Mistranslating serine tRNAs in Drosophila melanogaster
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse
ORGANISM(S): Drosophila Melanogaster (fruit Fly)
TISSUE(S): Whole Body
SUBMITTER: 
Matthew Berg
LAB HEAD: Judit Villén
PROVIDER: PXD052492 | Pride | 2024-06-15
REPOSITORIES: Pride
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Project description:Mistranslation, the mis-incorporation of an amino acid not specified by the “standard” genetic code, occurs in all cells. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. The goal of this study was to detect and quantify mistranslation from a serine tRNA variant with proline UGG anticodon and G26A secondary mutation engineered in yeast
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Project description:Mistranslation, the mis-incorporation of an amino acid not specified by the “standard” genetic code, occurs in all cells. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. The goal of this study was to detect mistranslation from two different tRNA variants. The first variant, tRNA-Pro-G3:U70, has a mutation in its acceptor stem creating a G3:U70 base pair which is the key identity element for the alanine tRNA synthetase. This tRNA should be charged with alanine and mis-incorporate alanine at proline codons. The second variant, tRNA-Ser-UCU,G26A, is a serine tRNA with an arginine anticodon and a G26A secondary mutation to dampen function and prevent lethal levels of mistranslation. This tRNA should mis-incorporate serine at arginine codons.
2021-05-17 | PXD025934 | Pride
Project description:Transfer RNAs (tRNAs) are the adaptor molecules required for reading of the genetic code and the accurate production of proteins. tRNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code, into nascent proteins. While genome sequencing has identified putative mistranslating tRNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. Here, we create a Drosophila melanogaster model for mistranslation by integrating a serine tRNA variant that mistranslates serine for proline (tRNA(Ser)[UGG, G26A]) into the fly genome. Using mass spectrometry, we find that tRNA(Ser)[UGG, G26A] misincorporates serine for proline at a frequency of ~ 0.6% per codon. This model will enable studies into the synergistic effects of mistranslating tRNA variants and disease-causing alleles.
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Project description:Transfer RNAs (tRNAs) maintain translational fidelity through strict charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic code is incorporated into a protein. Since alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in alanine tRNAs and the anticodon plays no role in charging, alanine tRNA variants with anticodon mutations have the potential to mistranslate alanine. Our goal was to quantify mis-incorporation of alanine into proteins in Saccharomyces cerevisiae strains expressing one of 57 different alanine tRNA anticodon variants. Using mass spectrometry, we observed mistranslation for 45 of the variants when expressed on single-copy plasmids.
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Project description:Altering the genetic code for applications in synthetic biology and genetic code expansion involves engineered tRNAs that incorporate amino acids that differ from what is defined by the “standard” genetic code. Since these engineered tRNA variants can be lethal due to proteotoxic stress, regulating their expression is necessary to achieve high levels of the resulting novel proteins. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II) transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4 or TetR-VP16 activated promoters downstream of a mistranslating tRNA serine variant that mis-incorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using mass spectrometry, we determine th frequency of mistranslation in both the induced and repressed conditions of the galactose inducible and tetracycline inducible systems.
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Project description:By physically linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some natural human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. Recent work showed a naturally occurring tRNASer variant decodes phenylalanine codons with serine and inhibits protein synthesis. We hypothesized that human tRNA variants that mis-read glycine (Gly) codons with alanine (Ala) will disrupt protein homeostasis. The A3G mutation occurs naturally in tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3:U70). Because AlaRS does not recognize the anticodon, the human tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNAGly and tRNAAla variants had no effect on protein synthesis in mammalian cells under normal growth conditions, however, tRNAGlyGCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at several Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC 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.
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Project description:Mistranslation, the mis-incorporation of an amino acid not specified by the “standard” genetic code, occurs in all cells. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. The goal of this study was to assess the transcriptome changes in yeast cells expressing two different mistranslating tRNA variants which mistranslate at similar frequencies (of ~ 3%) but result in different substitutions, namely alanine at proline codons or serine at arginine codons.
2021-05-11 | GSE174145 | GEO
Project description:tRNAs are encoded by a large gene family, usually with several isogenic tRNAs interacting with the same codon. Mutations in the anticodon region of other tRNAs can overcome specific tRNA deficiencies. Phylogenetic analysis suggests that such mutations have occurred in evolution, but the driving force is unclear. We show that in yeast suppressor mutations in other tRNAs are able to overcome deficiency of the essential TRT2-encoded tRNAThrCGU at high temperature (40°C). Surprisingly, these tRNA suppressor mutations were obtained after whole-genome transformation with DNA from thermotolerant Kluyveromyces marxianus or Ogataea polymorpha strains, but from which the mutations did apparently not originate. We suggest that transient presence of donor DNA in the host facilitates proliferation at high temperature and thus increases the chances for occurrence of spontaneous mutations suppressing defective growth at high temperature. Whole-genome sequence analysis of three transformants revealed only four to five non-synonymous mutations of which one causing TRT2 anticodon stem stabilization and two anticodon mutations in non-threonyl-tRNAs, tRNALysCUU and tRNAeMetCAU, were causative. Both anticodon mutations suppressed lethality of TRT2 deletion and apparently caused the respective tRNAs to become novel substrates for threonyl-tRNA synthetase. LC-MS/MS data could not detect any significant mistranslation and RT-qPCR results contradicted induction of the unfolded protein response. We suggest that stress conditions have been a driving force in evolution for the selection of anticodon-switching mutations in tRNAs as revealed by phylogenetic analysis. Importance of the work In this work we have identified for the first time the causative elements in a eukaryotic organism introduced by applying whole-genome transformation and responsible for the selectable trait of interest, i.e. high temperature tolerance. Surprisingly, the whole-genome transformants contained just a few SNPs, which were unrelated to the sequence of the donor DNA. In each of three independent transformants, we have identified a SNP in a tRNA, either stabilizing the essential tRNAThrCGU at high temperature or switching the anticodon of tRNALysCUU or tRNAeMetCAU into CGU, which is apparently enough for in vivo recognition by threonyl-tRNA synthetase. LC-MS/MS analysis indeed indicated absence of significant mistranslation. Phylogenetic analysis showed that similar mutations have occurred throughout evolution and we suggest that stress conditions may have been a driving force for their selection. The low number of SNPs introduced by whole-genome transformation may favor its application for improvement of industrial yeast strains.
2021-09-10 | PXD024655 | Pride
Project description:The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or post-translational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and provide support for the concept that mistranslation is a mechanism for cellular resiliency against environmental stress.
2023-10-24 | PXD041335 | Pride
Project description:Mistranslation describes the misincorporation of an amino acid into a nascent polypeptide. Mistranslation has diverse effects on multicellular eukaryotes and is implicated in several human diseases. We introduced a mistranslating serine transfer RNA (tRNA) into Drosophila melanogaster that misincorporates serine at proline codons and found that it affected male and female flies differently. Here, we compare the transcriptomic response of male and female flies to mistranslation to identify cellular pathways that underlie this sex-specific response. Both males and females downregulated genes associated with metabolism in response to proline-to-serine mistranslation. Only males downregulated genes associated with developmental processes and response to negative stimuli such as infection, whereas only females downregulated aerobic respiration and ATP synthesis genes. Both sexes upregulated genes associated with gametogenesis but females upregulated cell cycle and DNA maintenance genes, suggesting that mistranslation may compromise genome integrity in females. This transcriptomic analysis advances our understanding of how males and females respond to mistranslation and has important implications for future studies that examine the influence of mistranslation on disease.
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