scholarly journals Engineered transfer RNAs for suppression of premature termination codons

2018 ◽  
Author(s):  
John D. Lueck ◽  
Jae Seok Yoon ◽  
Alfredo Perales-Puchalt ◽  
Adam L. Mackey ◽  
Daniel T. Infield ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Premature Termination ◽  
Translation Termination ◽  
Genetic Disorders ◽  
Ribosome Profiling ◽  
Inherited Disease ◽  
Transfer Rnas ◽  
Premature Termination Codons ◽  
Nonsense Codons

ABSTRACTPremature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within the cystic fibrosis transmembrane conductance regulator (CFTR).

scholarly journals Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

2019 ◽  
Author(s):  
Jamie R Wangen ◽  
Rachel Green
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Ribosome Profiling ◽  
Termination Codon ◽  
Stop Codons ◽  
Genome Wide ◽  
A Genome

AbstractStop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418 treatment globally alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

scholarly journals Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jamie R Wangen ◽  
Rachel Green
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Ribosome Profiling ◽  
Termination Codon ◽  
Stop Codons ◽  
Genome Wide ◽  
A Genome

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418-induced miscoding alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

scholarly journals Molecular Basis for Rab Prenylation

2000 ◽  
Vol 150 (1) ◽  
pp. 89-104 ◽  
Author(s):  
Christelle Alory ◽  
William E. Balch
Keyword(s):  
Mammalian Cells ◽  
Normal Growth ◽  
Kinetic Properties ◽  
Temperature Sensitive ◽  
Rab Gtpases ◽  
Inherited Disease ◽  
Vesicular Traffic ◽  
Null Strain

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

scholarly journals Infrequent Translation of a Nonsense Codon Is Sufficient to Decrease mRNA Level

1999 ◽  
Vol 10 (3) ◽  
pp. 515-524 ◽  
Author(s):  
Alla Buzina ◽  
Marc J. Shulman
Keyword(s):  
Mammalian Cells ◽  
Translation Termination ◽  
Mrna Level ◽  
Chain Gene ◽  
Nonsense Mutations ◽  
Nonsense Codon ◽  
Residual Level ◽  
Nonsense Codons ◽  
High Level ◽  
In Cis

In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin μ heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of μ mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only ∼4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.

scholarly journals CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors

2019 ◽  
Author(s):  
Choongil Lee ◽  
Dong Hyun Jo ◽  
Gue-Ho Hwang ◽  
Jihyeon Yu ◽  
Jin Hyoung Kim ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Dna Sequence ◽  
Clinical Utility ◽  
Premature Termination ◽  
Genetic Disorders ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
Potential Clinical Utility ◽  
Clinically Significant ◽  
Adenine Base

AbstractA nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read-through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.

scholarly journals Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

2017 ◽  
Vol 114 (13) ◽  
pp. 3479-3484 ◽  
Author(s):  
Alireza Baradaran-Heravi ◽  
Jürgen Niesser ◽  
Aruna D. Balgi ◽  
Kunho Choi ◽  
Carla Zimmerman ◽  
...  
Keyword(s):  
Genetic Disorders ◽  
Xenograft Model ◽  
Minor Component ◽  
Premature Termination Codon ◽  
Tumor Xenograft ◽  
Nonsense Suppression ◽  
Nonsense Mutations ◽  
Nonsense Codons ◽  
Dynamics Simulations

Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders.

scholarly journals At Least One Intron Is Required for the Nonsense-Mediated Decay of Triosephosphate Isomerase mRNA: a Possible Link between Nuclear Splicing and Cytoplasmic Translation

1998 ◽  
Vol 18 (9) ◽  
pp. 5272-5283 ◽  
Author(s):  
Jing Zhang ◽  
Xiaolei Sun ◽  
Yimei Qian ◽  
Jeffrey P. LaDuca ◽  
Lynne E. Maquat
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Triosephosphate Isomerase ◽  
Translation Termination ◽  
Nonsense Mediated Decay ◽  
Nonsense Codon ◽  
Translation Initiation Codon ◽  
Exon Junctions ◽  
Nonsense Codons

ABSTRACT Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3′-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3′-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3′-most intron from pre-mRNA “marks” the 3′-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the “mark” mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5′ untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.

scholarly journals Suppression of aminoglycoside-induced premature termination codon readthrough by the TRP channel inhibitor AC1903

