Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

Abstract

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-TCPseq 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.

Unraveling the influences of sequence and position on yeast uORF activity using massively parallel reporter systems and machine learning

“…Our work also provides insight into how uORF features impact their ability to induce NMD. Reinitiation after uORF translation can protect human mRNA from NMD (Lindeboom et al, 2016;Zhang and Maquat, 1997), and short uORFs are known to reinitiate more efficiently than long ones (Gunišová et al, 2017(Gunišová et al, , 2016 likely due to increased retention of initiation factors (Bohlen et al, 2020;Wagner et al, 2020). Consistent with this, we found short uORFs induce less NMD than long uORFs.…”

Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity

“…In line with a recent report that the human eIF3 complex remains attached to 80S ribosomes in early elongation (Wagner et al 2020), we observed the bulk of read clusters comprising EIF-3.G(WT) and EIF-3.G(C130Y) footprints mapping between (-)150 to (+)200 nucleotides of the start codon ( Fig. 4D).…”

Phosphorylation of Ribosomal Protein S6 differentially affects mRNA translation based on ORF length

“…We used selective ribosome footprinting (Bohlen et al, 2020, Oh, Becker et al, 2011, Wagner et al, 2020) to determine the position on endogenous mRNAs of ribosomes containing phosphorylated Ribosomal Protein S6 (RPS6) (Figure 1A). Immunoprecipitation (IP) of ribosomes with phospho-RPS6 antibody was strongly reduced when RPS6 phosphorylation was inhibited with Torin1 (Figure 1B), indicating that the IP is specific, and that in these cells RPS6 is mainly phosphorylated by S6K downstream of mTOR.…”

“…Selective ribosome footprinting is a method that identifies the location of specific ribosome sub-populations on endogenous mRNAs in human cells (Bohlen, Fenzl et al, 2020, Schibich, Gloge et al, 2016, Wagner, Herrmannova et al, 2020). This method employs immunoprecipitation to isolate ribosomes bound to specific factors such as initiation factors or chaperones, followed by sequencing of their footprints to identify their locations.…”