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. Overall design: 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)