Eukaryotic ribosome recognizes 3 context and stimulates stop codon readthrough

It is known that the nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3 contexts have been described that are unfavourable for translation termination; however, the exact molecular mechanism that mediates their effect remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3 stop codon contexts. Our results revealed that ribosomes can independently recognize certain contexts and ignore stop codons that are followed by these sequences. Moreover, the efficiency of translation termination at the weak 3 contexts was almost equal to the one at the standard context. We propose that weak 3 contexts interact with the 18S rRNA provoking a conformational change in the U-turn-like structure of the stop codon in the A site of ribosome. This change makes incorporation of the near-cognate tRNA more preferable than recognition of the stop codon by the release factors and increases readthrough.

Download Full-text

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

Nucleic Acids Research ◽

10.1093/nar/gku663 ◽

2014 ◽

Vol 42 (15) ◽

pp. 10061-10072 ◽

Cited By ~ 68

Author(s):

Sandra Blanchet ◽

David Cornu ◽

Manuela Argentini ◽

Olivier Namy

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Stop Codon ◽

Reporter System ◽

Yeast Saccharomyces Cerevisiae ◽

Systematic Analysis ◽

Stop Codons ◽

Nucleotide Context ◽

Stop Codon Readthrough

AbstractStop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNAGln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.

Download Full-text

Ribosome recycling factor ABCE1 depletion inhibits nonsense-mediated mRNA decay by promoting stop codon readthrough

10.1101/870097 ◽

2019 ◽

Cited By ~ 1

Author(s):

Giuditta Annibaldis ◽

René Dreos ◽

Michal Domanski ◽

Sarah Carl ◽

Oliver Mühlemann

Keyword(s):

Mrna Decay ◽

Stop Codon ◽

Translation Termination ◽

Ribosome Profiling ◽

List Type ◽

Ribosome Recycling Factor ◽

Stop Codons ◽

Nonsense Mediated Mrna Decay ◽

Ribosome Disassembly ◽

Stop Codon Readthrough

SUMMARYNonsense-mediated mRNA decay (NMD) is an essential post-transcriptional surveillance pathway in vertebrates that appears to be mechanistically linked with translation termination. To gain more insight into this connection, we interfered with translation termination by depleting human cells of the ribosome recycling factor ABCE1, which resulted in an upregulation of many but not all endogenous NMD-sensitive mRNAs. Notably, the suppression of NMD on these mRNAs occurs at a step prior to their SMG6-mediated endonucleolytic cleavage. Ribosome profiling revealed that ABCE1 depletion results in ribosome stalling at stop codons and increased ribosome occupancy in 3’ UTRs, indicative of enhanced stop codon readthrough or re-initiation. Using reporter genes, we further demonstrate that the absence of ABCE1 indeed increases the rate of readthrough, which would explain the observed NMD inhibition, since enhanced readthrough has been previously shown to render NMD-sensitive transcripts resistant to NMD by displacing NMD triggering factors like UPF1 and exon junction complexes (EJCs) from the 3’ UTR. Collectively, our results show that improper ribosome disassembly interferes with proper NMD activation.HighlightsABCE1 knockdown suppresses NMD of many NMD-sensitive mRNAsThe observed NMD inhibition occurs at a stage prior to SMG6-mediated cleavage of the mRNAABCE1 depletion enhances ribosome occupancy at stop codons and in the 3’ UTRABCE1 depletion enhances readthrough of the stop codonEnhanced readthrough inhibits NMD, presumably by clearing the 3’ UTR of NMD factors

Download Full-text

Destabilization of Eukaryote mRNAs by 5′ Proximal Stop Codons Can Occur Independently of the Nonsense-Mediated mRNA Decay Pathway

Cells ◽

10.3390/cells8080800 ◽

2019 ◽

Vol 8 (8) ◽

pp. 800 ◽

Cited By ~ 1

Author(s):

Barbara Gorgoni ◽

Yun-Bo Zhao ◽

J. Krishnan ◽

Ian Stansfield

Keyword(s):

Mrna Stability ◽

Mrna Decay ◽

Closed Loop ◽

Stop Codon ◽

Translation Termination ◽

Translation System ◽

Translation Elongation ◽

Reading Frame ◽

Stop Codons ◽

Nonsense Mediated Mrna Decay

In eukaryotes, the binding of poly(A) binding protein (PAB) to the poly(A) tail is central to maintaining mRNA stability. PABP interacts with the translation termination apparatus, and with eIF4G to maintain 3′–5′ mRNA interactions as part of an mRNA closed loop. It is however unclear how ribosome recycling on a closed loop mRNA is influenced by the proximity of the stop codon to the poly(A) tail, and how post-termination ribosome recycling affects mRNA stability. We show that in a yeast disabled for nonsense mediated mRNA decay (NMD), a PGK1 mRNA with an early stop codon at codon 22 of the reading frame is still highly unstable, and that this instability cannot be significantly countered even when 50% stop codon readthrough is triggered. In an NMD-deficient mutant yeast, stable reporter alleles with more 3′ proximal stop codons could not be rendered unstable through Rli1-depletion, inferring defective Rli1 ribosome recycling is insufficient in itself to trigger mRNA instability. Mathematical modelling of a translation system including the effect of ribosome recycling and poly(A) tail shortening supports the hypothesis that impaired ribosome recycling from 5′ proximal stop codons may compromise initiation processes and thus destabilize the mRNA. A model is proposed wherein ribosomes undergo a maturation process during early elongation steps, and acquire competency to re-initiate on the same mRNA as translation elongation progresses beyond the very 5′ proximal regions of the mRNA.

