Structural and molecular insights into specialized translation mediated by the ribosome mRNA-binding channel [RNA-Seq]

The ribosome mRNA channel is central for translation, but its involvement in specific regulatory mechanisms remains unexplored. Employing Cryo-EM on ribosomal complexes bound to Kozak and TISU mRNAs from wild-type and RPS26 mutant cells (RPS26dC), we demonstrate that RPS26/eS26 adopts distinct conformations, providing a structural basis for its opposing effect on their activity. Translatome studies of wild-type and RPS26dC revealed AUG-context-dependent rearrangements in key intermediates of the 48S and full 80S initiation complexes and slower scanning. Downregulated mRNAs are enriched with AUG upstream nucleotides and a C at position -1 that contacts 18S rRNA nucleotide G1207, a contact lost in the mutant structure. The top downregulated mRNAs are replication-dependent histones, which, despite having short 5'UTRs and suboptimal Kozak, exhibit robust translation activity that is RPS26/eS26-dependent. We identified a translational enhancer in H2B 5'UTR, spanning nucleotide -16 to -9, overlapping the predicted RPS26/eS26-binding sites. Intriguingly, the H2B mRNA-ribosome complex structure adopts a conformation distinct from the Kozak and TISU. Exploiting these features, we designed a high-efficiency translational cassette with minimal leaky scanning, improving efficacy and safety for mRNA-based therapy. These findings underscore the central importance of the ribosome's mRNA-binding channel in selective translation regulation and its potential for therapeutic applications. Overall design: Ribo-seq profling of WT human embryonic kidney cells (HEK293T) and RPS26dC HEK293T cells