Hidden stop codons dictate mRNA fate by ambushing ribosomal frameshifting
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PROVIDER: PRJNA1295776 | ENA |
REPOSITORIES: ENA
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Project description:In eukaryotes, the intrinsic mRNA stability is generally dictated by codon usage bias, the uneven preferences for synonymous codons, in a translation-dependent manner. However, the conserved mechanism underlining this phenomenon remains elusive. Hidden stop codons (HSCs), which are stop codons located in the +1 or -1 frame relative to canonical open reading frames (ORF), are sidespread across all genomes but have long been overlooked. We demonstrate that in both fungi and mammals, HSCs play an important role in regulating mRNA decay across the genome by immediately terminating out-of-frame translations promoted by nonoptimal codons, primarily through +1 ribosomal frameshifting. In the filamentous fungus Neurospora, partially deleting HSCs via synonymous substitutions in the clock gene frequency disrupts the circadian rhythm due to increased mRNA stability. Similarly, in human cells, impaired translation termination caused by rapid depletion of eRF1 leads to globally increased stability of mRNAs enriched with non-optimal codons and HSCs, which are in part degraded through the NMD pathway via UPF1. Given that from yeasts to humans the occurrence of HSCs is genome-widely associated with the presence of nonoptimal codons, these findings suggest that in eukaryotes, HSCs might coevolve with codon usage to modulate mRNA stabilities via immediately terminating different levels of ribosomal frameshifting events promoted by nonoptimal codons.
2025-07-31 | GSE303542 | 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:mtRF1 recognises non-canonical stop codons in human mitochondria
| PRJNA896508 | ENA
Project description:Stop codon readthrough (SCR) has important biological implications but remains largely uncharacterized. Here, we identify 1,009 SCR events in plants using a proteogenomic strategy. Plant SCR candidates tend to have shorter transcript lengths and fewer exons and splice variants than non-SCR transcripts. Mass spectrometry evidence shows that stop codons involved in SCR events can be recoded as 20 standard amino acids, some of which are also supported by suppressor transfer RNA analysis. We also observe multiple functional signals in 34 maize extended proteins and characterize the structural and subcellular localization changes in the extended protein of BASIC TRANSCRIPTION FACTOR 3. Furthermore, the SCR events exhibit non-conserved signature and the extensions likely undergo protein-coding selection. Overall, our study not only characterizes that SCR events are commonly present in plants but also identifies the unprecedented recoding plasticity of stop codons, which provides important insights into the flexibility of genetic decoding.
2024-02-02 | PXD038762 | Pride
Project description:Stop codon readthrough (SCR) has important biological implications but remains largely uncharacterized. Here, we identify 1,009 SCR events in plants using a proteogenomic strategy. Plant SCR candidates tend to have shorter transcript lengths and fewer exons and splice variants than non-SCR transcripts. Mass spectrometry evidence shows that stop codons involved in SCR events can be recoded as 20 standard amino acids, some of which are also supported by suppressor transfer RNA analysis. We also observe multiple functional signals in 34 maize extended proteins and characterize the structural and subcellular localization changes in the extended protein of BASIC TRANSCRIPTION FACTOR 3. Furthermore, the SCR events exhibit non-conserved signature and the extensions likely undergo protein-coding selection. Overall, our study not only characterizes that SCR events are commonly present in plants but also identifies the unprecedented recoding plasticity of stop codons, which provides important insights into the flexibility of genetic decoding.
2024-02-02 | PXD035958 | Pride
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:Here, we use a novel technique for locating regions of N6-adenosine methylation (m6A) throughout the transcriptome and present a profile of m6A sites in the mouse brain. Our use of methylated RNA immunoprecipitation combined with RNA-seq (MeRIP-Seq) identifies thousands of RNAs which contain m6A sites. In addition, we find that regions of m6A formation are particularly enriched near stop codons, which might provide clues into the potential funciton of this highly prevalent RNA modificaiton. Examination of m6A sites in murine brain RNA and human embryonic kidney cells.
