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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE301926 | GEO | 2025/07/08

REPOSITORIES: GEO

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

Project description: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.

2025-07-08 | GSE301928 | GEO

Translation regulation of specific mRNAs by RPS26 C-terminal RNA-binding tail integrates energy metabolism and AMPK-mTOR signaling

Project description:Increasing evidence suggests that ribosome composition and modifications contribute to translation control. Much less is known about the regulatory roles of mRNA binding by ribosomal proteins to specific mRNA translation and ribosome specialization. Here we used CRISPR-Cas9, to mutate the RPS26 C-terminal tail (RPS26dC) predicted to bind AUG upstream nucleotides at the exit channel. RPS26 binding to positions -10 to -16 of short 5’UTR mRNAs exerts positive and negative effects on translation directed by Kozak and TISU, respectively. Consistent with that, shortening the 5′UTR from 16 to 10 nt diminished Kozak and enhanced TISU-driven translation. As TISU is resistant and Kozak is sensitive to energy stress, we examined stress responses and found that the RPS26dC mutation confers resistance to glucose starvation and mTOR inhibition. Furthermore, the basal mTOR activity is reduced while AMPK is activated in RPS26dC cells, mirroring energy-deprived WT cells. Likewise, the translatome of RPS26dC cells is correlated to glucose-starved WT cells. Our findings uncover the central roles of RPS26 C-terminal RNA binding in energy metabolism, in the translation of mRNAs bearing specific features and in the translation tolerance of TISU genes to energy stress.

2023-04-12 | GSE217180 | GEO

Study of the eIFG1-eIF1 interaction by TCP-seq on translation initiation in HEK 293T cells

Project description:Start codon recognition by the 48S complex is a critical step in translation. However, understanding the in vivo initiation and its regulation at a global scale is limited. Here, we analyzed translation complex profiling (TCP-seq) data to determine the impact of eIF4G1-eIF1 inhibition and the 48S organization. Our analysis provides the first global view of leaky scanning and reveal the central roles of mRNA features and eIF4G1-eIF1 in its regulation. Specifically, non-leaky genes are enriched with a Kozak bearing a C at -1 position while those with short 5’ UTR with TISU. Additionally, the stability of the 48S complex and its integrity during scanning are impaired upon eIF4G1-eIF1 inhibition. Detailed analysis of initiation site footprints revealed three main classes conserved from yeast to human. Our analysis provides a general overview of AUG selection and evidence for conformational rearrangements in vivo.

2024-03-27 | GSE226132 | GEO

Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements

Project description:The “Kozak sequence”, immediately upstream of the start codon, regulates the translational efficiency of an mRNA. Nevertheless, how this sequence is recognized remains unexplored. Here, we describe a novel approach to separate two ribosome populations from the same cells and use this method, and RNA-seq, to identify the mRNAs bound to ribosomes with and without Rps26, a protein linked to the pathogenesis of Diamond Blackfan Anemia (DBA). These analyses reveal that Rps26 contributes to recognition of the Kozak sequence in well-translated mRNAs, and that Rps26-deficient ribosomes preferentially translate mRNA from select stress response pathways. Surprisingly, we demonstrate that exposure of cells to these stresses leads to the formation of Rps26-deficient ribosomes and to the translation of their targets. Thus, Rps26-deficient ribosomes play aberrant pathogenic roles in DBA as well as physiological roles in augmenting the well-characterized stress transcriptional response with a heretofore unknown translational stress response, thereby creating a feed forward loop in gene-expression.

2017-07-30 | GSE86203 | GEO

Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors

Project description:Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation is regulated differently from canonical translation is poorly understood. We thus used start codon-selective reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of non-AUG reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA are resistant to cycloheximide (CHX), a translation inhibitor which slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes cause queuing of scanning pre-initiation complexes (PIC), preferentially enhancing otherwise poor recognition of non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. Moreover, PIC queuing can cause translation from an AUG codon in a poor context to become less sensitive to CHX. We further find that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence, but not those targeting newly initiated ribosomes. In total, these data indicate that ribosome queuing enables mRNAs with poor initiation context, namely those from non-AUG start codons, to be resistant to pharmacological inhibitors.

