A novel fluorescent reporter sensitive to serine mis-incorporation

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging amino acids onto cognate tRNAs during protein synthesis. In histidyl-tRNA synthetase (HARS), autosomal dominant mutations V133F, V155G, Y330C, S356N in the HARS catalytic domain cause Charcot Marie Tooth Disease Type 2 W (CMT2W), while RNA-binding domain mutation Y454S causes recessive Usher Syndrome Type IIIB (USH3B). In a yeast model, all human HARS variants complemented a genomic deletion of the yeast ortholog hts1 at high expression levels. CMT2W associated mutations, but not Y454S, resulted in reduced growth. We show mistranslation of histidine to glutamine and threonine in V155G and S356N but not Y330C mutants in yeast. Mistranslating V155G and S356N mutants lead to accumulation of insoluble proteins, which was rescued by histidine. Mutants V133F and Y330C showed the most significant growth defect and decreased HARS abundance in cells. Here, histidine supplementation led to insoluble protein aggregation and further reduced viability, indicating histidine toxicity associated with these mutants. V133F proteins displayed reduced thermal stability in vitro, which was rescued by tRNA. Our data will inform future treatment options for HARS patients, where histidine supplementation may either have a toxic or compensating effect depending on the nature of the causative HARS variant.

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“…4E ). A reduction in fluorescence can indicate either reduced protein abundance, or indicate misfolded, non-fluorescent proteins ( 29 ).…”

Section: Resultsmentioning

confidence: 99%

Histidine supplementation can escalate or rescue HARS deficiency in a Charcot–Marie–Tooth disease model

Qiu

Kenana

Beharry

et al. 2022

Human Molecular Genetics

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“…The bands corresponding to mCherry were identified at 37 kDa, excised from the gel, placed in sterile 1.5 ml centrifuge tubes containing 5% acetic acid, and submitted for mass spectrometry analysis at the Biological Mass Spectrometry Laboratory (The University of Western Ontario, London, Canada). Trypsin was used to digest mCherry and the Orbitrap Elite Velos Pro mass spectrometer (ThermoFisher) was used in FT/IT/CID configuration to identify amino acids mis-incorporated in mCherry as described before [24].…”

Section: (D) Protein Purification and Mass Spectrometrymentioning

confidence: 99%

“…While these Gly codons can be misread by both tRNA Gly GCC A3G and by the mutant tRNA Ala ACC , the tRNA Gly CCC A3G variant should only be able to misread GGG Gly codons. Rather than engineer an mCherry protein that is sensitive to a particular kind of codon mis-reading, as we have done elsewhere for Ser mis-incorporation [24], here we used mCherry as a measure of protein production, which can be slowed generally as a hallmark of mistranslation in mammalian cells [22,26,27].…”

Section: (B) Protein Synthesis In Mistranslating Cellsmentioning

confidence: 99%

“…The rate or level of mistranslation can also contribute to toxicity or growth defects associated with mistranslation [24,40]. Two synthetic tRNA variants, one that causes more frequent errors with Ser mis-incorporation at Ala codons and another that causes less frequent Ser mistranslation at Leu codons, were characterized in murine NIH3T3 cells [28].…”

Section: (B) Amino Acid Similarity May Alleviate Mistranslation Toxic...mentioning

confidence: 99%

“…The mCherry construct contains only one other Gly codon (GGU), and we found evidence that both tRNA Ala and the tRNA Gly variants direct Ala mis-incorporation at Gly56 (GGU) (electronic supplementary material, figure S4). In on-going studies, we are working towards quantitative methods relying on mistranslation sensitive fluorescent reporters [24] as well as absolute quantitation approaches with mass spectrometry [42] to accurately establish the level of Ala mis-incorporation Gly codons. In our future work, these approaches will also allow us to quantify the level of Ala mis-incorporation at each of the four Gly codons.…”

Section: (B) Amino Acid Similarity May Alleviate Mistranslation Toxic...mentioning

confidence: 99%

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Perseverance of protein homeostasis despite mistranslation of glycine codons with alanine

Hasan

Lant

O’Donoghue

2023

Phil. Trans. R. Soc. B

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By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNA Ser variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNA Gly variants (tRNA Gly CCC , tRNA Gly GCC ) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNA Ala AGC G35C (tRNA Ala ACC ) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNA Gly and tRNA Ala variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNA Gly GCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNA Gly GCC A3G and tRNA Ala AGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.

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Engineered mischarged transfer RNAs for correcting pathogenic missense mutations

Hou,

Zhang,

McGilvray

et al. 2024

Molecular Therapy

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A novel fluorescent reporter sensitive to serine mis-incorporation

Cited by 8 publications

References 49 publications

Histidine supplementation can escalate or rescue HARS deficiency in a Charcot–Marie–Tooth disease model

Histidine supplementation can escalate or rescue HARS deficiency in a Charcot–Marie–Tooth disease model

Perseverance of protein homeostasis despite mistranslation of glycine codons with alanine

Engineered mischarged transfer RNAs for correcting pathogenic missense mutations

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