Selective Translation Complex Profiling in yeast and human Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes [Yeast]

Translational control targeting mainly the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast Translational Complex Profile sequencing (TCP-seq), related to ribosome profiling, and adopted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ses) in addition to 80S ribosomes (80Ses), revealed that mammalian and yeast 40Ses distribute similarly across 5’UTRs indicating considerable evolutionary conservation. We further developed a variation called Selective TCP-seq (Sel-TCP-seq) enabling selection for 40Ses and 80Ses associated with an immuno-targeted factor in yeast and human. Sel- TCP-seq demonstrated that eIF2 and eIF3 travel along 5’UTRs with scanning 40Ses to successively dissociate upon start codon recognition. Manifesting the Sel-TCP-seq versatility for gene expression studies, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes. 3 sets; set1: 3 replicates for 40S and 80S TCP-seq, 1 replicate for each eIF3a, eIF3c and eIF2beta bound 40S Sel-TCP-seq and 1 replicate each for eIF3a and eIF3c bound 80S Sel-TCP-seq (1-1, 1-2, 1-3); set2+3: 2 more replicates (in addition to set1) of 80S TCP-seq and eIF3a bound 80S Sel-TCP-seq (2-1, 3-1)

翻译调控（Translational control）主要靶向翻译起始阶段，是基因表达（gene expression）调控的核心环节。全面解析该调控过程的所有维度，亟需实现实质性的技术突破。
本研究对与核糖体谱（ribosome profiling）相关的酵母翻译复合物谱测序（Translational Complex Profile sequencing, TCP-seq）技术进行优化改造，并将其适配至哺乳动物细胞体系。人类TCP-seq除可捕获80S核糖体（80S ribosomes, 80Ses）外，还能捕捉40S亚基（40S subunits, 40Ses）的保护足迹。通过该技术，研究团队发现哺乳动物与酵母的40S亚基在5'非翻译区（5’UTRs）的分布模式高度相似，表明二者存在显著的进化保守性（evolutionary conservation）。
本研究进一步开发了一种技术变体，命名为选择性TCP-seq（Selective TCP-seq, Sel-TCP-seq），可在酵母与人类细胞中富集与免疫靶向因子结合的40S亚基与80S核糖体。借助Sel-TCP-seq技术，研究证实真核起始因子2（eIF2）与真核起始因子3（eIF3）可与正在扫描信使RNA（mRNA）的40S亚基一同沿5'非翻译区移动，并在识别起始密码子（start codon）后逐步解离。
为展现Sel-TCP-seq在基因表达研究中的多功能性，本研究还鉴定出四种处于不同构象状态的48S起始中间体，为ATF4与GCN4信使RNA（mRNA）的翻译调控提供了全新见解，并证实了起始因子复合物（initiation factor complexes）的共翻译组装（co-translational assembly）过程。
本研究共包含3组测序数据集：组1包含40S与80S TCP-seq的3次生物学重复，以及分别结合eIF3a、eIF3c与eIF2β的40S亚基Sel-TCP-seq各1次重复，还有分别结合eIF3a与eIF3c的80S核糖体Sel-TCP-seq各1次重复（样本编号依次为1-1、1-2、1-3）；组2与组3在组1的基础上，新增了80S TCP-seq与结合eIF3a的80S核糖体Sel-TCP-seq的2次额外生物学重复（样本编号分别为2-1与3-1）。