Analysis of the role of RPS26 during translation in HEK293T RPS26 p.100_M115 del cells by 48S footprinting (TCP-seq)

The ribosome mRNA channel is central for translation, but its role in specific regulatory mechanisms remains unexplored. Using Cryo-EM ribosomal complexes bound to Kozak and TISU mRNAs from WT and RPS26dC mutant cells, we demonstrate that RPS26 adopts distinct conformations, providing the basis for its opposing effects on their activity. Translatome studies of the RPS26dC mutant revealed sequence-dependent rearrangements of the 48S and 80S and slower scanning. Downregulated mRNAs are enriched with AUG upstream nucleotides and a -1C that contacts 18S G1207, a contact that is lost in the mutant structure. The top downregulated mRNAs are replication-dependent histones. Despite very short 5’UTRs and suboptimal Kozak, histones 5’UTRs exhibit strong translation activity that is RPS26-dependent. We identified a translational enhancer in H2B 5’UTR spanning nucleotide -16 to -9, matching RPS26 binding sites. Intriguingly, the H2B-ribosome complex adopts a different conformation compared to Kozak and TISU. Exploiting these features, we designed a translational cassette with high efficiency and minimal leaky scanning, improving efficacy and safety for mRNA therapy. These findings underscore the importance of the ribosome mRNA binding channel for selective regulation and therapeutic applications.