scholarly journals Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

2016 ◽  
Vol 113 (44) ◽  
pp. 12508-12513 ◽  
Author(s):  
Bijoyita Roy ◽  
Westley J. Friesen ◽  
Yuki Tomizawa ◽  
John D. Leszyk ◽  
Jin Zhuo ◽  
...  
Keyword(s):  
Premature Termination Codon ◽  
Nonsense Suppression ◽  
Base Pairs ◽  
Inherited Disorders ◽  
Nonsense Mutations ◽  
Nonsense Codon ◽  
Codon Positions ◽  
A Site ◽  
Cognate Trna ◽  
Selection Of

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

scholarly journals Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3

2015 ◽  
Vol 112 (10) ◽  
pp. 3038-3043 ◽  
Author(s):  
Bijoyita Roy ◽  
John D. Leszyk ◽  
David A. Mangus ◽  
Allan Jacobson
Keyword(s):  
Amino Acids ◽  
Full Length ◽  
Nonsense Suppression ◽  
Truncated Protein ◽  
Premature Termination Codons ◽  
Codon Positions ◽  
Nonsense Codons ◽  
Readthrough Protein ◽  
A Site ◽  
Cognate Trna

Premature termination codons (PTCs) in an mRNA ORF inactivate gene function by causing production of a truncated protein and destabilization of the mRNA. Readthrough of a PTC allows ribosomal A-site insertion of a near-cognate tRNA, leading to synthesis of a full-length protein from otherwise defective mRNA. To understand the mechanism of such nonsense suppression, we developed a yeast system that allows purification and sequence analysis of full-length readthrough products arising as a consequence of endogenous readthrough or the compromised termination fidelity attributable to the loss of Upf (up-frameshift) factors, defective release factors, or the presence of the aminoglycoside gentamicin. Unlike classical “wobble” models, our analyses showed that three of four possible near-cognate tRNAs could mispair at position 1 or 3 of nonsense codons and that, irrespective of whether readthrough is endogenous or induced, the same sets of amino acids are inserted. We identified the insertion of Gln, Tyr, and Lys at UAA and UAG, whereas Trp, Arg, and Cys were inserted at UGA, and the frequency of insertion of individual amino acids was distinct for specific nonsense codons and readthrough-inducing agents. Our analysis suggests that the use of genetic or chemical means to increase readthrough does not promote novel or alternative mispairing events; rather, readthrough effectors cause quantitative enhancement of endogenous mistranslation events. Knowledge of the amino acids incorporated during readthrough not only elucidates the decoding process but also may allow predictions of the functionality of readthrough protein products.

scholarly journals Screening Readthrough Compounds to Suppress Nonsense Mutations: Possible Application to β-Thalassemia

2020 ◽  
Vol 9 (2) ◽  
pp. 289 ◽  
Author(s):  
Monica Borgatti ◽  
Emiliano Altamura ◽  
Francesca Salvatori ◽  
Elisabetta D’Aversa ◽  
Nicola Altamura
Keyword(s):  
Genetic Disease ◽  
Chelation Therapy ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Nonsense Suppression ◽  
Current Status ◽  
Nonsense Mutations ◽  
Nonsense Mediated Mrna Decay ◽  
Mrna Destabilization ◽  
Premature Translation Termination

Several types of thalassemia (including β039-thalassemia) are caused by nonsense mutations in genes controlling globin production, leading to premature translation termination and mRNA destabilization mediated by the nonsense mediated mRNA decay. Drugs (for instance, aminoglycosides) can be designed to suppress premature translation termination by inducing readthrough (or nonsense suppression) at the premature termination codon. These findings have introduced new hopes for the development of a pharmacologic approach to cure this genetic disease. In the present review, we first summarize the principle and current status of the chemical relief for the expression of functional proteins from genes otherwise unfruitful for the presence of nonsense mutations. Second, we compare data available on readthrough molecules for β0-thalassemia. The examples reported in the review strongly suggest that ribosomal readthrough should be considered as a therapeutic approach for the treatment of β0-thalassemia caused by nonsense mutations. Concluding, the discovery of molecules, exhibiting the property of inducing β-globin, such as readthrough compounds, is of great interest and represents a hope for several patients, whose survival will depend on the possible use of drugs rendering blood transfusion and chelation therapy unnecessary.

