The control of mRNA translation into proteins is critical for the adaptation of eukaryotic cells to environmental changes and stress conditions. Nutrient stress responses in yeast (Saccharomyces cerevisiae) are prototypical to eukaryotes and are known to cause large-scale translational reprogramming, downregulating translation of most messenger (m)RNAs while upregulating translation of others. Glucose starvation is one of the better studied nutrient stresses in yeast as it causes rapid changes, mainly during the initiation phase of translation. While this effect has been described before, methods to obtain insights into particular initiation intermediates, and the dynamics of the response, were limiting. To understand how translational control operates, we aim to provide detailed data for the response to glucose starvation, a common stress condition where dysregulation can be critical in multiple diseases including cancer.
Glucose starvation induces rapid loss of the translation initiation factor eIF4A from initiation complexes, followed by relocation of these stalled initiation intermediates into cytoplasmic bodies. However, the mRNA-wide loss of eIF4A, its dynamics upon the induction of the stress, and its specific effects on translation across the transcriptome remain unknown.
To address these problems, we refined TCP-seq, which is based on ribosome footprinting (Archer SK et al. Nature 2016 535:570-574; Shirokikh NE et al. Nat. Protoc. 2017 12:697-731). TCP-seq captures all ribosome-mRNA complexes, including initiation intermediates. In glucose starvation/restoration time-course experiments, we found that the translation response time, measured by changes in polysome profiles, was much shorter than anticipated, approaching 20 seconds. We selected three conditions that resulted in minimal, intermediate or maximal polysome disassembly, to obtain samples for TCP-seq. Further, we aim to use yeast strains harbouring tagged versions of eIF4A, eIF4E, eIF4G and Pab1 for selective footprinting of complexes involving these factors and uncover their mRNA-specific distribution and its dynamics across the transcriptome during glucose starvation.