Mitochondrial translation termination, recycling, and rescue in in-frame and out-of-frame contexts
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE288420 | GEO | 2026/06/05
REPOSITORIES: GEO
Similar Datasets
Project description:Mitochondrial translation system highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG having no cognate tRNAs in human mitochondria. Their locations at the end of COX1 and ND6 open reading frames, respectively, suggests they function as stop codons. However, while canonical stop codons, UAA and UAG, are recognized by mtRF1a in mitochondria, the release mechanism at AGA and AGG remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 production. Finally, we set up an in vitro reconstituted mitochondrial translation system, which confirms the specific release activity of mtRF1 on AGA and AGG codons. Together, our study uncovers the mechanism of translation termination in mitochondria and provides first insights into the consequences of its failure.
2022-11-16 | PXD034342 | Pride
Project description:The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial translation, however, exhibits deviations from the standard genetic code including reassigning of two arginine codons to stop codons. Translation termination at these non-canonical stop codons requires a protein factor to release the newly synthesized polypeptide chain. Currently it is not known how, and by which release factor, these stop codons are recognized. Here, we used biochemical experiments on knockout mutants in human cells in combination with cryo-electron microscopy to establish that the unusual mitochondrial release factor 1 (mtRF1) detects the non-canonical stop codons. We show that loss of this factor leads to stalling of mitochondrial ribosomes on non-canonical stop codons. As a result, reduced levels of cytochrome C oxidase subunit 1 of the oxidative phosphorylation complex IV are synthesized leading to a defect in mitochondrial respiration. We further show that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual distortion in the mRNA conformation and that the ribosomal RNA importantly participates in the specific recognition of the non-canonical stop codons.
2023-05-09 | GSE216779 | GEO
Project description:The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial translation deviates from the standard genetic code which includes the reassignment of two arginine codons into stop codons {Jukes, 1993 #438}. Translation termination at these non-canonical stop codons requires a protein factor to release the newly synthesized polypeptide chain, however, the identity of this factor is not known currently{Nadler, 2021 #406}. Here, we used gene editing and ribo-profiling in combination with cryo-electron microscopy to establish that the unusual mitochondrial release factor 1 (mtRF1) detects the non-canonical stop codons. We show that loss of mtRF1 leads to stalling of mitochondrial ribosomes on non-canonical stop codons and consequent reduced translation of cytochrome C oxidase subunit 1 that results in decreased mitochondrial respiration. We show that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual distortion in the mRNA conformation and that the ribosomal RNA importantly participates in the specific recognition of the non-canonical stop codons.
2023-03-21 | GSE216979 | GEO
Project description:Translation termination is an essential cellular process that is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins fom ribosomes, and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. SRI-41315 promotes retention of eRF1 on ribosomes and leads to a higher frequency of translation termination at near-cognate stop codons. In this study, the systematic effect of SRI-41315 on translation termination at cognate and near-cognate stop codons is evaluated using a rabbit reticulocyte lysate-based in vitro translation system with endogenous transcript as well as reporter transcripts containing defined near-cognate stop codon.
2023-12-31 | GSE248946 | GEO
Project description:Mitochondrial gene expression is essential for oxidative phosphorylation. Mitochondrial-encoded mRNAs are translated by dedicated mitochondrial ribosomes (mitoribosomes), whose regulation remains elusive. In the baker´s yeast Saccharomyces cerevisiae, nuclear-encoded mitochondrial translational activators (TAs) facilitate transcript-specific translation by a yet unknown mechanism. Here, we investigated the function of TAs containing RNA-binding pentatricopeptide repeats (PPRs) using selective mitoribosome profiling and cryo-EM structural analysis. These analyses revealed that TAs exhibit strong selectivity for mitoribosomes initiating on their target transcripts. Moreover, TA-mitoribosome footprints indicated that TAs recruit mitoribosomes proximal to the start codon. Two cryo-EM structures of mRNA-TA complexes bound to post-initiation/pre-elongation-stalled mitoribosomes revealed the general mechanism of TA action. Specifically, the TAs bind to structural elements in the 5' UTR of the client mRNA as well as to the mRNA exit channel to align the mRNA in the small subunit during initiation. Our findings provide a structural basis for understanding how mitochondria achieve transcript-specific translation initiation without relying on general sequence elements to position ribosomes at start codons.
