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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE294217 | GEO | 2026/07/15

REPOSITORIES: GEO

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Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling.The study includes 32 samples, comprised of 16 mRNA-Seq samples and 16 ribosome footprint profiling samples, derived from biological replicates of 3 mutant strains, ded1-cs, ded1-ts and tif1-ts, and the corresponding wild-type strains. The tif1-ts mutant and its wild-type counterpart were analyzed at 30°C and 37°C.

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

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