Ribosome profiling unveils translational regulation of metabolic enzymes in primary CD4⁺ Th1 cells

Highlights

• Translation controls the metabolism of human CD4⁺ Th1 cells.

• Translation regulates glucose, fatty acids and pentose phosphates pathways.

• Translational control discriminates between reversible and irreversible reactions.

• Translational targets are mRNAs with structured 5′ untranslated regions.

Abstract

The transition from a naïve to an effector T cell is an essential event that requires metabolic reprogramming. We have recently demonstrated that the rapid metabolic changes that occur following stimulation of naïve T cells require the translation of preexisting mRNAs. Here, we provide evidence that translation regulates the metabolic asset of effector T cells. By performing ribosome profiling in human CD4⁺ Th1 cells, we show that the metabolism of glucose, fatty acids and pentose phosphates is regulated at the translational level. In Th1 cells, each pathway has at least one enzyme regulated at the translational level and selected enzymes have high translational efficiencies. mRNA expression does not predict protein expression. For instance, PKM2 mRNA is equally present in naïve T and Th1 cells, but the protein is abundant only in Th1. 5′-untranslated regions (UTRs) may partly account for this regulation. Overall we suggest that immunometabolism is controlled by translation.

Introduction

Protein synthesis is the decoding of mRNAs into proteins, alias translation. Translational control leads to the selection, starting from the global mRNA pool, of those transcripts requiring to be decoded into proteins (Truitt and Ruggero, 2016). The impact of translation on gene expression has been estimated to be at least as important as transcription (Schwanhausser et al., 2012), meaning that different mRNAs are differentially translated. Translation is divided into four phases: initiation, elongation, termination and recycling. Initiation is the rate-limiting event in the selection of a specific mRNA. At initiation, eIFs (eukaryotic Initiation Factors) perform mechanistic steps under the coordination of signaling pathways (Loreni et al., 2014) and interplay with regulatory elements in the untranslated regions (UTRs) of mRNAs, thus resulting in specific stimulation or inhibition of translation (Hinnebusch et al., 2016). One major pathway controlling initiation of translation is the mTORC1 pathway. mTORC1 stimulates the activity of the eIF4F complex which is necessary for efficient translation of mRNAs containing structured 5’ UTRs (Loreni et al., 2014). The relevance of mTORC1 in lymphocyte biology is well established (Zeng and Chi, 2017).

CD4⁺ T cells are essential for survival (Jung and Paauw, 1998). The transition from naïve T cells to effector T cells requires a complete metabolic rewiring that includes the activation of a glycolytic and fatty acid synthesis program, essential steps for nucleotide biosynthesis and, hence, for cellular growth. It has been traditionally thought that metabolic reprogramming correlates with transcriptional rewiring (Hough et al., 2015). Given the central role of mTORC1 in translation (Loreni et al., 2014), lipid synthesis (Lamming and Sabatini, 2013) and T cell differentiation (Zeng and Chi, 2017), we recently asked whether translational control plays a major role in T cell activation. We found that eukaryotic Initiation Factor 6 (eIF6) is fundamental for the acquisition of effector functions by CD4⁺ T cells (Manfrini et al., 2017a, 2017b) and that the transition from quiescence to the activated status requires translational control of the lipid biosynthetic pathway (Ricciardi et al., 2018). Overall, these data confirm the hypothesis that translational control is a major player in T cell activation (Piccirillo et al., 2014) and metabolism (Biffo et al., 2018; Brina et al., 2015; Miluzio et al., 2016). Whether translational control of metabolism affects also other stages of T cell differentiation is still unknown.

Ribosome profiling is a technology that allows the direct analysis of ribosomal footprints on cellular mRNAs, thus providing a detailed snapshot of translational control (Ingolia et al., 2011). Herein, by using this technique, we investigated the impact of translational regulation on gene expression in CD4⁺ Th1 cells. Although ribosome profiling has been recently performed in primary mouse T cells (Moore et al., 2018; Myers et al., 2019), our study stands as the first-ever ribosome profiling performed in primary human T cells. Intriguingly, we found that the metabolism of T cells is controlled also at the translational level, confirming that transcriptional studies largely fail in predicting the presence of metabolic enzymes.