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 Suppression of Nonsense Mutations by New Emerging Technologies

2020 ◽  
Vol 21 (12) ◽  
pp. 4394 ◽  
Author(s):  
Pedro Morais ◽  
Hironori Adachi ◽  
Yi-Tao Yu
Keyword(s):  
Clinical Trials ◽  
New Technologies ◽  
Genetic Disorders ◽  
Protein Translation ◽  
Premature Termination Codon ◽  
Nonsense Suppression ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Nonsense Mutations ◽  
The Impact

Nonsense mutations often result from single nucleotide substitutions that change a sense codon (coding for an amino acid) to a nonsense or premature termination codon (PTC) within the coding region of a gene. The impact of nonsense mutations is two-fold: (1) the PTC-containing mRNA is degraded by a surveillance pathway called nonsense-mediated mRNA decay (NMD) and (2) protein translation stops prematurely at the PTC codon, and thus no functional full-length protein is produced. As such, nonsense mutations result in a large number of human diseases. Nonsense suppression is a strategy that aims to correct the defects of hundreds of genetic disorders and reverse disease phenotypes and conditions. While most clinical trials have been performed with small molecules, there is an increasing need for sequence-specific repair approaches that are safer and adaptable to personalized medicine. Here, we discuss recent advances in both conventional strategies as well as new technologies. Several of these will soon be tested in clinical trials as nonsense therapies, even if they still have some limitations and challenges to overcome.

scholarly journals Polarity effects in the hisG gene of salmonella require a site within the coding sequence.

Genetics ◽  
1988 ◽  
Vol 118 (2) ◽  
pp. 193-202
Author(s):  
M S Ciampi ◽  
J R Roth
Keyword(s):  
Point Mutations ◽  
Polar Effect ◽  
Single Site ◽  
Base Pairs ◽  
Nonsense Mutations ◽  
Coding Sequence ◽  
Polarity Effect ◽  
A Site ◽  
Polarity Effects ◽  
Insertion Mutations

Abstract A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.

scholarly journals Nonsense suppression position effect implicates poly(A)-binding protein in the regulation of translation termination

2019 ◽  
Author(s):  
Chan Wu ◽  
Bijoyita Roy ◽  
Feng He ◽  
Allan Jacobson
Keyword(s):  
Translation Termination ◽  
Binding Protein ◽  
Position Effect ◽  
Critical Role ◽  
Yeast Cells ◽  
Nonsense Suppression ◽  
Regulation Of Translation ◽  
Nonsense Codon ◽  
A Site ◽  
Site Recognition

SUMMARYReadthrough of translation termination codons, also known as nonsense suppression, is a relatively inefficient process mediated by ribosomal A site recognition and insertion of near-cognate tRNAs. Multiple factors influence readthrough efficiency, including nonsense codon specificity and context. To determine whether nonsense codon position in a gene influences the extent of readthrough, we generated a series of LUC nonsense alleles and quantitated both readthrough and termination efficiencies at each nonsense codon in yeast cells lacking nonsense-mediated mRNA decay (NMD) activity. Readthrough efficiency for premature termination codons (PTCs) manifested a marked dependence on PTC proximity to the mRNA 3’-end, decreasing progressively across the LUC ORF but increasing with 3’-UTR lengthening. These effects were eliminated, and translation termination efficiency decreased considerably, in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and suggest that inefficient termination is the trigger for NMD.

scholarly journals Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay.

1995 ◽  
Vol 15 (4) ◽  
pp. 2231-2244 ◽  
Author(s):  
S Zhang ◽  
M J Ruiz-Echevarria ◽  
Y Quan ◽  
S W Peltz
Keyword(s):  
Mrna Decay ◽  
Sequence Motif ◽  
Nonsense Mutations ◽  
Stem Loop ◽  
Cis Acting ◽  
Stem Loop Structure ◽  
Nonsense Codon ◽  
Nonsense Mediated Mrna Decay

In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay. We have been investigating the cis-acting sequences involved in this decay pathway. Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3' of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization. The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3' of a nonsense codon, promote rapid decay of its mRNA. Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements. In addition, four copies of this motif can function as an independent downstream element. The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present. Our results indicate the sequences 5' of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon. We also suggest that the sequences 3' of the motif modulate the activity of the downstream element by forming RNA secondary structures. Consistent with this view, a stem-loop structure positioned 3' of the sequence motif can enhance the activity of the downstream element. This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover. The role of these sequences in mRNA decay is discussed.

