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).eIF5A（真核翻译延伸因子5A）是存在于所有真核生物中的必需翻译延伸因子，也是目前已知唯一一种需经过hypusination（羟腐胺修饰）这一翻译后修饰的蛋白质。本研究采用5Pseq技术（Pelechano等，2015，PMID 26046441），对酿酒酵母（S. cerevisiae）eIF5A敲低细胞的核糖体动态进行了全面分析。该技术通过对共翻译降解过程中产生的5’磷酸化mRNA中间体进行测序，实现对核糖体动态的研究。由于eIF5A是酵母的必需蛋白，我们使用了两株eIF5A温度敏感型菌株，其编码eIF5A蛋白的高表达基因TIF51A（HYP2）中分别携带两类突变：一株带有单个脯氨酸83位→丝氨酸突变（tif51A-1），另一株带有半胱氨酸39位→酪氨酸与甘氨酸118位→天冬氨酸的双突变（tif51A-3）（Li等，2011，PMID: 24923804）。