Study of ribosome dynamics of rpl3[W255C] and its interactions with ribosome-associated chaperones

Proteostasis is a fundamental network of cellular pathways that ensures the optimal concentration and composition of correctly folded proteins within cells in normal and stress conditions. Among key components of this network are the molecular chaperones, which mediate protein folding but also act as modulators of protein synthesis. We have reported on a functional link between translation and de novo folding of proteins in the yeast Saccharomyces cerevisiae by uncovering a specific synthetic-lethal interaction between apparent unrelated mutant variants, the uL3[W255C] variant of the ribosomal protein uL3 and the null mutants of Zuo1 and Ssz1. Zuo1 and Ssz1 are components of the chaperone system named as ribosome-associated complex. Here, we performed a genome-wide analysis of ribosome dynamics by 5PSeq (Pelechano et al. 2015 PMID 26046441) in strains harbouring either wild-type uL3 or mutant uL3[W255C] in the presence or absence of Zuo1 or Ssz1. This method allows the study of ribosome dynamics, by sequencing 5’ phosphorylated mRNA co-translational degradation intermediates. Our results indicate that the rpl3[W255C] mutant is slightly impaired in translation elongation, defect that is significantly enhanced when combined with the deprivation of either Zuo1 or Ssz1.蛋白质稳态（Proteostasis）是一类核心细胞通路网络，可保障细胞在正常及应激条件下，胞内正确折叠蛋白质的最优浓度与组成。该网络的关键组分包括分子伴侣（molecular chaperones），其不仅介导蛋白质折叠过程，还可作为蛋白质合成的调控因子。我们此前曾在酿酒酵母（Saccharomyces cerevisiae）中报道过翻译与蛋白质新生折叠之间的功能关联：通过揭示看似无关的突变体变体间的特异性合成致死互作——即核糖体蛋白uL3的uL3[W255C]变体与Zuo1、Ssz1的敲除突变体之间的合成致死互作。Zuo1与Ssz1属于被称为核糖体相关复合物（ribosome-associated complex）的分子伴侣系统组分。本研究通过5PSeq（Pelechano等，2015，PMID 26046441），对携带野生型uL3或突变型uL3[W255C]的菌株，在存在或缺失Zuo1或Ssz1的条件下开展了全基因组核糖体动态分析。该方法通过对共翻译降解的5'磷酸化mRNA中间产物进行测序，实现核糖体动态特征的研究。我们的结果显示，rpl3[W255C]突变体的翻译延伸过程存在轻度损伤，且当该突变与Zuo1或Ssz1缺失相结合时，该缺陷会显著加剧。