Eukaryotic translation

Gene expression patterns change dramatically depending on cell fate, metabolic status, tissue and age. The specific subsets of proteins expressed in each state allow cells to carry out the functions needed during either proliferation or differentiation, and to respond to environmental changes and stress conditions. 

Gene expression is not only regulated at the transcriptional level, but also at the post-transcriptional level via processes such as mRNA translation and mRNA turnover. Our team develops basic research on the translational controls that influence the regulation of gene expression and cell fate. Projects range from studies on the role of translation termination factors in the translational control of mRNAs carrying regulatory short open reading frames (uORF) in their 5' non-coding region (5'UTR) to studies on cytoplasmic mRNA turnover. More recently, we have become interested in how eukaryotic cells repair or eliminate RNA damages induced by genotoxic stresses.

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During the past years, our team has studied translation termination in eukaryotes using mammals as model organisms. We are now concerned by the potential role of translation termination factors in the control of cell growth and proliferation.

In eukaryotes, two release factors, eRF1 and eRF3, are required to complete protein synthesis. These factors associate in a complex which binds to the ribosome when a stop codon enters the ribosomal A site. eRF1 recognizes all three stop codons (UAA, UAG and UGA) at the decoding site and activates the peptidyl-transferase centre which triggers the hydrolysis of the peptidyl-tRNA and the release of the nascent polypeptide. eRF3, which belongs to the small GTPase family, stimulates eRF1 release activity in a GTP-dependent manner. In addition to its role in translation, several findings in yeast and mammals have related eRF3 to cell cycle regulation, proliferation and apoptosis. 

Highlights

Because of these observations, we have developed a project on the question of how eRF3 is involved in cell cycle regulation. RNA interference experiments targeting eRF3 mRNA showed that eRF3 depletion induces a cell cycle arrest at G1 phase correlated with a decrease in the global rate of translation and a hypophosphorylation of initiation factor 4E-binding protein (4E-BP) and ribosomal protein S6 kinase (S6K), two direct targets of the protein kinase mTOR. These results suggested that eRF3 depletion inhibits cell cycle progression through the inhibition of the mTOR signalling pathway which is a major effector of cell growth and proliferation via the regulation of protein synthesis (Chauvin et al., MCB 2007).

Further large scale analysis of mRNA profiles in eRF3a-depleted cells revealed an increased level of mRNAs expressed from promoters controlled by the activating transcription factor 4 (ATF4). We also showed that eRF3a depletion modifies ATF4 translational control at upstream regulatory ORFs (uORFs), thus increasing ATF4 ORF translation and confirmed the increase in ATF4 activity. Finally, we demonstrated that the increase in REDD1 expression, one of the upregulated targets of ATF4, is responsible for the inhibition of the mTOR pathway in eRF3a depleted cells.

Our results shed light on the molecular mechanisms connecting eRF3a depletion to mTOR pathway inhibition and give the first example of ATF4 activation that bypasses the signal transduction cascade leading to the phosphorylation of translation initiation factor eIF2 (Ait Ghezala et al., NAR 2012).

Future directions

  • Translation termination and mRNA stability

To extend our previous work on translation termination factors, we now aim to characterize the relationship between translation termination and the mRNA deadenylation process using both in vitro and cell culture systems.

  • Translation termination, uORF carrying mRNAs and cell fate

Following our work on ATF4 uORFs, we became interested in the translational regulation of mRNAs carrying uORFs. Currently, we are studying the involvement of these controls in the reprogramming of gene expression that governs cell fate.

  • Genotoxic stress and RNA damages

In their natural environments, cells do not undergo continuous cycle of replication, but instead are engaged in gene expression programs required to maintain their differentiated state. All along this non-replicative state, genotoxic stresses not only induce DNA lesions that produce mutant transcripts via a process known as transcriptional mutagenesis, but also damage RNA molecules potentially causing translation impairments. We thus would like to identify the essential functions that could counteract the toxic effect of RNA damages and their potential effect on cell fate and ageing.

Collaborations

  • Olivier Namy, Institut de Génétique et Microbiologie, UMR8621 CNRS-Université Paris-Sud Orsay
  • Djemel Hamdane, Chimie des Processus Biologiques, CNRS UMR 8229 Collège de France, Paris