Expriments on the Selection of Algal Subsrates by Polyzoan Larvae

1959 ◽  
Vol 36 (4) ◽  
pp. 613-631
Author(s):  
J. S. RYLAND
Keyword(s):  
Adverse Effect ◽  
Algal Species ◽  
Ecological Distribution ◽  
Substrate Preferences ◽  
Fucus Serratus ◽  
Chemical Factors ◽  
A Site ◽  
Marked Specificity ◽  
Selection Of ◽  
Actual Substrate

1. Many species of Polyzoa show marked specificity with regard to the substrate on which they occur. Epiphytic forms are often found mainly on one species of alga. 2. Experiments were performed in which a number of algal species were offered to polyzoan larvae as substrates for settlement. The disposition of algae, and the dishes containing them, was such that the layout conformed to a Youden Square design. This not only achieved economy of materials, but ensured a balanced experiment, made possible a statistical analysis of the results, and eliminated any possible effects of extraneous environmental factors. 3. The larvae showed marked substrate preferences when settling. In the littoral forms Alcyonidium hirsutum, A. polyoum and Flustrellidra hispida, the selection of algae accorded closely with their observed natural distributions: in each case highest settlement took place on Fucus serratus. It seems probable that positive selection plays an important role in determining the distribution of these species on the shore. Celleporella hyalina larvae were also selective, but the preferences were less clearly related to the ecological distribution of the adult. 4. Surface texture appears more important than contour as a factor influencing the choice made by larvae between algal substrates, although the physical and/or chemical factors responsible for the observed differences in attractiveness of algae are largely unknown. However, it is evident that the nature of the surface alters with age, and that this influences favourability. The presence of mucus has an adverse effect on settlement. Once the actual substrate has been chosen, the larvae respond to surface contour and, if possible, select a groove or concavity as a site for fixation.

scholarly journals P nucleotides in V(D)J recombination: a fine-structure analysis.

1993 ◽  
Vol 13 (2) ◽  
pp. 1078-1092 ◽  
Author(s):  
J T Meier ◽  
S M Lewis
Keyword(s):  
Fine Structure ◽  
Germ Line ◽  
Statistical Tests ◽  
Random Sequence ◽  
Genetic Locus ◽  
Gene Segment ◽  
Lymphoid Cells ◽  
Base Pairs ◽  
Nucleotide Data ◽  
Selection Of

Antigen receptor genes acquire junctional inserts upon assembly from their component, germ line-encoded V, D, and J segments. Inserts are generally of random sequence, but a small number of V-D, D-J, or V-J junctions are exceptional. In such junctions, one or two added base pairs inversely repeat the sequence of the abutting germ line DNA. (For example, a gene segment ending AG might acquire an insert beginning with the residues CT upon joining). It has been proposed that the nonrandom residues, termed "P nucleotides," are a consequence of an obligatory end-modification step in V(D)J recombination. P insertion in normal, unselected V(D)J joining products, however, has not been rigorously established. Here, we use an experimentally manipulable system, isolated from immune selection of any kind, to examine the fine structure of V(D)J junctions formed in wild-type lymphoid cells. Our results, according to statistical tests, show the following, (i) The frequency of P insertion is influenced by the DNA sequence of the joined ends. (ii) P inserts may be longer than two residues in length. (iii) P inserts are associated with coding ends only. Additionally, a systematic survey of published P nucleotide data shows no evidence for variation in P insertion as a function of genetic locus and ontogeny. Together, these analyses establish the generality of the P nucleotide pattern within inserts but do not fully support previous conjectures as to their origin and centrality in the joining reaction.

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