2012-12-31 | E-GEOD-34734 | biostudies-arrayexpress
Project description:Here, we use a novel technique for locating regions of N6-adenosine methylation (m6A) throughout the transcriptome and present a profile of m6A sites in the mouse brain. Our use of methylated RNA immunoprecipitation combined with RNA-seq (MeRIP-Seq) identifies thousands of RNAs which contain m6A sites. In addition, we find that regions of m6A formation are particularly enriched near stop codons, which might provide clues into the potential funciton of this highly prevalent RNA modificaiton.
2012-05-17 | GSE29714 | GEO
Project description:Codon usage bias, which refers to uneven use of synonymous codons, was shown to associate with mRNA stability from yeast to human. However, the underlying molecular basis is largely unknown. With bioinformatic analyses we unexpectedly found that codon usage bias is inversely correlated to density of out-of-frame stop codons (hidden stop codon or HSC). To understand the physiological function of HSCs, we use the frequency gene of Neurospora crassa to examine the role of HSCs in circadian rhythm. We show that deleting HSCs in the upstream of frequency (frq) open reading frame without altering FRQ sequence resulted in loss of circadian rhythm. Further analyses revealed that HSC deletion of frq resulted in elevated stability of frq mRNA, which consequently causes higher FRQ protein level. Combined various methods, we showed that rare codons in the upstream frq region generates significant amount of aberrant translation at +1 frame, possibly via ribosomal frameshifting. Consistently, at genome wide we showed that both in N. crassa and S. cerevisiae, the codon usage bias combined with numbers of HSC are inversely correlated to mRNA stability. Together, these data indicate that HSCs might act as an intrinsic mechanism to terminate aberrant ribosomal frameshifting events triggered by non-optimal codons, thus fine-tune mRNA stability.
2023-12-31 | GSE245041 | GEO
Project description:Readthrough of a translation termination codon is regulated by ribosomal A site recognition and insertion of near-cognate tRNAs. Small molecules exist that mediate the incorporation of amino acids at the stop codon and the production of full-length, often functional protein but defining the actual amino acid that is incorporated remains a challenging area. We report on the development of a human cell model that can be used to determine whether rules can be developed using mass spectrometry that defines the type of amino acid that is placed at a premature termination codon during readthrough mediated by an aminoglycoside. The first premature termination codon we analysed contained the relatively common cancer-associated termination signal at codon 213 in the p53 gene. Although we could detect a tryptic peptide with the incorporation of an R at codon 213 in the presence of the aminoglycoside, there were no other tryptic peptides detected across codon 213 that could be recovered so we needed to create a more robust artificial premature stop codon model. P53 expression plasmids were developed that incorporate a string of single synthetic UGA (opal) stop codons at S127P128A129 within the relatively abundant tryptic p53 peptide 122-SVTCTYSPALNK-132. The treatment of cells stably expressing the p53-UGA129 mutation, treated with Gentamycin, followed by immunoprecipitation and trypsinization of p53, resulted in the identification R, W, or C within the tryptic peptide at codon-UGA129; as expected based on the two base pairing of the respective anticodons to UGA with R being the most abundant using all three codons. By contrast, incorporating the amber or ochre premature stop codons, UAA129 or UAG129 resulted in the incorporation of a Y or Q amino acid, again as expected based on the two base pairings to the anticodons, with Q being the most abundant. The incorporation of these amino acids at codons 127, 128, or 129 generally results in a p53 protein that is predicted to be ‘unfolded’ or inactive as defined by Molecular Dynamic Simulations. As such, the data also highlight the need in the future to not only produce novel small molecules that can read through premature termination codons but also the need to design methods to insert the required amino acid at the position that could result in a ‘wild-type’ functional protein.
2024-10-29 | PXD039307 | Pride