2019-06-05 | GSE125086 | GEO

Saccharomyces cerevisiae

Project description:Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements

| PRJNA340886 | ENA

eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide

Project description:The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at the NCCs initiating N-terminal extensions on GRS1 and ALA1 mRNAs, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.

2019-10-18 | GSE138599 | GEO

Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine

Project description:mRNA function is influenced by modifications that modulate canonical nucleobase behavior. We show that a single modification mediates distinct impacts on mRNA translation in a position-dependent manner. While cytidine acetylation (ac4C) within protein-coding sequences stimulates translation, ac4C within 5’UTRs impacts protein synthesis at the level of initiation. 5’UTR acetylation promotes initiation at upstream sequences, competitively inhibiting annotated start codons. Acetylation further directly impedes initiation at optimal AUG contexts: ac4C within AUG-flanking Kozak sequences reduced initiation in base-resolved transcriptome-wide HeLa results, and in vitro utilizing substrates with site-specific ac4C incorporation. Cryo-EM of mammalian 80S initiation complexes revealed ac4C in the -1-position adjacent to an AUG start codon disrupts an interaction between C and hypermodified t6A at nucleotide 37 of the initiator tRNA. These findings demonstrate the impact of RNA modifications on nucleobase function at a molecular level and introduce mRNA acetylation as a factor regulating translation in a location-specific manner.

2022-06-08 | GSE162043 | GEO

Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver [RNASeq]

Project description:Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. While rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in wild-type and Bmal1 deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic genes expression, Bmal1 deletion having surprisingly more impact at the post-transcriptional level. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5â-TOP sequences and for genes involved in mitochondrial activity and harboring a TISU motif. The increased translation efficiency of 5â-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion impacts also amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation. RNA-Seq from total RNA of mouse liver during the dirunal cycle. Time-series mRNA profiles of wild type (WT) and Bmal -/- mice under ad libitum and night restriced feeding regimen were generated by deep sequencing.

2015-11-10 | E-GEOD-73552 | biostudies-arrayexpress

Boric acidon intercepts 80S ribosome migration from AUG-stop by stable eRF1 immobilization

Project description:Upon the environmental changes, cells flexibly and rapidly alter the gene expression by the translation control. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is upregulated through the upstream open reading frame (uORF), which comprises only AUG and stop codons, in response to the shortage of boric acid. However, the molecular details of how the minimum uORF controls the translation of downstream main ORF depending on the boric acid concentration remained unclear. Here we showed that the 80S ribosome (80S) assembled at AUG-stop migrates into the subsequent RNA segment for downstream translation initiation and that the presence of boric acid impedes the process by stable confinement of eukaryotic release factor 1 (eRF1) on 80S on AUG-stop. Ribosome profiling along the in vitro-translated reporter mRNA revealed that the number of the ribosomes on AUG-stop and that along the downstream are anti-correlated in response to boric acid, reflecting the 80S scanning leaving AUG-stop for translation initiation from downstream ORF. Moreover, cryo-electron microscopy (cryo-EM) analysis revealed that, in the presence of boric acid, the initiator methionyl-tRNA (Met-tRNAi) in the P site stably contacts eRF1 in the A site on AUG-stop. In contrast, the absence of boric acid led to weaker eRF1 density, suggesting that eRF1 cannot be stably positioned without boric acid. Consistent with the structural features, the hydrolysis of Met-tRNAi on AUG-stop was accelerated by boric acid, which is likely to be the checkpoint for the subsequent 80S scanning of the downstream. Our results provide a molecular insight into the translation regulation by a minimum and environment-responsive uORF.

2023-09-20 | GSE189222 | GEO

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