq) and processed (RPKM) files of all 57 high-throughput sequenced samples.