Download Full-text

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

Biomolecules ◽

10.3390/biom10060911 ◽

2020 ◽

Vol 10 (6) ◽

pp. 911 ◽

Cited By ~ 3

Author(s):

Kseniya A. Lashkevich ◽

Valeriya I. Shlyk ◽

Artem S. Kushchenko ◽

Vadim N. Gladyshev ◽

Elena Z. Alkalaeva ◽

...

Keyword(s):

Stop Codon ◽

Translation Termination ◽

Protein Biosynthesis ◽

Inhibitory Effect ◽

In Vitro Translation ◽

Nonsense Suppression ◽

Translation System ◽

Free System ◽

Termination Rate

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.

Download Full-text

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

Nucleic Acids Research ◽

10.1093/nar/gkz929 ◽

2019 ◽

Vol 47 (21) ◽

pp. 11326-11343 ◽

Cited By ~ 1

Author(s):

Kristýna Poncová ◽

Susan Wagner ◽

Myrte Esmeralda Jansen ◽

Petra Beznosková ◽

Stanislava Gunišová ◽

...

Keyword(s):

Translational Control ◽

Stop Codon ◽

Translation Termination ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation ◽

C Terminus ◽

Context Specific ◽

Stop Codon Readthrough

Abstract Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

Download Full-text

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020599118 ◽

2021 ◽

Vol 118 (2) ◽

pp. e2020599118

Author(s):

Martin Y. Ng ◽

Hong Li ◽

Mikel D. Ghelfi ◽

Yale E. Goldman ◽

Barry S. Cooperman

Keyword(s):

Protein Synthesis ◽

Stop Codon ◽

European Medicines Agency ◽

Translation System ◽

Release Factor ◽

Factor Activity ◽

Eukaryotic Translation ◽

Nonsense Codons ◽

Cognate Trna

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through–inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell’s protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

Download Full-text

Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

10.1101/2020.11.11.377739 ◽

2020 ◽

Author(s):

Alexey Shuvalov ◽

Ekaterina Shuvalova ◽

Nikita Biziaev ◽

Elizaveta Sokolova ◽

Konstantin Evmenov ◽

...

Keyword(s):

Stop Codon ◽

Translation Termination ◽

Translation System ◽

Release Factor ◽

Viral Mrnas ◽

Codon Recognition ◽

Free Translation ◽

Stop Codon Recognition ◽

A Cell

ABSTRACTThe Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

Download Full-text

Metabolic stress promotes stop-codon readthrough and phenotypic heterogeneity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013543117 ◽

2020 ◽

Vol 117 (36) ◽

pp. 22167-22172

Author(s):

Hong Zhang ◽

Zhihui Lyu ◽

Yongqiang Fan ◽

Christopher R. Evans ◽

Karl W. Barber ◽

...

Keyword(s):

Phenotypic Diversity ◽

Stop Codon ◽

Single Cells ◽

Metabolic Stress ◽

Aminoglycoside Antibiotic ◽

Bacterial Cells ◽

Excess Carbon ◽

Stop Codons ◽

Release Factors ◽

Stop Codon Readthrough

Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity.

Download Full-text

Distinct Paths To Stop Codon Reassignment by the Variant-Code Organisms Tetrahymena and Euplotes

Molecular and Cellular Biology ◽

10.1128/mcb.26.2.438-447.2006 ◽

2006 ◽

Vol 26 (2) ◽

pp. 438-447 ◽

Cited By ~ 39

Author(s):

Joe Salas-Marco ◽

Hua Fan-Minogue ◽

Adam K. Kallmeyer ◽

Lawrence A. Klobutcher ◽

Philip J. Farabaugh ◽

...

Keyword(s):

Stop Codon ◽

Translation Termination ◽

Yeast Cells ◽

Universal Genetic Code ◽

Stop Codons ◽

Codon Recognition ◽

Ciliate Species ◽

Stop Codon Recognition ◽

Euplotes Octocarinatus

ABSTRACT The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.

Download Full-text

Mutations in Eukaryotic Release Factors 1 and 3 Act as General Nonsense Suppressors in Drosophila

Genetics ◽

10.1093/genetics/165.2.601 ◽

2003 ◽

Vol 165 (2) ◽

pp. 601-612

Author(s):

Anna T Chao ◽

Herman A Dierick ◽

Tracie M Addy ◽

Amy Bejsovec

Keyword(s):

Stop Codon ◽

Translation Termination ◽

Nonsense Suppression ◽

Release Factor ◽

Nonsense Mutations ◽

Stop Codons ◽

Larval Stages ◽

Mutant Phenotypes ◽

Polypeptide Chains ◽

Nonsense Suppressors

Abstract In a screen for suppressors of the Drosophila winglessPE4 nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens.

Download Full-text