2025-07-16 | GSE282943 | GEO
Project description:Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 allele of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.
2017-03-09 | GSE88725 | GEO
Project description:Proline-rich antimicrobial peptide apidaecin (Api) inhibits bacterial protein synthesis in a distinctive way. In vitro biochemical and structural studies showed that Api binds in the nascent peptide exit tunnel of the ribosome that has completed translation of the gene and has released the newly-synthesized protein. Once in the tunnel, Api traps the release factors on the post-termination ribosome leading to cessation of translation. The mode of Api action in the bacterial cell, however, had remained unknow. By analyzing distribution of ribosomes on mRNAs in Api-treated cells using Ribo-seq approach we uncovered a range of effects that stem from the unique mechanism of Api action. Exposure of bacterial cells to Api results in arrest of translation at stop codons, likely in a post-release state, due to the immediate Api action, as well as in a pre-release state due to the depletion of available release factors. In addition, arrest of translation at the end of the open reading frame (ORF) leads to a pronounced queuing of the translating ribosomes behind the one arrested at the stop codon. One of the major consequences of the Api action is a dramatically increased stop codon bypass by the ribosomes paused in a pre-release state, leading to accumulation of proteins with C-terminal extensions, as confirmed by whole-cell proteomics analysis. Stop codon bypass, that occurs either in 0-frame, by misincorporation of a near-cognate aminoacyl-tRNA at the stop codon, or via frameshifting allows for a fraction of the translating ribosomes to reach the 3’ ends of mRNA transcripts. The pervasive stalling of pre-release ribosomes at the stop codons and at the RNA transcripts ends triggers activation of the cellular ribosome rescue systems which, likely due to Api action as well, remain ineffective. While major Api effects are directed towards the ribosome at the end of the ORFs, cells exposed to Api show a somewhat increased ribosome occupancy of the start codons. Understanding the unique mode of Api to inhibit translation termination in the cell can help envisioning the development of treatments for genetic diseases and research tools for genome exploration.
2020-10-05 | PXD019012 | Pride
Project description:The proline-rich antimicrobial peptide apidaecin (Api) inhibits bacterial protein synthesis in a distinctive way. In vitro biochemical and structural studies showed that Api binds in the nascent peptide exit tunnel of a ribosome that has completed translation of a gene and traps the release factors on the ribosome. By analyzing the distribution of ribosomes on mRNAs in Api-treated cells using Ribo-seq, we uncovered a range of effects that stem from the unique mechanism of Api action. Exposure of bacterial cells to Api results in arrest of ribosomes at stop codons, likely in a post-release state due to direct Api action, as well as in a pre-release state due to the depletion of available release factors. In addition, Api action leads to a pronounced queuing of translating ribosomes behind those arrested at stop codons. One of the major consequences of Api action is a dramatically increased level of stop codon bypass by ribosomes paused in a pre-release state, leading to an accumulation of proteins with C-terminal extensions, as confirmed by whole-cell proteomics analysis. Stop codon bypass occurs either in 0-frame, by misincorporation of a near-cognate aminoacyl-tRNA at the stop codon, or via frameshifting, allowing for a fraction of the translating ribosomes to reach the 3’ ends of mRNA transcripts. The pervasive stalling of pre-release ribosomes at the stop codons and at the RNA transcripts ends triggers activation of the cellular ribosome rescue systems which, likely due to Api action as well, remain ineffective. Understanding the unique mode of Api action as translation termination inhibitor in the cell can advance the development of treatments for infection and genetic diseases as well as new research tools for genome exploration.
2020-10-02 | GSE150034 | GEO
Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo or serve as alternative initiation factors. Ribosome profiling of strains missing these factors revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3’UTR, as evidenced by peaks in the footprint data and 3’UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3’UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in the recycling of 40S ribosomal subunits at stop codons and translation reinitiation.
2018-08-30 | GSE108942 | GEO
Project description:Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play crucial roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focused on exploring the function of GTPBP8, the human homolog of EngB. We found that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells showed the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis revealed that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit16S rRNA. Our study highlights the crucial role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.
2024-06-19 | GSE256250 | GEO