2021 ◽  
Author(s):  
Alireza Baradaran-Heravi ◽  
Claudia C Bauer ◽  
Isabelle B Pickles ◽  
Sara Hosseini-Farahabadi ◽  
Aruna Balgi ◽  
...  
Keyword(s):  
Premature Termination ◽  
Genetic Disorders ◽  
Premature Termination Codon ◽  
Cation Channels ◽  
Trpc Channels ◽  
Nonsense Mutations ◽  
Variable Effect ◽  
Premature Termination Codons ◽  
Ic50 Values ◽  
Nonsense Mediated Mrna Decay

Nonsense mutations, which occur in ~11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as gentamicin and G418 permit PTC readthrough and so may address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we addressed the hypothesis that TRP non-selective cation channels contribute to the variable effect of aminoglycosides by controlling their cellular uptake. To attempt to identify the channel type involved, we tested AC1903, a 2-aminobenzimidazole derivative recently reported to selectively inhibit TRPC5 cation channels. AC1903 consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cell line and patient-derived JEB01 keratinocytes. In an effort to validate the suggested role of TRPC5, we tested an independent and more potent inhibitor called Pico145, which affects channels containing TRPC1, TRPC4 and TRPC5 but not other TRPCs or other channels. Unexpectedly, Pico145 was completely without effect, suggesting that AC1903 may work through other or additional targets. Consistent with this suggestion, AC1903 inhibited multiple TRPC channels including homomeric TRPC3, TRPC4, TRPC5, TRPC6 as well as concatemeric TRPC4-C1 and TRPC5-C1 channels, all with low micromolar IC50 values. It also inhibited TRPV4 channels but had weak or no effects on TRPV1 and no effect on another non-selective cation channel, PIEZO1. Overall, our study reveals a suppressor of aminoglycoside-mediated PTC readthrough (i.e., AC1903) but suggests that this compound has previously unrecognised effects. These effects require further investigation to determine the molecular mechanism by which AC1903 suppresses aminoglycoside uptake and PTC readthrough.

scholarly journals Engineered transfer RNAs for suppression of premature termination codons

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
John D. Lueck ◽  
Jae Seok Yoon ◽  
Alfredo Perales-Puchalt ◽  
Adam L. Mackey ◽  
Daniel T. Infield ◽  
...  
Keyword(s):  
Premature Termination ◽  
Transfer Rnas ◽  
Premature Termination Codons

scholarly journals Maintenance of Transcription-Translation Coupling by Elongation Factor P

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Sara Elgamal ◽  
Irina Artsimovitch ◽  
Michael Ibba
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Premature Termination ◽  
Transcription Termination ◽  
Elongation Factor ◽  
Gene Clusters ◽  
Ribosome Profiling ◽  
Tight Coupling ◽  
Elongation Factor P

ABSTRACT Under conditions of tight coupling between translation and transcription, the ribosome enables synthesis of full-length mRNAs by preventing both formation of intrinsic terminator hairpins and loading of the transcription termination factor Rho. While previous studies have focused on transcription factors, we investigated the role of Escherichia coli elongation factor P (EF-P), an elongation factor required for efficient translation of mRNAs containing consecutive proline codons, in maintaining coupled translation and transcription. In the absence of EF-P, the presence of Rho utilization ( rut ) sites led to an ~30-fold decrease in translation of polyproline-encoding mRNAs. Coexpression of the Rho inhibitor Psu fully restored translation. EF-P was also shown to inhibit premature termination during synthesis and translation of mRNAs encoding intrinsic terminators. The effects of EF-P loss on expression of polyproline mRNAs were augmented by a substitution in RNA polymerase that accelerates transcription. Analyses of previously reported ribosome profiling and global proteomic data identified several candidate gene clusters where EF-P could act to prevent premature transcription termination. In vivo probing allowed detection of some predicted premature termination products in the absence of EF-P. Our findings support a model in which EF-P maintains coupling of translation and transcription by decreasing ribosome stalling at polyproline motifs. Other regulators that facilitate ribosome translocation through roadblocks to prevent premature transcription termination upon uncoupling remain to be identified. IMPORTANCE Bacterial mRNA and protein syntheses are often tightly coupled, with ribosomes binding newly synthesized Shine-Dalgarno sequences and then translating nascent mRNAs as they emerge from RNA polymerase. While previous studies have mainly focused on the roles of transcription factors, here we investigated whether translation factors can also play a role in maintaining coupling and preventing premature transcription termination. Using the polyproline synthesis enhancer elongation factor P, we found that rapid translation through potential stalling motifs is required to provide efficient coupling between ribosomes and RNA polymerase. These findings show that translation enhancers can play an important role in gene expression by preventing premature termination of transcription.

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