Study of ribosome dynamics after eIF5A depletion in budding yeast

eIF5A is an essential translation elongation factor present in all eukaryotes, and the only known protein to follow a post-translational modification called hypusination. Here, we performed a wide analysis of ribosome dynamics in S. cerevisiae eIF5A depleted cells using 5Pseq (Pelechano et al. 2015 PMID 26046441). This method allows the study of ribosome dynamics, by sequencing 5’ phosphorylated mRNA co-translational degradation intermediates. Since eIF5A is an essential protein in yeast, we used two eIF5A temperature-sensitive strains containing a single Pro83 to Ser mutation (tif51A-1) and double Cys39 to Tyr and Gly118 to Asp mutations (tif51A-3) in the highly expressed gene TIF51A (HYP2) that encodes eIF5A protein (Li et al. 2011 PMID: 24923804). For our 5PSeq experiments we incubated the wild-type and the two eIF5A mutant cells at 37°C for 4 hours, conditions in which there is almost no drop in cell viability for both mutants but it can be observed an important reduction in eIF5A protein. We performed three independent 5PSeq experiments with each of the strains tif51A-1 (hyp2-1) and tif51A-3 (hyp2-3) and compared with two independent experiments with the isogenic wild-type BY4741 strain.